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Sample records for enzyme immunoassay technique

  1. Advanced techniques in immunoassay

    International Nuclear Information System (INIS)

    Toth, G.

    1982-01-01

    A brief overview of the development history of radioimmunoassay and related techniques with their theory and practice are given. A comparison of radioimmunoassay (RIA), enzyme immunoassay (EIA), spin immunoassay (SIA), sequential saturation analysis (SSA) etc., based on their main parameters, and their fields of application and recent trends are presented. (Sz.J.)

  2. Thermophilic Campylobacter spp. in turkey samples: evaluation of two automated enzyme immunoassays and conventional microbiological techniques

    DEFF Research Database (Denmark)

    Borck, Birgitte; Stryhn, H.; Ersboll, A.K.

    2002-01-01

    Aims: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. Methods and Results: A total of 286 samples (faecal, meat...

  3. Evaluation of enzyme immunoassay techniques for diagnosis of the most common intestinal protozoa in fecal samples.

    Science.gov (United States)

    Gaafar, Maha R

    2011-08-01

    This study was designed to evaluate the antigen capture enzyme immunoassays (EIAs) Triage parasite panel and TechLab Entamoeba histolytica II in detecting Giardia intestinalis, Cryptosporidium sp, and Entamoeba histolytica in fecal samples in comparison to microscopy, and in differentiating Entamoeba histolytica from Entamoeba dispar. The Triage EIA was evaluated using 100 stool specimens that were tested by standard ova and parasite examination, including staining with both trichrome and modified acid-fast stains. Differentiation between E. histolytica and E. dispar was performed using TechLab. Microscopic examination revealed that 19% of the samples were positive for Giardia, 4% for Cryptosporidium, and 1% for E. histolytica/E. dispar, and other parasites were found in 5%. By Triage, 23% of the samples were infected with Giardia, 5% with Cryptosporidium, and 2% with E. histolytica/E. dispar. Triage showed a sensitivity of 100% and specificity of 91.5%. The TechLab assay was negative for both samples diagnosed as E. histolytica/E. dispar by Triage, which suggested that they were E. dispar. Both tests showed no cross-reactivity with other intestinal protozoa. These results indicate that antigen detection by EIA has the potential to become a valuable tool, capable of making stool diagnostics more effective. Copyright © 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  4. The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A after renal transplantation

    Directory of Open Access Journals (Sweden)

    Xiao-Hui Luo

    2011-05-01

    Full Text Available The feasibility and the clinical value of the enzyme-multiplied immunoassay technique (EMIT monitoring of blood concentrations of cyclosporine A (CsA in patients treated with CsA were investigated after kidney transplantation. The validation method was performed to the EMIT determination of CsA blood concentration, the CsA whole blood ‘trough concentrations (C0 of patients in different time periods after renal transplantation were monitored, and combined with the clinical complications, the statistical results were analyzed and compared. EMIT was precise, accurate and stable, also with a high quality control. The mean postoperative blood concentration of CsA was as follows: 12 months, (185.6 ± 28.1ng/mL. The toxic reaction rate of CsA blood concentration within the recommended therapeutic concentration was 14. 1%, significantly lower than that of the none-recommended dose group (37.2% (P < 0.05; the transplantation rejection rate was 4.4%, significantly lower than that of the none-recommended dose group (22.5% (P < 0.05. Using EMIT to monitor the blood concentration of CsA as the routine laboratory method is feasible, and is able to reduce the CsA toxicity and rejection significantly, leading to achieving the desired therapeutic effect. Keywords: enzyme-multiplied immunoassay technique, renal transplantation, cyclosporin A, blood concentration monitoring

  5. [Automated analyzer of enzyme immunoassay].

    Science.gov (United States)

    Osawa, S

    1995-09-01

    Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

  6. Survey of immunoassay techniques for biological analysis

    International Nuclear Information System (INIS)

    Burtis, C.A.

    1986-10-01

    Immunoassay is a very specific, sensitive, and widely applicable analytical technique. Recent advances in genetic engineering have led to the development of monoclonal antibodies which further improves the specificity of immunoassays. Originally, radioisotopes were used to label the antigens and antibodies used in immunoassays. However, in the last decade, numerous types of immunoassays have been developed which utilize enzymes and fluorescent dyes as labels. Given the technical, safety, health, and disposal problems associated with using radioisotopes, immunoassays that utilize the enzyme and fluorescent labels are rapidly replacing those using radioisotope labels. These newer techniques are as sensitive, are easily automated, have stable reagents, and do not have a disposal problem. 6 refs., 1 fig., 2 tabs

  7. Immunoassay separation technique

    International Nuclear Information System (INIS)

    1977-01-01

    A method for effecting the immunoassay of a multiplicity of samples, each possibly containing an antigen or an antibody to be assayed, is discussed. Each sample is incubated with a solution containing a detectable antigen or antibody to form a multiplicity of mixtures, each mixture containing as components antigen-antibody, non-complexed antigen and non-complexed antibody. At least one of the components of the said mixture is separated by adsorption. There after, quantity of detectable antigen or antibody is detected in one of the non-adsorbed portions of the mixture. An improvement, compared to other techniques, is the continuous and sequential separation of at least one component, which is intended to be separated from each said multiplicity of mixtures

  8. Review of the biochemical basis of enzyme immunoassays

    International Nuclear Information System (INIS)

    Klingler, W.

    1982-01-01

    The ever increasing number of radioimmunological determination poses problems allied with the handling of radioactive substances. In recent years various non-radioactive methods have been developed, among which the enzyme immunoassay is already in routine use. Homogeneous and heterogeneous enzyme immunoassays are described. Criteria for enzymes, substrates and enzyme-substrate reactions are listed. (orig.) [de

  9. Determining total thyroxine with the aid of the homogeneous enzyme immunoassay

    International Nuclear Information System (INIS)

    Vogt, W.

    1978-01-01

    Total thyrozine values obtained with the aid of the homogeneous enzyme immunoassay are compared with those delivered by the RIA and the efficiency of the EMIT technique is evaluated. Some results obtained via Enzymun-T4, a heterologous enzyme immunoassay, are also given. (VJ) 891 VJ [de

  10. Enzyme immunoassay for DDT analysis in Lebanese soils

    International Nuclear Information System (INIS)

    Bashour, I.; Dagher, S.; Shammas, G.; Sukkariyah, B.; Kawar, N.

    2000-01-01

    Full text: The use of enzyme-linked immunosorbent assay (ELISA) technique in estimating pesticide residue in soils is a faster, less expensive and easier method to use than the gas chromatography (GC) analysis technique..In the test, DDT pesticide residues in the simple compete with enzyme (horseradish peroxidase)-labeled DDT for a limited number of antibody binding sites on the inside surfaces of the test wells; the envirologix plate kit was tested for the measurement of total DDT in virgin and fortified (0-1000 ng g exp-1) soil samples of different properties from Lebanon. Extraction of DDT from soil was done by shaking the samples for 16 hours on a mechanical shaker with 90% methanol without any clean-up steps. Then the samples were allowed to stand for 30 minutes and an aliquot was taken from the clear supernatant. The DDT in the extract was measured in triplicate by GC and ELISA. The results indicated that the two techniques were highly correlated (r2 =0.9671-0.9973). Differences in soils physical and chemical properties did not accuracy of the detection limits of ELISA when compared to GC-ECD results. Immunoassay technique is a suitable method for rapid and accurate measurement of DDT residue in mineral Lebanese soils

  11. Preparation by irradiation of a solid support for enzyme immunoassay

    International Nuclear Information System (INIS)

    Kumakura, M.; Kaetsu, I.

    1984-01-01

    Reagents (immobilized anti-α-fetoprotein discs) having a porous structure were prepared for enzyme immunoassay of α-fetoprotein by radiation polymerization at low temperatures. Discs were attached to sticks for easy handling. The activity (determined by absorbance at 492 nm) of the discs varied with the hydrophilic properties and size of the discs. The discs are sufficiently sensitive and precise for enzyme immunoassay of α-fetoprotein. Anti-AFP dissolved in PBS solution was mixed with a monomer solution of hydroxyethyl methacrylate and hydroxypropyl methacrylate. The mixture was frozen to -78 0 C and gamma irradiated. (Auth.)

  12. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    International Nuclear Information System (INIS)

    Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

    1981-01-01

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10 5 and greater than or equal to 3 X 10 6 USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10 5 and greater than or equal to10 4 USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described

  13. Determination of digoxin by enzyme immunoassay and radioimmunoassay

    International Nuclear Information System (INIS)

    Mueller, H.; Braeuer, H.; Foerster, G.; Reinhardt, M.

    1978-01-01

    The results of parallel determinations of digoxin in the sera of non selected patients (n=104) by enzyme immunoassay (EMIT.EIA) and radioimmunoassay (J-125 labeled RIA) were compared with each other. The determinations revealed considerably different concentrations; the values determined by EIA were statistical lower (for EIA 1,09+'0,99ng/ml, for RIA 1,34+'1,01ng/ml, p [de

  14. Comparative determination of phenytoin by spectrophotometry, gas chromatography, liquid chromatography, enzyme immunoassay, and radioimmunoassay

    International Nuclear Information System (INIS)

    Castro, A.; Ibanez, J.; DiCesare, J.L.; Adams, R.F.; Malkus, H.

    1978-01-01

    Sera from patients being treated with phenytoin were analyzed for the drug by spectrophotometry, gas chromatography, radioimmunoasay, enzyme immunoassay, and liquid chromatography. The assay values obtained were intercompared statistically. Enzyme immunoassay and liquid chromatography appear to be attractive alternatives to the more traditional methods of spectrophotometry and gas chromatography. Our radioimmunoassay data correlated poorly with results by the four other methods

  15. Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

    Science.gov (United States)

    Groome, N P

    1981-10-01

    The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

  16. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    Science.gov (United States)

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

    2016-07-01

    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  17. Evidence for carcinoembryonic antigen using radioimmunoassay and enzyme immunoassay

    International Nuclear Information System (INIS)

    Kungda Gao, L.

    1980-01-01

    A commercially available radioimmunoassay for the determination of carcinoembryonic antigen (CEA) was initially compared with an enzyme immunoassay (EIA). Considerable differences were found between the individual value. For three patients suffering from carcinomas of the digestive tract a better indication of the disease was given in the RIA than in the EIA. A further 110 patients with various illnesses were examined for serum CEA-levels using RIA. A method for measuring CEA in feces by RIA was developed. The normal range lies below 300 ng/ml. This assay could be of significance for the early recognition of colo-rectal carcinoma. In part II of this dissertation CEA was isolated from colo-rectal carcinomas using three different gel filtration media. It was only possible to obtain almost pure CEA (24 μg CEA per μg protein) by one of the methods. Six guinea pigs were immunized with the isolated CEA and all developed antibodies. The isolated CEA was labelled with 125 I and an own RIA saturation sequence and double antibody separation was developed. One of the antisera was able to distinguish without overlap 7 healthy patients from 7 suffering from colo-rectal carcinomas in non-extracted serum. (orig./MG) [de

  18. Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

    Science.gov (United States)

    Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

    2014-06-01

    In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6 days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4 °C with no additive up to 30 days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

  19. Comparison of enzyme immunoassays for the diagnosis of bovine brucellosis

    International Nuclear Information System (INIS)

    Nielsen, K.; Kelly, L.; Gall, D.; Balsevicius, S.; Bosse, J.; Kelly, W.; Nicoletti, P.

    1998-01-01

    The indirect enzyme immunoassay for measurement of bovine antibody to Brucella abortus was tested on 15,716 Canadian sera to assess the specificity. These sera were also tested by the buffered plate antigen test. Two ELISA formats were used for assessment of data: the targeting procedure using a positive control serum allowed to develop to an optical density of 1.0 and the use of a positive control serum to determine relative positivity at a set time. Two different cut-off values were also assessed for each assay. A total of 763 sera gave reactions above established cut-off values in the ELISA while 216 were positive in the buffered plate antigen test (BPAT). A modification of the indirect ELISA employed divalent cation chelating agents (EDTA/EGTA) incorporated into the serum incubation stage to eliminate some non-specific reactions. This method was applied only to the 763 indirect ELISA reactor sera and it eliminated all but 93 or 37, depending on the cut-off selected, of the reactions. Sensitivity was assessed by testing 424 sera from Brucella abortus culture positive cattle. The indirect ELISA classified all 424 sera as positive by either method of data handling and with or without addition of EDTA/EGTA for a specificity estimate of 100%. In the BPAT, 412 sera gave a positive agglutination reaction. Ten percent of the 15,716 sera were randomly selected and tested by two different competitive ELISAs and by the complement fixation test (CFT). One competitive ELISA used Brucella abortus O-polysaccharide as the antigen and an enzyme conjugated monoclonal antibody to the O-polysaccharide for competition and detection. Of the sera tested, 34 gave false positive reactions. On a retest, the false positive reactions were reduced to 2. The second competitive ELISA used lipopolysaccharide as the antigen, a different monoclonal antibody but also specific for the O-polysaccharide for competition and commercially available goat anti-mouse IgG enzyme conjugate for detection

  20. A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

    Science.gov (United States)

    van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

    2015-05-01

    A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost

  1. Nitrocellulose membrane-based enzyme-linked immunoassay for dengue serotype-1 IgM detection

    International Nuclear Information System (INIS)

    Leon, S.; Guevara, C.; Chunga, A.

    1999-01-01

    To evaluate the sensitivity and specifity of a nitrocellulose membrane-based immunoassay for dengue IgM, with respect to capture enzyme immunoassay, for the diagnosis of dengue virus infection. 101 serum samples were processed and divided into 2 groups: 53 from dengue serotype 1 (DEN1) infected patients, and 48 from healthy subjects. Both groups were tested with a nitrocellulose membrane-based IgM capture enzyme immunoassay (NMB-EIA) and also with an ELISA as referential pattern. NMB-EIA testing detected IgM anti-DEN1 in 94,34% of samples from infected patients, and in 14,58% of control samples, whereas ELISA fails to report false positive or false negative results: NMB-EIA appears to be a good alternative for dengue infection diagnosis. (authors)

  2. Ervaringen met een solid phase enzyme immunoassay voor het aantonen van gonorroe bij promiscue vrouwen

    NARCIS (Netherlands)

    Ulsen; J.van*; Michel; M.F.*; Strik; R.van*; Joost; T.H.van*; Stolz; E.*; Eijk; R.V.W.van

    1985-01-01

    De Gonozyme test (Abbott Laboratories), een nieuwe enzyme immunoassay (EIA) voor het aantonen van Neisseria gonorrhoeae werd geevalueerd in een grote groep promiscue vrouwen. Als de EIA werd uitgevoerd met materiaal afkomstig van de cervix, bedroeg de prevalentie van gonorroe 8,2%. Vergeleken

  3. False-negative syphilis treponemal enzyme immunoassay results in an HIV-infected case-patient.

    Science.gov (United States)

    Katz, Alan R; Komeya, Alan Y; Tomas, Juval E

    2017-06-01

    We present a case report of a false-negative syphilis treponemal enzyme immunoassay test result in an HIV-infected male. While treponemal tests are widely considered to be more sensitive and specific than non-treponemal tests, our findings point to potential challenges using the reverse sequence syphilis screening algorithm.

  4. Competitive enzyme immunoassay for human chorionic somatomammotropin using the avidin-biotin system

    International Nuclear Information System (INIS)

    Rappuoli, R.; Leoncini, P.; Tarli, P.; Neri, P.

    1981-01-01

    Human chorionic somatomammotropin (HCS) is determined by an enzyme immunoassay where HCS competes with biotin-labeled HCS for insolubilized anti-HCS antibodies. Enzyme-labeled avidin is then used to reveal the amount of bound HCS. The system proves to be sensitive (1 ng/ml of HCS can be detected) and results agree with radioimmunoassay determinations (correlation coefficient = 0.979). Kinetics of the avidin-biotin reaction and coating of polystyrene wells are also investigated

  5. Comparative studies on the determination of alphafetoprotein by enzyme immunoassay and by radioimmunoassay

    International Nuclear Information System (INIS)

    Haller, G.; Linneke, P.; Voss, P.; Jeske, W.

    1987-01-01

    Alphafetoprotein (AFP) was determined in serum of pregnant women in the tenth till sixteenth week of pregnancy by means of two enzyme immunoassays (Enzymun-Test AFP, Boehringer Mannheim, FRG and AFP EIA 'Dessau' 1000, Research Institute for Vaccine Dessau, GDR) and a radioimmunoassay (Radioimmunoassay Kit, AFP-PR, CIS, France). Parallel determinations in sera of 438 patients, who had come to surveillance for the first consultation were estimated. A comparison between the methods showed a good correlation. (author)

  6. Evaluation of automated enzyme immunoassays for five anticonvulsants and theophylline adapted to a centrifugal analyzer.

    Science.gov (United States)

    Urquhart, N; Godolphin, W; Campbell, D J

    1979-05-01

    We report a clinical evaluation of the enzyme immunoassay (EMIT) performed with the GEMSAEC centrifugal analyzer as compared to gas-liquid and liquid chromatography for anticonvulsant drugs and theophylline, respectively. A good correlation was obtained for all drugs, although some difficulties were experienced with one lot of reagent for ethosuximide. The analyzer has an economic advantage if many samples are being analyzed for few drugs in each sample.

  7. Comparison of Salivary and Serum Enzyme Immunoassays for the Diagnosis of Helicobacter pylori Infection

    Directory of Open Access Journals (Sweden)

    John M Embil

    1998-01-01

    Full Text Available Infection with Helicobacter pylori has been established as an important risk factor for the development of peptic ulcer disease, gastritis and gastric cancer. The diagnosis of H pylori infection can be established by invasive or noninvasive techniques. Two noninvasive enzyme immunoassays (EIAs for antibody detection – HeliSal and Pylori Stat – were compared with histology. Both assays detect immunoglobulin (Ig G directed against purified H pylori antigen. The test populations consisted of 104 consecutive patients scheduled for upper gastrointestinal endoscopy. Of these patients, 97 (93% had symptoms compatible with peptic ulcer disease. Saliva and serum were collected simultaneously at the time of endoscopy. Salivary EIA had a sensitivity of 66%, specificity of 67%, positive predictive value of 67% and negative predictive value of 66% compared with the serum EIA, where the results were 98%, 48%, 64% and 96%, respectively. Although the salivary EIA is an appealing noninvasive test, it was not a sensitive and specific assay. The serum EIA also lacked specificity, but was highly sensitive with a good negative predictive value. Although a negative serum EIA rules out H pylori infection, a positive result must be interpreted in the clinical context and confirmed with a more specific measure.

  8. Chemiluminescence enzyme immunoassay based on magnetic nanoparticles for detection of hepatocellular carcinoma marker glypican-3

    Directory of Open Access Journals (Sweden)

    Qian-Yun Zhang

    2011-08-01

    Full Text Available Glypican-3 (GPC3 is reported as a great promising tumor marker for hepatocellular carcinoma (HCC diagnosis. Highly sensitive and accurate analysis of serum GPC3 (sGPC3, in combination with or instead of traditional HCC marker alpha-fetoprotein (AFP, is essential for early diagnosis of HCC. Biomaterial-functionalized magnetic particles have been utilized as solid supports with good biological compatibility for sensitive immunoassay. Here, the magnetic nanoparticles (MnPs and magnetic microparticles (MmPs with carboxyl groups were further modified with streptavidin, and applied for the development of chemiluminescence enzyme immunoassay (CLEIA. After comparing between MnPs- and MmPs-based CLEIA, MnPs-based CLEIA was proved to be a better method with less assay time, greater sensitivity, better linearity and longer chemiluminescence platform. MnPs-based CLEIA was applied for detection of sGPC3 in normal liver, hepatocirrhosis, secondary liver cancer and HCC serum samples. The results indicated that sGPC3 was effective in diagnosis of HCC with high performance. Keywords: Magnetic nanoparticle, Magnetic microparticle, Chemiluminescence enzyme immunoassay, Glypican-3, Hepatocellular carcinoma

  9. A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

    Science.gov (United States)

    Yu, Fei; Wu, Yongjun; Yu, Songcheng; Zhang, Huili; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B.

    With alkaline phosphatase (ALP)-adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR-McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350-1000 pg/mL and the detection limit of 0.24 ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%-96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

  10. The influence of intravenous canrenoate on the determination of digoxin in serum by radio- and enzyme-immunoassay

    International Nuclear Information System (INIS)

    Rietbrock, N.; Lichey, J.; Borner, K.; Freie Univ. Berlin

    1979-01-01

    Ten patients were kept on a constant maintenance dose of digoxin. During a baseline period of 6 days, blood samples were taken daily for analysis of digoxin in serum. On the 6th day the maintenance dose of digoxin was withheld and a single intravenous dose of 200mg potassium-canrenoate (AldactoneR) was administered to all patients. Digoxin in serum was determined by a classical radioimmunoassay with 125 I-digoxin and solid phase technique (RIA-NEN) and partly by a heterogenous enzyme-immunoassay (EnzymunR-Digoxin, Boehringer, Mannheim). Results of the radioimmunoassay indicated a rise of apparent serum digoxin levels with an average maximum of 201% of the mean baseline value 30 min after injection of canrenoate and a gradual return to the baseline value within 6 to 10 hours. Contrary to the radioimmunoassay there was no interference when using the enzyme-immunoassay in a subgroup of identical serum samples: serum digoxin levels remained constant throughout the test. Interference of determinations of digoxin in serum by spironolactone and its metabolites appear to be related to two factors: 1. The mode of administration and the amount of interfering drug, 2. the specifity of the digoxin antibody used in the kit. (orig.) [de

  11. Enzyme immunoassay

    DEFF Research Database (Denmark)

    Feldt-Rasmussen, B; Dinesen, B; Deckert, M

    1985-01-01

    variation of the 24 h urine albumin excretion of different days was high in patients with incipient diabetic nephropathy (51.5%) and was only slightly reduced by taking the variation of creatinine excretion into account (39.5%). No correlation was found between albumin excretion, and HbA1c or urine glucose...

  12. Radiolabelling for immunoassay

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    Since the early 1960s labelled compounds employed in immunoassay techniques, both radioimmunoassay and immunoradiometric assay, have involved radioisotopes typically 3 H (tritium) and 125 Iodine. With the advent of increasingly stringent governmental regulations regarding usage and disposal of radioisotopes and the impetus of research towards improved immunoassay sensitivity following the discovery of monoclonal antibodies and their application to excess reagent immunometric assay methodology, radioisotopic labels are gradually being replaced by non-isotopic labels: enzyme, fluorescence and chemiluminescence

  13. Detection of virus-specific intrathecally synthesised immunoglobulin G with a fully automated enzyme immunoassay system

    Directory of Open Access Journals (Sweden)

    Weissbrich Benedikt

    2007-05-01

    Full Text Available Abstract Background The determination of virus-specific immunoglobulin G (IgG antibodies in cerebrospinal fluid (CSF is useful for the diagnosis of virus associated diseases of the central nervous system (CNS and for the detection of a polyspecific intrathecal immune response in patients with multiple sclerosis. Quantification of virus-specific IgG in the CSF is frequently performed by calculation of a virus-specific antibody index (AI. Determination of the AI is a demanding and labour-intensive technique and therefore automation is desirable. We evaluated the precision and the diagnostic value of a fully automated enzyme immunoassay for the detection of virus-specific IgG in serum and CSF using the analyser BEP2000 (Dade Behring. Methods The AI for measles, rubella, varicella-zoster, and herpes simplex virus IgG was determined from pairs of serum and CSF samples of patients with viral CNS infections, multiple sclerosis and of control patients. CSF and serum samples were tested simultaneously with reference to a standard curve. Starting dilutions were 1:6 and 1:36 for CSF and 1:1386 and 1:8316 for serum samples. Results The interassay coefficient of variation was below 10% for all parameters tested. There was good agreement between AIs obtained with the BEP2000 and AIs derived from the semi-automated reference method. Conclusion Determination of virus-specific IgG in serum-CSF-pairs for calculation of AI has been successfully automated on the BEP2000. Current limitations of the assay layout imposed by the analyser software should be solved in future versions to offer more convenience in comparison to manual or semi-automated methods.

  14. The Use of Recombinant Hemagglutinine Protein of Rinderpest Virus in Enzyme Immunoassay

    OpenAIRE

    BULUT, Hakan; BOLAT, Yusuf

    2003-01-01

    In this study, Rinderpest virus (RPV) recombinant hemagglutinine protein (rH) fused with protein A region of Staphylococcus aureus was expressed in Escherichia coli and purified by IgG affinity chromatography. rH protein was also used to establish enzyme immunoassay. Therefore, to prevent IgG binding to the protein A the wells coated with the rH proteins were blocked by human serum. Afterwards, RPV antigens were added to the wells to evaluate this assay. To this end, serum from mice immunized...

  15. Enzyme immunoassay of oestrogen receptors in needle biopsies from human liver

    DEFF Research Database (Denmark)

    Becker, U; Andersen, J; Poulsen, H S

    1991-01-01

    For quantitative assessments of sex hormone receptors in liver tissue, ligand binding assays are inconvenient, as they require large biopsies (0.5-1.0 g). The present study shows that it is possible to measure oestrogen receptors (ER) quantitatively in needle biopsy specimens as small as 10 mg...... by modifications of a commercial enzyme immunoassay employing monoclonal antibodies. Sucrose gradient centrifugation and the dextran charcoal method served as reference methods. A consecutive series of needle biopsies from patients suspected of liver disease were investigated. The biopsies (n = 37) had a median...

  16. The management of isolated positive syphilis enzyme immunoassay results in HIV-negative patients attending a sexual health clinic.

    Science.gov (United States)

    Thorley, Nicola; Adebayo, Michael; Smit, Erasmus; Radcliffe, Keith

    2016-08-01

    An unconfirmed positive treponemal enzyme immunoassay (enzyme immunoassay positive, Treponema pallidum particle agglutination negative and rapid plasma reagin negative) presents a clinical challenge to distinguish early syphilis infection from false-positive results. These cases are referred for syphilis line assay (INNO-LIA) and recalled for repeat syphilis serology. We performed a retrospective audit to establish the proportion of HIV-negative cases with unconfirmed positive enzyme immunoassay results, the proportion of these cases that received an INNO-LIA test and repeat syphilis serology testing and reviewed the clinical outcomes; 0.35% (80/22687) cases had an unconfirmed positive treponemal enzyme immunoassay result. Repeat syphilis serology was performed in 80% (64/80) cases, but no additional cases of syphilis were identified. Eighty-eight per cent (70/80) received an INNO-LIA test; 14% (5/37) unconfirmed enzyme immunoassay-positive cases with no prior history of syphilis were confirmed on INNO-LIA assay, supporting a diagnosis of latent syphilis. As a confirmatory treponemal test, the INNO-LIA assay may be more useful than repeat syphilis serological testing. © The Author(s) 2016.

  17. Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities.

    Science.gov (United States)

    Warkentin, Theodore E; Sheppard, Jo-Ann I; Chu, F Victor; Kapoor, Anil; Crowther, Mark A; Gangji, Azim

    2015-01-01

    Repeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera-when first testing SRA-negative-continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive. © 2015 by The American Society of Hematology.

  18. Evaluation and comparison of radio-, fluorescence, and enzyme-linked immunoassays for serum thyroxine

    International Nuclear Information System (INIS)

    Kaplan, L.A.; Gau, N.; Fearn, J.; Steain, E.A.; Chen, I.W.; Maxon, H.; Volle, C.

    1981-01-01

    We have compared three analytical systems for the measurement of serum thyroxine: enzyme-linked immunoassay (EIA), fluorescent immunoassay (FIA) and a radioimmunoassay (RIA). These were evaluated with respect to their precision, accuracy, analytical sensitivity and sample throughput. The RIA is more sensitive than the EIA (10 μg/L vs. 35 μg/L). Both systems have excellent precision (X=86 μg/L, C.V.sub(RIA)=C.V.sub(EIA)=4.6 percent). Both the EIA and RIA demonstrate good accuracy with recovery of between 97-98 percent of added thyroxine. The FIA has an apparent sensitiviity between that of the RIA and EIA (25 μg/L), but a precision consistently lower than the other two systems (C.V. =7.4 percent, X=86 μg/L). Patients' results by RIA compared well with those from EIA (r=0.91,P 0.05). Although not fully automated, the EIA performed on the Abbott ABA-100 analyzer has a sample throughput equal to the automated RIA system (Micromedic, Concept 4)

  19. Study on the determination of human placental lactogen (HPL) using an enzyme-immunoassay. Comparison with a commercial radio-immunoassay in the course of normal pregnancies

    International Nuclear Information System (INIS)

    Schneider, B.

    1982-01-01

    A novel enzyme-immunoassay (EIA) for determining human placental lactogen (HPL) was studied for its practicability and quality. The precision of the system in series was tested by using a serum taken each in the 19th, 29th and 40th pregnancy week. A normal range graph between the 10th and the 40th pregnancy week (10 sera per pregnancy week) was established from 310 sera of normal-course pregnancies. The graph practically agreed with the known RIA-established graphs. When comparing with a radio-immunoassay for HPL of routine application and known quality criteria, r=0.93 indicated a close correlation of the values found. (orig./MG) [de

  20. Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

    Science.gov (United States)

    Thurman, E.M.; Aga, D.S.

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  1. Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

    Science.gov (United States)

    Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

    1993-08-01

    A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

  2. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

  3. Detection of hidden hazelnut protein in food by IgY-based indirect competitive enzyme-immunoassay

    NARCIS (Netherlands)

    Baumgartner, S.; Bremer, M.G.E.G.; Kemmers - Voncken, A.E.M.; Smits, N.G.E.; Haasnoot, W.; Banks, J.; Reece, P.; Danks, C.; Tomkies, V.; Immer, U.; Schmitt, K.; Krska, R.

    2004-01-01

    The development of an indirect competitive enzyme-immunoassay for the detection of hidden hazelnut protein in complex food matrices is described. A sensitive and selective polyclonal antibody was raised by immunisation of laying hens with protein extracts from roasted hazelnuts. In contrast to

  4. Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Nielsen, E.M.; Stryhn, H.

    1999-01-01

    We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests...

  5. Quantification of patient specific assay interference in different formats of enzyme linked immunoassays for therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Grebenchtchikov, N.J.; Geurts-Moespot, A.; Heijmen, L.; Laarhoven, H.W.M. van; Herpen, C.M.L. van; Thijs, A.M.J.; Span, P.N.; Sweep, F.C.

    2014-01-01

    BackgroundThe use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are

  6. Quantification of patient-specific assay interference in different formats of enzyme-linked immunoassays for therapeutic monoclonal antibodies

    NARCIS (Netherlands)

    Grebenchtchikov, Nicolai; Geurts-Moespot, Anneke J.; Heijmen, Linda; van Laarhoven, Hanneke W. M.; van Herpen, Carla M. L.; Thijs, Annemarie M. J.; Span, Paul N.; Sweep, Fred C. G. J.

    2014-01-01

    The use of therapeutic monoclonal antibodies for clinical purposes has significantly increased in recent years, and so has the need to monitor antibody concentrations. This may be achieved using the well-established enzyme linked immunoassay (ELISA) methods; however, these assays are subject to a

  7. Comparative digoxin determination in serum by means of a radioimmuno- and an enzyme immunoassay

    International Nuclear Information System (INIS)

    Reinhard, M.

    1981-01-01

    Two immunologic measuring methods for the quantitative digoxin determination in serum were compared. One method bases on the principle of radioisotope dilution, the second one on the principle of enzyme inhibition. The radioimmunoassay served as reference method. The limit of detection for RIA is 0.23 ng/ml, for EIA 0.40 ng/ml. For both methods the measuring range extends up to approx. 5.5 ng/ml. The degree of precision in series is 8.2% for RIA, 10.8% for EIA. Day-to-day precision is 4.4% for RIA, 15.2% for EIA. On comparison, the 59 serum samples of patients who received digoxin did not show any systematic difference. The results obtained can be transformed by means of the equations Csub(EIA) = 0.041 ng/ml + 0.936 Csub(RIA). In pathologic sera, however, there are significant differences disfavoring EIA, because due to high color concentrations or turbidities these sera do not permit any or any exact extinction measurements. The enzyme immunoassay should not be used with such sera. With regard to practicability the EIA corresponds more or less to RIA. The EIA can essentially be economized by using semi-microcuvettes and applying only the half of the recommended enzyme and antibody volume. (orig.) [de

  8. Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

    Science.gov (United States)

    Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

    2010-02-15

    A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

  9. Erythema migrans and serodiagnosis by enzyme immunoassay and immunoblot with three borrelia species.

    Science.gov (United States)

    Stanek, G; Breier, F; Menzinger, G; Schaar, B; Hafner, M; Partsch, H

    1999-12-10

    There is wide divergence of opinion between physicians regarding the use of serological measures for the diagnosis and treatment of erythema migrans, the hallmark of Lyme borreliosis. We studied the outcome of an enzyme immunoassay and immunoblot (Western blot) used on the sera of patients who had suffered tick bite and erythema migrans, and had been subsequently treated with various antibiotics. Ninety-nine consecutive patients presenting with erythema migrans after tick bite were prospectively recruited at the outpatient department of two Vienna City hospitals and at the consultation office for Lyme borreliosis of the Institute of Hygiene. University Vienna. Blood samples were taken before antibiotic treatment and 3 and 6 months thereafter. Blood samples from 100 blood donors served as controls. Antibodies against Borrelia burgdorferi sensu lato were determined by enzyme immunoassay (IgG and IgM EIA) and by IgG immunoblot. The latter was performed with isolates of B. alzelii (H2) B. burgdorferi sensu stricto (Le) and B. garinii (W) from Austrian patients. The 4 interpretation criteria for immunoblot results were: A (3 bands out of 8), B (2 bands out of 9), C and D (1 band out of 6). In all patients, the erythema resolved within the treatment period. No complications secondary to the borrelia infection were registered. After treatment there was no significant change in titre, nor was there a difference in the immunoblot pattern between the first, second and third serum samples. Serum antibodies to B. burgdorferi were positive by EIA in 22.9% (IgG) and 2.5% (IgM). Immunoblot results offered by borrelia species and by the interpretation criteria, ranging between 8.3% (criterion A, strain Le) and 44.2% (criterion D, strain H2). By EIA, control samples were IgG and IgM positive in 5% and 1%, respectively. Positive immunoblot results with strain H2 were found in 9%, 13%, 18%, and 20% by the criteria A through D respectively. After antibiotic treatment of erythema

  10. Application of a Newly Developed High-Sensitivity HBsAg Chemiluminescent Enzyme Immunoassay for Hepatitis B Patients with HBsAg Seroclearance

    OpenAIRE

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-01-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstr...

  11. Design of a single-step immunoassay principle based on the combination of an enzyme-labeled antibody release coating and a hydrogel copolymerized with a fluorescent enzyme substrate in a microfluidic capillary device.

    Science.gov (United States)

    Wakayama, Hideki; Henares, Terence G; Jigawa, Kaede; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-11-21

    A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

  12. Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.

    Science.gov (United States)

    Liu, Xing; Tang, Zongwen; Duan, Zhenhua; He, Zhenyun; Shu, Mei; Wang, Xianxian; Gee, Shirley J; Hammock, Bruce D; Xu, Yang

    2017-03-01

    A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC 50 of 0.64ng/mL and a linear range (IC 20 -IC 80 ) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.

    Science.gov (United States)

    Ordronneau, P; Abdullah, L H; Petrusz, P

    1991-09-13

    In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.

  14. [Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and anatoxins].

    Science.gov (United States)

    Burkin, M A; Gal'vidis, I A; Iakovleva, I V; Sviridov, V V

    2007-01-01

    Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG1 monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse anatoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and anatoxins. The detection sensitivity of diphtheria toxin/anatoxin equaled 0.0005 Lf/ml; tetanus toxin and anatoxin were detected with sensitivities of 20 LD50/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum anatoxins (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E anatoxin (0.001 UI/ml).

  15. Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

    Science.gov (United States)

    Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

    1994-03-01

    Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

  16. Effects of saliva collection using cotton swab on cortisol enzyme immunoassay.

    Science.gov (United States)

    Kozaki, Tomoaki; Hashiguchi, Nobuko; Kaji, Yumi; Yasukouchi, Akira; Tochihara, Yutaka

    2009-12-01

    Cotton swabs are among the most commonly used devices for collecting saliva, but various studies have reported that their use impacts the results of salivary cortisol assays. These studies, however, estimated this impact by comparing the average of the concentration and/or scatter plots. In the present study, we estimated the impact of cotton swabs on the results of salivary cortisol enzyme immunoassay (EIA) by Bland-Altman plot. Eight healthy males (aged 20-23 years) provided four saliva samples on different days to yield a total of 32 samples. Saliva samples were collected directly in plastic tubes using plastic straws and then pipetted onto cotton swabs (cotton saliva collection) and into clear sterile tubes (passive saliva collection). There was a lower correlation between cotton and passive saliva collection. Individually, four subjects showed a negative correlation between passive and cotton saliva collection. A Bland-Altman plot indicated that cotton swabs causes a proportional bias on the EIA assay result. Our findings indicate a considerable effect of using cotton swabs for saliva collection, and subject-specific variability in the impact. A Bland-Altman plot further suggests possible reasons for this effect.

  17. A sensitive chemiluminescence enzyme immunoassay based on molecularly imprinted polymers solid-phase extraction of parathion.

    Science.gov (United States)

    Chen, Ge; Jin, Maojun; Du, Pengfei; Zhang, Chan; Cui, Xueyan; Zhang, Yudan; She, Yongxin; Shao, Hua; Jin, Fen; Wang, Shanshan; Zheng, Lufei; Wang, Jing

    2017-08-01

    The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74-18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1.12, 1.17, and 0.85 for apple, rice, orange and cabbage in samples pretreated with the mixture of PSA and C 18 . However, the ratio of sample (apple, rice, orange, and cabbage) matrix-matched standard-MIPs curve slope rate to the solvent standard curve was 1.05, 0.92, 1.09, and 1.05 in samples pretreated with MIPs, respectively. The results demonstrated that the matrices of the samples greatly interfered with the detection of parathion residues by CLEIA. The MIPs bound specifically to the parathion in the samples and eliminated the matrix interference effect. Therefore, the CLEIA method have successfully applied MIPs in sample pretreatment to eliminate matrix interference effects and provided a new sensitive assay for agro-products. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. The clinical application of fluorescent-enzyme immunoassay to detect human thyroid peroxidase autoantibody quantitatively

    International Nuclear Information System (INIS)

    Chai Jinyan; Fang Peihua; Li Ning; Zhang Yanli

    2010-01-01

    Objective: To establish a fluorescent-enzyme immunoassay (FEIA) with recombinant human thyroid peroxidase (hTPO) as the antigen. Methods: Sera of 326 healthy people, 119 cases of Hashimoto's thyroiditis (HT), 116 cases of Graves disease (untreated), 28 cases of nodular goiter, 10 ca-ses of subacute thyroiditis and 6 cases of simple goiter were measured by the FEIA with recombinant hTPO as the antigen. Rrank sum test and χ 2 -test were used in inter-groups. Results: (1) Concentration above 4000 U/L was considered to be positive. (2) The intra-assay coefficient of variation (CV) and the inter-as-say CV were 4.59% ∼ 6.52% and 17.37% ∼ 17.45%. (3) The values measured by the FEIA were positively correlated with the values measured by hTPO antibody (hTPOAb) commercial kit (r=0.80, P 2 = 53.45, 39.30, 15.41 and 21.74, all P < 0.01). Conclusions: The method can be applied in the serum measurement of thyroid disease. HT presented the highest positive rate, therefore, the FEIA was an effective method in diagnosing HT. (authors)

  19. Evaluation of electrochemiluminescene immunoassay and enzyme-linked immunosorbent assay for serum HBsAb detection

    International Nuclear Information System (INIS)

    Ma Caiyun; Jiang Li; Ge Gaoxia; Zhang Xiaojie

    2005-01-01

    Electrochemiluminescene immunoassay (ECLIA) and enzyme-linked immunosorbent (ELISA) were used to detect the different concentrations of serum HBsAb, in order to compare the results of ECLIA and ELISA. The result showed that intra-assay coefficient of variation of ECLIA was about 0.95% for high value, 1.13% for mean values and 2.63% for low value, while that of ELISA was about 5.76%, 12.8% and 15.9%, respectively. The interassay coefficient of ECLIA was about 4.03% for high values, 4.93% for mean values and 7.34% for low values, while that of ELISA was about 10.1%, 19.6% and 25.2%, respectively. The analytical sensitivity of ECLIA was about 4.0IU/L, while that of ELISA is about 19.0IU/L. Only in 3 samples, the results measured by ECLIA and ELISA were different (ECLIA: positive; ELISA: negative) among 165 samples. It is concluded that the met hod of ECLIA is more efficient than ELISA for detection of HBsAb in serum. (authors)

  20. A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

    Science.gov (United States)

    Feng, Xiao-Li; Ren, Hong-Lin; Li, Yan-Song; Hu, Pan; Zhou, Yu; Liu, Zeng-Shan; Yan, Dong-Ming; Hui, Qi; Liu, Dong; Lin, Chao; Liu, Nan-Nan; Liu, Yan-Yan; Lu, Shi-Ying

    2014-08-15

    Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Clinical significance of quantitative analysis of thyroid peroxidase antibody (TPOAb) with chemiluminescence enzyme immunoassay

    International Nuclear Information System (INIS)

    Zhu Cuiying; Wang Qing; Huang Gang

    2004-01-01

    Objective: The only method of laboratory diagnosis for autoimmune thyroid diseases used to be serum TGA and TMA detections. Morerecently, quantitative analysis of TPOAb has been introduced. To assess the relative sensitivity of these tests , positive rates detected with the respective tests were compared. Methods: Serum TGA, TMA (with RIA) and TPOAb (with chemiluminescence enzyme immunoassay) were simultaneously detected in 998 cases of thyroid diseases (hyperthyroidism 307, Hashimoto's disease 193, simple goiter 498). For complementary sake, fine needle aspiration cytology was obtained in a number of cases including all the patients with Hashimoto's disease. Results: Positive detection rate of TPOAb in three groups of patients (hyperthyroidism, Hashimoto's, simple goiter) was 81.76%, 96.89 % and 42.97% respectively. With TMA, the positive rate was only 54.72%, 65.80%, 22.09% respectively. About one third more cases would be detected with the newer method. Conclusion: For the laboratory detection of auto immune thyroid diseases, quantitative analysis of TPOAb is much wore sensitive than the conventional TMA detection. (authors)

  2. Enzyme immunoassays for the diagnosis of bovine brucellosis. Trial in Latin America

    International Nuclear Information System (INIS)

    Gall, D.; Nielsen, K.; Colling, A.; Marino, O.; Moreno, E.; Perez, B.; Samartino, L.

    1998-01-01

    The results of a field trail conducted in Latin America with two indirect (IELISA) and two competitive (CELISA) enzyme immunoassays for the detection of bovine antibody to Brucella abortus are reported. One of the CELISA formats performed most accurately. The relative sensitivity of this assay was 97.47%, the relative specificity for unexposed cattle was 98.32% and the specificity in cattle vaccinated with B. abortus strain 19 was 96.51%. The same assay format under Canadian conditions had a sensitivity of 100%, a specificity of 99.90% and a specificity of 97.7% in a strain 19 vaccinated population. Overall, the CELISA performed as expected and the results were not dissimilar to the results obtained in the Canadian study thus providing further evidence that this CELISA can in many instances differentiate infected cattle from those that are vaccinated or infected with a cross-reacting organism while still giving very low false positive or false negative results. (author)

  3. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    International Nuclear Information System (INIS)

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay

  4. Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

    OpenAIRE

    Turunen, H J

    1983-01-01

    A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of th...

  5. Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

    Science.gov (United States)

    Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

    2011-01-01

    A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

  6. Two competitive enzyme immunoassays for the detection of IgG class antibodies to hepatitis a antigen

    Directory of Open Access Journals (Sweden)

    Claudia Lamarca Vitral

    1991-06-01

    Full Text Available Two competitive enzyme immunoassays (EIA techniques were developed: in the first (COMP-1, test sera were added together with HAV antigen on anti-HAV IgG-coated wells followed by an anti-HA VHRP conjugate; in the second (COMP-2, test sera and anti-HA VHRP conjugate competed for HAV epitopes previously adsorbed to anti-HA V IgG-coated wells. Both procedures used tetramethylbenzidine (TMB as a substrate. Both competitive tests were shown to be reproducible and suitable for routine diagnosis and research purposes.Foram desenvolvidos dois ensaios imunoenzimáticos (EIA competitivos: no primeiro (COMP-1 colocou-se numa placa sensibilizada com anti-HAVIgG as amostras teste juntamente como antígeno HA Vea seguir o conjugado anti-HA VHRP; no segundo (COMP-2, as amostras teste e o conjugado anti-HAV HRP competem pelos epitopos do antígeno HAV previamente absorvido na placa sensibilizada do anti-HAV IgG. O substrato utilizado foi tetrametilbenzidina (TMB. Ambas as técnicas mostraram ser produtíveis e aplicáveis para fins de diagnóstico e pesquisa.

  7. Aspergillus Galactomannan Enzyme Immunoassay and Quantitative PCR for Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid

    Science.gov (United States)

    Musher, Benjamin; Fredricks, David; Leisenring, Wendy; Balajee, S. Arunmozhi; Smith, Caitlin; Marr, Kieren A.

    2004-01-01

    Invasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillus species from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillus antigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for additional costly and potentially morbid diagnostic evaluation. We evaluated the performance of a galactomannan enzyme immunoassay (GM EIA; Bio-Rad) by using a range of index cutoffs to define positivity and a quantitative PCR (qPCR) assay for the detection of Aspergillus species from BAL samples of patients with proven and probable IPA (case patients; n = 49) and without IPA (control patients; n = 50). The sensitivity of the GM EIA was 61% with an index cutoff of 1.0 and 76% with an index cutoff of 0.5; the corresponding specificities were 98 and 94%, respectively. The sensitivity and specificity of qPCR assay were 67 and 100%, respectively. The sensitivity with 22 culture-negative BAL specimens from patients with IPA was 41% for GM EIA with an index cutoff of 1.0, 59% for GM EIA with an index cutoff of 0.5, and 36% for qPCR assay. GM EIA indices and DNA quantities corresponded to BAL fungal burdens, with culture-positive samples having larger amounts of antigen and DNA compared to culture-negative samples. GM EIA and qPCR assay add to the sensitivity of BAL for diagnosing IPA in high-risk patients, with excellent specificity. Adjunctive use of these tests may reduce dependence on invasive diagnostic procedures. PMID:15583275

  8. The Significance of Isolated Reactive Treponemal Enzyme Immunoassay in the Diagnosis of Early Syphilis.

    Science.gov (United States)

    Caswell, Rachel J; Hathorn, Emma; Manavi, Kaveh

    2016-06-01

    The Treponemal test algorithm for syphilis screening is widely used. A diagnostic challenge between identifying early syphilis versus a false positive signal occurs in cases where the treponemal enzyme immunoassay (EIA) is reactive and confirmatory T. pallidum particle agglutination assay is negative. We investigated the diagnostic outcome of isolated reactive EIA in patients attending a sexual health clinic. Results of syphilis serology tests carried out at Birmingham Whittall Street Clinic between August 10, 2010, and November 31, 2014, were reviewed. Cases with isolated EIA were routinely invited for repeat syphilis serology. Outcomes of patients with isolated EIA were reviewed and the proportion with confirmed positive syphilis serology on their repeat test identified. The number of isolated EIA cases needed to retest to identify 1 case of early syphilis was calculated. A total of 121,724 syphilis screening tests were performed. Among the 1561 individual patients with reactive EIA sera, 316 (20% of total reactive tests) had isolated reactive EIA. Repeat syphilis serology results of 163 patients were reviewed; 106 patients remained with isolated reactive EIA, 50 had negative EIA test and 7 (4.3%) had confirmed reactive EIA. Of the 7 patients, 2 had evidence of early syphilis infection. The number of isolated EIA needed to retest to identify 1 case of early syphilis was 81.5 (95% confidence interval, 22.9-671.4). Routine recall of patients with isolated EIA sera is not warranted. Risk of acquisition or presence of early syphilis should be assessed independently and irrespective of a negative syphilis screening test or isolated EIA.

  9. Evaluation of Correlation between Pretest Probability for Clostridium difficile Infection and Clostridium difficile Enzyme Immunoassay Results.

    Science.gov (United States)

    Kwon, Jennie H; Reske, Kimberly A; Hink, Tiffany; Burnham, C A; Dubberke, Erik R

    2017-02-01

    The objective of this study was to evaluate the clinical characteristics and outcomes of hospitalized patients tested for Clostridium difficile and determine the correlation between pretest probability for C. difficile infection (CDI) and assay results. Patients with testing ordered for C. difficile were enrolled and assigned a high, medium, or low pretest probability of CDI based on clinical evaluation, laboratory, and imaging results. Stool was tested for C. difficile by toxin enzyme immunoassay (EIA) and toxigenic culture (TC). Chi-square analyses and the log rank test were utilized. Among the 111 patients enrolled, stool samples from nine were TC positive and four were EIA positive. Sixty-one (55%) patients had clinically significant diarrhea, 19 (17%) patients did not, and clinically significant diarrhea could not be determined for 31 (28%) patients. Seventy-two (65%) patients were assessed as having a low pretest probability of having CDI, 34 (31%) as having a medium probability, and 5 (5%) as having a high probability. None of the patients with low pretest probabilities had a positive EIA, but four were TC positive. None of the seven patients with a positive TC but a negative index EIA developed CDI within 30 days after the index test or died within 90 days after the index toxin EIA date. Pretest probability for CDI should be considered prior to ordering C. difficile testing and must be taken into account when interpreting test results. CDI is a clinical diagnosis supported by laboratory data, and the detection of toxigenic C. difficile in stool does not necessarily confirm the diagnosis of CDI. Copyright © 2017 American Society for Microbiology.

  10. Development and application of radioimmunoassay and enzyme immunoassays in microbiological and immunological diagnosis. 3. Comparative studies for the detection of virus antibodies with passive hemagglutination test, radioimmunoassay and enzyme immunoassay, resp

    Energy Technology Data Exchange (ETDEWEB)

    Lauf, H; Struy, H; Morenz, J [Medizinische Akademie, Magdeburg (German Democratic Republic)

    1982-06-01

    Radioimmuno- and enzyme immunoassays (solid phase RIA and ELISA) developed by the authors for the determination of antibodies of adeno-2- and parainfluenza-1-viruses are described and the detection sensibility for antibodies is compared with that of the conventional passive hemagglutination test. The sensibility of the radioimmunoassay for the detection of IgG antibodies against adeno-2-viruses is nearly 10 times higher than that of the passive hemagglutination. RIA and ELISA show no essential differences in their detection sensibilities in the detection of IgG antibodies against parainfluenza-1-viruses.

  11. Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle, buffaloes, and goats.

    Science.gov (United States)

    Yadav, R; Mohan, K; Kumar, V; Sarkar, M; Nitu, K; Meyer, H H D; Prakash, B S

    2013-08-01

    A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Sources of variation in an enzyme-linked immunoassay of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae).

    Science.gov (United States)

    Tabachnick, W J; Mecham, J O

    1991-03-01

    An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).

  13. Immunoassay technique

    International Nuclear Information System (INIS)

    Goodearl, P.D.

    1981-01-01

    For quantitatively assaying antigen in a fluid, a mixture thereof with further antigen, labelled for example by substitution or addition of a radioactive molecule is caused to flow along a paper wick or other carrier on which antibodies to the antigen have been immobilised, e.g. by activating the paper by application of cyanogen bromide, then bringing it into contact with a solution of the antibodies. The paper wick or other carrier is developed by causing a glycerol/water solution to rise through the wick by capillary action, and there is measured the distance travelled along the wick by the labelled antigen. (author)

  14. An enzyme immunoassay to quantify neurofilament light chain in cerebrospinal fluid.

    NARCIS (Netherlands)

    Geel, W.J.A. van; Rosengren, L.E.; Verbeek, M.M.

    2005-01-01

    Neurofilament light chain is a component of the axonal cytoskeleton. The concentration of the neurofilament light chain in cerebrospinal fluid may reflect axonal damage or the extent of white matter damage. In this study we describe a sensitive immunoassay for the detection of neurofilament light

  15. Microfluidic Platform for Enzyme-Linked and Magnetic Particle-Based Immunoassay

    Directory of Open Access Journals (Sweden)

    Dorota G. Pijanowska

    2013-06-01

    Full Text Available This article presents design and testing of a microfluidic platform for immunoassay. The method is based on sandwiched ELISA, whereby the primary antibody is immobilized on nitrocelluose and, subsequently, magnetic beads are used as a label to detect the analyte. The chip takes approximately 2 h and 15 min to complete the assay. A Hall Effect sensor using 0.35-μm BioMEMS TSMC technology (Taiwan Semiconductor Manufacturing Company Bio-Micro-Electro-Mechanical Systems was fabricated to sense the magnetic field from the beads. Furthermore, florescence detection and absorbance measurements from the chip demonstrate successful immunoassay on the chip. In addition, investigation also covers the Hall Effect simulations, mechanical modeling of the bead–protein complex, testing of the microfluidic platform with magnetic beads averaging 10 nm, and measurements with an inductor-based system.

  16. Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay.

    Science.gov (United States)

    Abdelshafi, Nahla A; Panne, Ulrich; Schneider, Rudolf J

    2017-04-01

    This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm 2 of one banknote by swabbing 1cm 2 : 0, 14, and 21pg/cm 2 . Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Competitive photometric enzyme immunoassay for fullerene C60 and its derivatives using a fullerene conjugated to horseradish peroxidase

    International Nuclear Information System (INIS)

    Hendrickson, Olga D.; Smirnova, Natalya I.; Zherdev, Anatoly V.; Dzantiev, Boris B.; Sveshnikov, Peter G.

    2016-01-01

    The article describes a highly sensitive single-step microplate enzyme immunoassay of the ELISA type for fullerene C 60 and its derivatives. Monoclonal anti-fullerene antibodies and a conjugate between fullerene and horseradish peroxidase were used as specific reagents. A direct competitive ELISA was carried out that was based on antibodies immobilized in the well of a microtiter plate, a peroxidase-labeled antigen, and detection via the dye formed from 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Both pristine fullerene C 60 and its water-soluble forms can be determined. The detection limits are 1.5 ng∙mL −1 for fullerene C 60 , and between 0.1 and 1.3 ng∙mL −1 for its derivatives. This ELISA format allows for almost two-fold reduction of the time needed for the assay in comparison to indirect scheme with labeled antibodies. (author)

  18. [Establishment of chemiluminescent enzyme immunoassay for detecting antibodies against foot-and-mouth disease virus serotype O in swine].

    Science.gov (United States)

    Cui, Chen; Huang, Ligang; Li, Jing; Zou, Xingqi; Zhu, Yuanyuan; Xie, Lei; Zhao, Qizu; Yang, Limin; Liu, Wenjun

    2016-11-25

    Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

  19. Novel enzyme immunoassay system for simultaneous detection of six subclasses of antiphospholipid antibodies for differential diagnosis of antiphospholipid syndrome.

    Science.gov (United States)

    Nojima, Junzo; Motoki, Yukari; Hara, Kazusa; Sakata, Toshiyuki; Ichihara, Kiyoshi

    2017-06-01

    : Antiphospholipid syndrome, which often complicates systemic lupus erythematosus (SLE), features high occurrence of arterial and/or venous thrombosis and recurrent fetal loss. However, which antibody subclass contributes to which clinical event remains uncertain. We newly developed an up-to-date enzyme immunoassay system using the AcuStar automated analyzer (Instrumentation Laboratory, Bedford, Massachusetts, USA) for parallel detection of six subclasses of antiphospholipid antibodies (aPLs): anticardiolipin antibodies (aCL) of IgG, IgM, and IgA and anti-β2-glycoprotein I antibodies (aβ2GPI) of IgG, IgM, and IgA. They were measured in 276 healthy volunteers and 138 patients with SLE: 45 with thromboembolic complications (29 arterial; 16 venous) and 93 without. Lupus anticoagulant activity in their plasma was measured according to the guidelines recommended by the Subcommittee on Lupus Anticoagulant/Phospholipid-Dependent Antibodies. aCL/β2GPI was measured with a standard ELISA kit commonly used in Japan. The positive results of IgG aCL, IgA aCL, and IgG aβ2GPI were closely associated with thromboembolic complications, whereas IgM aCL and IgM aβ2GPI were not. receiver operating characteristic analysis revealed that the accuracy of predicting thromboembolic complications based on the composite test results of the former three antibodies were obviously higher than by each alone. Regarding agreement with the test results of lupus anticoagulant activity, IgG aβ2GPI showed the closest match. Patients with SLE frequently possess various combinations of the six aPL subclasses, and this antibody spectrum is closely associated with thromboembolic events in these patients. This new automated enzyme immunoassay system allows simultaneous analysis of the profile of aPL subclasses for the differential diagnosis of antiphospholipid antibody syndrome in its early stage.

  20. Comparison of conventional culture methods and two commercial enzyme immunoassays for detection of Salmonella in porcine fecal samples and cecal contents

    DEFF Research Database (Denmark)

    Wegener, Henrik Caspar; Baggesen, Dorte Lau

    1997-01-01

    Two commercial enzyme immunoassays, designated EIA-1 and EIA-2, for the detection of salmonella in feces and cecal contents were compared to conventional culture methods. Out of 362 cecal content samples, 35 were positive by EIA-1 and 30 were positive by EIA-2 and conventional methods. Out of 189...

  1. A three-parameter langmuir-type model for fitting standard curves of sandwich enzyme immunoassays with special attention to the α-fetoprotein assay

    NARCIS (Netherlands)

    Kortlandt, W.; Endeman, H.J.; Hoeke, J.O.O.

    In a simplified approach to the reaction kinetics of enzyme-linked immunoassays, a Langmuir-type equation y = [ax/(b + x)] + c was derived. This model proved to be superior to logit-log and semilog models in the curve-fitting of standard curves. An assay for α-fetoprotein developed in our laboratory

  2. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

    2015-01-01

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL −1 with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO 2 particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors

  3. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell–core silica nanospheres based on enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

    2015-08-05

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL{sup −1} with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. - Highlights: • A versatile ELISA-based immunoassay for PCV2 antibody was developed. • Enzyme and CdSe QDs modified SiO{sub 2} particles were used to improve sensitivity. • The simultaneous three ELISA-based techniques enhanced the detection reliability. • The biosensors strategy could provide a new avenue to ELISA-based sensors.

  4. Evaluation of a magnetic particles-based chemiluminescence enzyme immunoassay for Golgi protein 73 in human serum.

    Science.gov (United States)

    Liu, Xiangyi; Wan, Xiaohua; Lu, Sheng; Zhang, Lijun; Yu, Shaohua; Lu, Xinxin

    2015-05-20

    Golgi protein 73 (GP73) is regarded as a potential serum biomarker for early diagnosis of hepatocellular carcinoma (HCC). We developed a rapid magnetic particles-based chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of serum GP73. Fluorescein isothiocyanate (FITC) and alkaline phosphatase (ALP) were used to label 2 different monoclonal antibodies to GP73. Serum GP73 was captured with labeled antibodies and formed a sandwiched immunoreaction. The magnetic particles (MPs) coated with anti-FITC antibody were used as a means of separation of the GP73 protein from other serum proteins. After adding the enzyme substrate solution, the relative light unit (RLU) was measured. A MPs-CLEIA for serum GP73 was established and evaluated. The RLU was directly proportional to the concentration of GP73. The method linearity was 5-600 μg/l. Limit of the blank was 2.19 μg/l. The intra- and inter-assay imprecision was 73-0.89), and the sensitivity and specificity, with cut-off value of 115.6 μg/l, were 75.4% and 92.1%, respectively. The proposed method demonstrates an acceptable performance for quantifying serum GP73. This assay could be appropriate for routine use in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids.

    Science.gov (United States)

    Vidal, Juan C; Bertolín, Juan R; Bonel, Laura; Asturias, Laura; Arcos-Martínez, M Julia; Castillo, Juan R

    2016-06-05

    Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h). Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B{sub 1} detection in cereal

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Mei [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Xu, Yang, E-mail: xuyang@ncu.edu.cn [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Liu, Xing [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); College of Food Science and Technology, Hainan University, No. 58 Renmin Avenue, Haikou 570228 (China); Li, Yanping; He, Qinghua [Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Tu, Zhui [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Fu, Jinheng [Jiangxi-OAI Joint Research Institute, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047 (China); Gee, Shirley J.; Hammock, Bruce D. [Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States)

    2016-06-14

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB{sub 1} was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB{sub 1} were 2.69 and 0.35 ng mL{sup −1}, respectively, with a linear range of 0.93–7.73 ng mL{sup −1}. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL{sup −1}, and the IC{sub 50} was 0.89 ± 0.09 ng mL{sup −1} with a linear range of 0.29–2.68 ng mL{sup −1}. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB{sub 1} contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. - Highlights: • Ab2β−Nb−AP has the potential to replace chemically-coupled probes. • Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly. • We developed a green and rapid one-step competitive enzyme immunoassay. • The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.

  7. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B_1 detection in cereal

    International Nuclear Information System (INIS)

    Shu, Mei; Xu, Yang; Liu, Xing; Li, Yanping; He, Qinghua; Tu, Zhui; Fu, Jinheng; Gee, Shirley J.; Hammock, Bruce D.

    2016-01-01

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB_1 was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB_1 were 2.69 and 0.35 ng mL"−"1, respectively, with a linear range of 0.93–7.73 ng mL"−"1. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL"−"1, and the IC_5_0 was 0.89 ± 0.09 ng mL"−"1 with a linear range of 0.29–2.68 ng mL"−"1. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB_1 contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. - Highlights: • Ab2β−Nb−AP has the potential to replace chemically-coupled probes. • Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly. • We developed a green and rapid one-step competitive enzyme immunoassay. • The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.

  8. Evaluation of eight enzyme immunoassays for detection of immunoglobulin G against Helicobacter pylori

    NARCIS (Netherlands)

    Thijs, JC; Kleibeuker, JH; vanZwet, AA; Berrelkamp, RJP; Meijer, B.C

    Eight commercial enzyme-linked immunosorbent assays (ELISAs) were used to test sera taken from 102 patients in whom Helicobacter pylori infection status had been determined by means of biopsy culture, PCR, histology, and urease production and by C-13 urea breath test. By those means, 61 patients had

  9. Non-radiometric immunoassays fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) and radioimmunoassay (RIA) for evaluation of thyroid function in normal and hypothyroid dogs

    Energy Technology Data Exchange (ETDEWEB)

    Jerico, M.M.; Larsson, C.E. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina Veterinaria e Zootecnia. Dept. de Clinica Medica]. E-mail: marciajerico@hotmail.com; Mendonca, B.B. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina . Lab. de Hormonios e Genetica Molecular; Otsuka, M. [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina Veterinaria e Zootecnia. Hospital Veterinario; Maganin Junior, A. [Canil da Policia Militar do Estado de Sao Paulo, SP (Brazil)

    2001-07-01

    We proposed the comparison of thyroxine (T4) and free thyroxine (FT4) measurements by fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) with radioimmunoassay (RIA) in thyroid function evaluation of normal (n=50) and hypothyroid dogs (n=9). T4 and FT4 serum concentrations were measured in basal conditions and 6 h after TRH stimulation (200 mug/IV). All our reference values are based on the 5th and 95th percentile. The reference values for basal T4 in healthy dogs were 0.50 to 2.35 mug/dL (FIA), 0.50 to 2.51 mug/dL (FEIA) and 0.35 to 0.74 mug/dL (RIA). After TRH, the values were >= 1.37 mug/dL (FIA), >= 0,26 mug/dL (FEIA) and >= 0.40 mug/dL (RIA). Basal FT4 values in healthy dogs were 0.65 to 2.20 ng/dL (FIA), 0.38 to 1.43 ng/dL (FEIA) and 0.10 to 1.24 ng/dL (RIA). After TRH, the values were >= 1.30 ng/dL (FIA), >= 0.77 ng/dL (FEIA) and >=0.50 ng/dL (RIA). In hypothyroid dogs, the mean +- SD for T4 in basal conditions and after TRH were 0.24 +- 0.20 mug/dL and 0.26 +- 0.20 mug/dL (FIA), 0.27 +- 0.12 mug/dL and 0.32 +- 0.51 mug/dL (FEIA) and 0.19 +- 0.30 mug/dL and 0,24 +- 0.09 mug/dL (RIA), respectively. In the same group the mean +- SD basal FT4 values and after TRH were 0.28 +- 0.33 ng/dL and 0.28 +- 0.39 ng/dL (FIA), 0,12 +- 0.26 ng/dL and 0.23 +- 0.56 ng/dL (FEIA) and 0,15 +- 0,15 ng/dL and 0,17 +- 0,28 ng/dL (RIA), respectively. Significant differences (p<0.05) between the normal and hypothyroid groups (Kruskall-Wallis test) were observed in the three methods and between basal and stimulated values in normal dogs (Wilcoxon test), by the three methods. The best sensitivity for diagnosing hypothyroidism was obtained through T4 values (100%), and the best specificity through FT4 values (100%), both determined by FIA after TRH stimulation. We conclude that T4 and FT4 measured by fluoroimmunoassay after TRH stimulation can be an excellent alternative. (author)

  10. Non-radiometric immunoassays (fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) and radioimmunoassay (RIA) for evaluation of thyroid function in normal and hypothyroid dogs

    International Nuclear Information System (INIS)

    Jerico, M.M.; Larsson, C.E.

    2001-01-01

    We proposed the comparison of thyroxine (T4) and free thyroxine (FT4) measurements by fluoroimmunoassay (FIA) and fluorometric enzyme immunoassay (FEIA) with radioimmunoassay (RIA) in thyroid function evaluation of normal (n=50) and hypothyroid dogs (n=9). T4 and FT4 serum concentrations were measured in basal conditions and 6 h after TRH stimulation (200 mug/IV). All our reference values are based on the 5th and 95th percentile. The reference values for basal T4 in healthy dogs were 0.50 to 2.35 mug/dL (FIA), 0.50 to 2.51 mug/dL (FEIA) and 0.35 to 0.74 mug/dL (RIA). After TRH, the values were >= 1.37 mug/dL (FIA), >= 0,26 mug/dL (FEIA) and >= 0.40 mug/dL (RIA). Basal FT4 values in healthy dogs were 0.65 to 2.20 ng/dL (FIA), 0.38 to 1.43 ng/dL (FEIA) and 0.10 to 1.24 ng/dL (RIA). After TRH, the values were >= 1.30 ng/dL (FIA), >= 0.77 ng/dL (FEIA) and >=0.50 ng/dL (RIA). In hypothyroid dogs, the mean +- SD for T4 in basal conditions and after TRH were 0.24 +- 0.20 mug/dL and 0.26 +- 0.20 mug/dL (FIA), 0.27 +- 0.12 mug/dL and 0.32 +- 0.51 mug/dL (FEIA) and 0.19 +- 0.30 mug/dL and 0,24 +- 0.09 mug/dL (RIA), respectively. In the same group the mean +- SD basal FT4 values and after TRH were 0.28 +- 0.33 ng/dL and 0.28 +- 0.39 ng/dL (FIA), 0,12 +- 0.26 ng/dL and 0.23 +- 0.56 ng/dL (FEIA) and 0,15 +- 0,15 ng/dL and 0,17 +- 0,28 ng/dL (RIA), respectively. Significant differences (p<0.05) between the normal and hypothyroid groups (Kruskall-Wallis test) were observed in the three methods and between basal and stimulated values in normal dogs (Wilcoxon test), by the three methods. The best sensitivity for diagnosing hypothyroidism was obtained through T4 values (100%), and the best specificity through FT4 values (100%), both determined by FIA after TRH stimulation. We conclude that T4 and FT4 measured by fluoroimmunoassay after TRH stimulation can be an excellent alternative. (author)

  11. Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps.

    Science.gov (United States)

    Harmsen, T; Jongerius, M C; van der Zwan, C W; Plantinga, A D; Kraaijeveld, C A; Berbers, G A

    1992-01-01

    A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA. PMID:1500523

  12. Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli.

    Science.gov (United States)

    Birnbaum, S; Bülow, L; Hardy, K; Danielsson, B; Mosbach, K

    1986-10-01

    We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.

  13. Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli

    International Nuclear Information System (INIS)

    Birnbaum, S.; Buelow, L.; Hardy, K.; Danielsson, B.; Mosbach, K.

    1986-01-01

    The authors have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 μg/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 0 C in the enzyme thermistor unit. Thus, immediate assay start up was possible

  14. Computer-assisted enzyme immunoassays and simplified immunofluorescence assays: applications for the diagnostic laboratory and the veterinarian's office.

    Science.gov (United States)

    Jacobson, R H; Downing, D R; Lynch, T J

    1982-11-15

    A computer-assisted enzyme-linked immunosorbent assay (ELISA) system, based on kinetics of the reaction between substrate and enzyme molecules, was developed for testing large numbers of sera in laboratory applications. Systematic and random errors associated with conventional ELISA technique were identified leading to results formulated on a statistically validated, objective, and standardized basis. In a parallel development, an inexpensive system for field and veterinary office applications contained many of the qualities of the computer-assisted ELISA. This system uses a fluorogenic indicator (rather than the enzyme-substrate interaction) in a rapid test (15 to 20 minutes' duration) which promises broad application in serodiagnosis.

  15. Ultrasensitive electrochemical immunoassay of staphylococcal enterotoxin B in food using enzyme-nanosilica-doped carbon nanotubes for signal amplification.

    Science.gov (United States)

    Tang, Dianping; Tang, Juan; Su, Biling; Chen, Guonan

    2010-10-27

    A new sandwich-type electrochemical immunoassay for ultrasensitive detection of staphylococcal enterotoxin B (SEB) in food was developed using horseradish peroxidase-nanosilica-doped multiwalled carbon nanotubes (HRPSiCNTs) for signal amplification. Rabbit polyclonal anti-SEB antibodies immobilized on the screen-printed carbon electrode (SPCE) and covalently bound to the HRPSiCNTs were used as capture antibodies and detection antibodies, respectively. In the presence of SEB analyte, the sandwich-type immunocomplex could be formed between the immobilized anti-SEB on the SPCE and anti-SEB-labeled HRPSiCNTs, and the carried HRP could catalyze the electrochemical reduction of H2O2 with the help of thionine. The high content of HRP in the HRPSiCNTs could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with the increase of SEB in the sample, and exhibited a dynamic range of 0.05-15 ng/mL with a low detection limit (LOD) of 10 pg/mL SEB (at 3σ). Intra- and interassay coefficients of variation were below 10%. In addition, the assay was evaluated with SEB spiked samples including watermelon juice, soymilk, apple juice, and pork food, receiving excellent correlation with results from commercially available enzyme-linked immunosorbent assay (ELISA).

  16. Comparison of a Recombinant-antigen Enzyme Immunoassay with Treponema pallidum Hemagglutination Test for Serological Confirmation of Syphilis

    Directory of Open Access Journals (Sweden)

    Rodríguez Islay

    2002-01-01

    Full Text Available A recombinant-antigen enzyme immunoassay (EIA, BioSCREEN TM anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38, secondary (n = 10, early latent (n = 28 and congenital syphilis (n = 2, patients with leptospirosis ( n= 8, infectious mononucleosis (n = 7, hepatitis (n = 9, diabetes mellitus (n = 11, rheumatoid arthritis (n = 13, leprosy (n = 11, tuberculosis (n = 9, HIV/Aids ( n= 12, systemic lupus erythematosus (n = 4, rheumatic fever (n = 3, old-persons (n = 9, pregnant women (n = 29 and blood donors (n = 164. The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  17. Detection of soluble antigens of Toxoplasma gondii by a four-layer modification of an enzyme immunoassay.

    Science.gov (United States)

    Turunen, H J

    1983-01-01

    A sensitive four-layer modification of an enzyme immunoassay for the detection of soluble antigens of Toxoplasma gondii is described. Microtiter plates were sensitized with rabbit anti-toxoplasma immunoglobulins (6 micrograms/ml) used as the primary antibodies; guinea pig anti-toxoplasma immunoglobulins (6 micrograms/ml) were used as the secondary trapping antibodies. Horseradish peroxidase-conjugated anti-guinea pig immunoglobulins were used as the indicator antibodies. The specificity of the antigen assay was confirmed by using guinea pig immunoglobulins from preimmunization sera. The sensitivity of the antigen assay was found to be at least 10 ng of antigen protein per ml. The suitability of the method for detecting antigens of T. gondii in different specimens was studied by experimental toxoplasma infection in mice. Antigenic components of T. gondii could be detected in different tissue specimens from infected animals from the first day after infection onwards. Toxoplasma antigen in serum and urine samples from infected mice reached detectable levels on day 2 after infection followed by a linear increase in antigen concentration in succeeding samples. This method might offer a valuable aid for a rapid etiological diagnosis also in human cases of acute toxoplasmosis. PMID:6345574

  18. [Fundamental evaluation of apolipoprotein B-48 by chemiluminescence enzyme immunoassay--identification of apolipoprotein B-48 with immunoblotting].

    Science.gov (United States)

    Sato, Itsuko; Fujioka, Yoshio; Hayashi, Fujio; Mukai, Masahiko; Kawano, Seiji; Ishikawa, Yuichi; Yamashita, Shizuya; Kumagai, Shunichi

    2007-06-01

    Apolipoprotein B-48 (apo B-48) is a constituent of chylomicrons and chylomicron remnants, and its fasting concentration has been reported to be a marker of postprandial hyperlipidemia, which is thought to be a risk factor of atherosclerosis. We evaluated the serum apo B-48 concentrations by chemiluminescence enzyme immunoassay (CLEIA), which was recently introduced as Lumipulse f fully automated immunosaasy analyzer by Fujirebio Inc (Tokyo, Japan), and performed immunoblotting on agarose gel electrophoresis with anti-apo B-48 antibody. Apo B-48 assay was intra-assay reproducible (CVs: 1.9-3.1%) and inter-assay reproducible (CVs: 2.2-4.4%). The assay range for apo B-48 was from 0.2 to 40.0 microg/ml. The effects of interfering substances such as free/conjugated birirubin, hemoglobin, Intrafat, ascorbic acid and rheumatoid factor were negligible. For storage, it was preferable to freeze, and to avoid frozen-thaw process as much as possible. Anti-apo B-48 antibody was reactive over a wide range from origin to the position of very-low-density lipoproteins in immunoblotting after agarose gel electrophoresis. Apo B-48 measurement by CLEIA was feasible to clinical use for the assessment of lipoprotein metabolism.

  19. Detection of Total Ergot Alkaloids in Cereal Flour and in Bread by a Generic Enzyme Immunoassay Method.

    Science.gov (United States)

    Gross, Madeleine; Curtui, Valeriu; Usleber, Ewald

    2018-05-01

    Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, α-ergocryptine, and ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

  20. Summary of field trials using the direct and competitive enzyme immunoassays for detection of antibody to brucella abortus

    International Nuclear Information System (INIS)

    Nielsen, K.; Gall, D.

    1998-01-01

    Two indirect and two competitive enzyme immunoassays for detection of antibody to Brucella abortus, validated elsewhere, were field tested in five different Latin American laboratories. Testing was performed according to standardised protocols using sera obtained in each area. Sera from B. abortus infected herds, from vaccinated (but serologically negative in a screening test) and non-vaccinated cattle were tested in each assay and compared to the results obtained with conventional diagnostic tests used for diagnosis of brucellosis in each country. Relative sensitivity and specificity values were calculated for each country as well as a weighted summary combining the data from all the participating laboratories. The result demonstrate that all ELISAs performed as well as, or better than, the conventional aerological tests. Given the inherent errors in the use of the latter in the diagnosis of brucellosis, it is recommended that the ELISAs described here be considered as replacements for the conventional tests. The CELISA using the lipopolysaccharide antigen with the competing monoclonal antibody M84, should be considered as the most useful because of cross-species and vaccination considerations. (author)

  1. Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.

    Science.gov (United States)

    Clem, T R; Yolken, R H

    1978-01-01

    A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories.

  2. Clostridium difficile Testing Algorithm: Is There a Difference in Patients Who Test Positive by Enzyme Immunoassay vs. Those Who Only Test Positive by Nucleic Acid Amplification Methodology?

    OpenAIRE

    Polak, Jonathan; Odili, Ogheneruona; Craver, Mary Ashleigh; Mayen, Anthony; Purrman, Kyle; Rahman, Asem; Sang, Charlie Joseph; Cook, Paul P

    2017-01-01

    Abstract Background Testing for Clostridium difficile infection (CDI) commonly involves checking for the presence of toxins A and B by enzyme immunoassay (EIA) or nucleic acid amplification (NAA). The former is very specific, but not very sensitive. The latter is very sensitive. Beginning in 2011, our hospital incorporated an algorithm that involved testing liquid stool specimens for glutamate dehydrogenase (GDH) and toxin by EIA. For discrepant results, the stool specimen was tested for the ...

  3. Enzyme immunoassay for rabies antibody in hybridoma culture fluids and its application to differentiation of street and laboratory strains of rabies virus.

    OpenAIRE

    Smith, J S; Sumner, J W; Roumillat, L F

    1984-01-01

    A rapid and sensitive enzyme immunoassay is described for detecting rabies antibody in hybridoma culture fluids. Glass fiber filter disks were used to immobilize gamma-irradiated mouse neuroblastoma cells infected with street or laboratory strains of rabies virus. Bound rabies-specific antibody was detected by reaction with horseradish peroxidase-labeled goat anti-mouse immunoglobulin G. The assay was performed in a 96-well filtration device developed by Cleveland et al. (J. Clin. Microbiol. ...

  4. Identification of Performance Problems in a Commercial Human Immunodeficiency Virus Type 1 Enzyme Immunoassay by Multiuser External Quality Control Monitoring and Real-Time Data Analysis▿ †

    OpenAIRE

    Kim, J.; Swantee, C.; Lee, B.; Gunning, H.; Chow, A.; Sidaway, F.; Sherlock, C.; Garceau, R.; Dimech, W.; Malloch, L.

    2009-01-01

    In June 2005, a pilot program was implemented in Canadian laboratories to monitor the performance of the Abbott human immunodeficiency virus types 1 and 2 (HIV-1/2) gO enzyme immunoassay (EIA). Two different external quality control (QC) reagents and a “real-time” software analysis program were evaluated. In November 2005, higher-than-expected calibrator rate values in these kits were first reported at the Ontario Ministry of Health (Etobicoke), followed by the Alberta Provincial Public Healt...

  5. [The efficiency of the enzyme immunoassay test system opisthorchiasis-CIC-EIA-best to detect circulating immune complexes containing opisthorchis antigens in the serum of patients with opisthorchiasis].

    Science.gov (United States)

    Starkova, T V; Poletaeva, O G; Kovrova, E A; Krasovskaia, N N; Tkachenko, T N; Masiago, A V; Ofitserov, V I; Tereshchenko, A Iu

    2011-01-01

    The efficacy of a kit of Opisthorchiasis-CIC-EIA-Best reagents was evaluated using 270 sera from patients in the study and control groups. The kit showed a sufficient sensitivity (not less than 87.2%) and a high specificity (not less than 97.9%). The use of the above kit of the reagents for enzyme immunoassay in practical healthcare enables one to increase detection rates among the infested subjects on comprehensive examination of those with suspected opisthorchiasis.

  6. Evaluation of the microparticle enzyme immunoassay Abbott IMx Select Chlamydia and the importance of urethral site sampling to detect Chlamydia trachomatis in women.

    OpenAIRE

    Brokenshire, M K; Say, P J; van Vonno, A H; Wong, C

    1997-01-01

    OBJECTIVE: To evaluate the commercial microparticle enzyme immunoassay (MEIA), Abbott IMx Select Chlamydia, for the detection of Chlamydia trachomatis in women and to compare its performance with endocervical cell culture. Also, to determine whether sampling the urethral site is an important part of chlamydial diagnosis in women. SETTING: The Auckland, Manukau, and Waitakere Sexual Health Clinics, Auckland, New Zealand and the Department of Clinical Microbiology, Auckland Hospital, Auckland, ...

  7. Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

    Science.gov (United States)

    Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

    2015-01-01

    Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry.

  8. Validation of enzyme immunoassay applied to the serological diagnosis of hog brucellosis

    International Nuclear Information System (INIS)

    Ramondino, Romina; Marticorena, Damian

    1997-01-01

    The application of an enzyme immunization test in the serological diagnostic of a disease demands its validation as a previous stage. A test validation requires to know: cut off, diagnostic and relative specificity and sensitivity and harmonization grade between the new test and the classical test in use. The purpose of this work is the following: a) To determine the cut off and to calculate the specificity of two immunization tests, indirect (I-ELISA) and competitive (C-ELISA) by the analysis of serum coming from Canada (country free of this disease); b) To calculate the values of relative sensitivity of the tests with serum coming from infected populations and diagnostic sensitivity with samples of serum from hogs with positive isolation to Brucella suis

  9. Development and application of radioimmunoassays and enzyme immunoassays in microbiological and immunological diagnosis. 2

    International Nuclear Information System (INIS)

    Mueller, W.A.; Struy, H.; Holzwarth, F.

    1982-01-01

    Comparative studies of indirect immunofluorescence test (IT), complement binding reaction (CBR), enzyme- and radioimmunoassay (ELISA, RIA) for the detection of toxoplasma antibodies in sera of 513 patients are reported. The precision dependent on time, showed coefficients of variation from 3% to 12% (IFT 3%, CBR 10%, ELISA 12%, RIA 7%). The correlation of IFT and ELISA as well as RIA was relatively unfavourable (coefficient of correlation IFT/ELISA r = 0.52, IFT/RIA r = 0.54, RIA/ELISA r = 0.60). The ELISA is the most sensitive method for the detection of antibodies. The specificity of the Toxo-ELISA has to be improved by application of suitable fractions of antigens. (author)

  10. An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor.

    Science.gov (United States)

    Joung, Hyou-Arm; Oh, Young Kyoung; Kim, Min-Gon

    2014-03-15

    Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3 ± 0.3 min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum. © 2013 Elsevier B.V. All rights reserved.

  11. Use of enzyme immunoassays in disease diagnosis, with particular reference to rinderpest

    International Nuclear Information System (INIS)

    Crowther, J.R.

    1986-01-01

    The wider principles of the use of enzyme linked immunosorbent assays (ELISA) in diagnosis are illustrated. The development of an antirinderpest antibody assay is explained in order to demonstrate the relative ease with which the test can be set up and used in various countries. The various stages of the test can be standardized in central laboratories; for example, antigen (inactivated) can be prepared and passively attached to microplates. Purchasing and titration of conjugates and selection of negative control sera could also be done centrally. Thus 'kits' for the initiation of diagnosis could be rapidly made available. When assays have been used in specific countries to study antibody populations in large numbers of animals, it may be found necessary to modify the above procedures; for example, different negative sera may be necessary, reflecting the mean (and distribution) of the country. Relatively simple standardization steps to titrate antigen, conjugates and sera would allow assays to be set up in individual countries with no need for outside standardization. However, it may be useful to obtain sero-negative and sero-positive standards from an outside source which could be used on a worldwide basis for interlaboratory standardization. The provision of washing solution, blocking buffers, plates, pipettes and readers is the only requisite for the versatility of ELISA to be fully realized in the study of many disease agents. (author)

  12. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: Implications for immunoassays

    DEFF Research Database (Denmark)

    Jørgensen, Kaj Anker; Karamperis, Nikolaos; Koefoed-Nielsen, Pernille Bundgaard

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  13. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays

    DEFF Research Database (Denmark)

    Karamperis, N; Koefoed-Nielsen, PB; Brahe, P

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  14. Influence of gamma radiation on antibodies fixation on polystyrene. Application to ELISA enzyme immunoassay

    International Nuclear Information System (INIS)

    Esterlin, S.

    1986-01-01

    This thesis is divided into two parts: the first part includes a description of the ELISA test, a comparative analysis of the main supports (polystyrene microtitration trays) used for this technique and an evaluation of gamma radiation effects on the quality of the supports. The study was carried out with the following antigens: Spiroplasma citri R8A2, a tymovirus and Tristeza virus. The second part dealt with the effects of gamma radiations on antibodies and antibody-support system (interaction immobilized immunoglobulins-plastic support) [fr

  15. Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

    Science.gov (United States)

    Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2003-01-01

    We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

  16. Detection of Campylobacter in Stool and Determination of Significance by Culture, Enzyme Immunoassay, and PCR in Developing Countries

    Science.gov (United States)

    Platts-Mills, James A.; Liu, Jie; Gratz, Jean; Mduma, Esto; Amour, Caroline; Swai, Ndealilia; Taniuchi, Mami; Begum, Sharmin; Peñataro Yori, Pablo; Tilley, Drake H.; Lee, Gwenyth; Shen, Zeli; Whary, Mark T.; Fox, James G.; McGrath, Monica; Kosek, Margaret; Haque, Rashidul

    2014-01-01

    Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level. PMID:24452175

  17. Usefulness of enzyme immunoassay (EIA) for screening of anti HIV antibodies in urinary specimens: A comparative analysis.

    Science.gov (United States)

    Sahni, A K; Nagendra, A; Roy, Partha; Patrikar, S

    2014-07-01

    Standard HIV testing is done using serum or plasma. FDA approved ELISA to screen urine for IgG antibodies to HIV-1 in 1996. It is a simple, noninvasive test and is appropriate for developing countries where health care personnel may not be professionally trained or where clean needles for drawing blood may not always be available. 436 individuals with high-risk behavior and strong clinical suspicion of HIV infection were screened for IgG antibodies to HIV-1 in urine by ELISA. Urine HIV testing was performed by enzyme immunoassay, at the ongoing Voluntary Confidential Counseling and Testing Center (VCCTC) at a large tertiary care microbiology lab. The individuals enrolled for the study had high-risk exposure to the virus and majorities were from a state with a high incidence of HIV infection. In all individuals, both serum and urine were tested for IgG antibodies to HIV-1. Overall, 135 individuals (30.96%) were HIV-positive, of whom 96 (71%) had never previously tested positive; 87% of those who tested positive received their results, and most were referred for medical care. Sensitivity, specificity and predictive values of HIV-1 urine ELISA test kit were determined. Sensitivity was found to be 89.6%; 95% CI [82.9-94.0], specificity 97.3%; 95% CI [94.6-98.8], positive predictive value 93.8%; 95% CI [87.8-97.1] and negative predictive value 95.4%; 95% CI [92.3-97.4]. Efficiency, sensitivity, and specificity of the urine-based screening for HIV-1 test kits were excellent as compared to the reference test.

  18. The utility of repeat enzyme immunoassay testing for the diagnosis of Clostridium difficile infection: A systematic review of the literature

    Directory of Open Access Journals (Sweden)

    P S Garimella

    2012-01-01

    Full Text Available Over the last 20 years, the prevalence of healthcare-associated Clostridium difficile (C. diff disease has increased. While multiple tests are available for the diagnosis of C. diff infection, enzyme immunoassay (EIA testing for toxin is the most used. Repeat EIA testing, although of limited utility, is common in medical practice. To assess the utility of repeat EIA testing to diagnose C. diff infections. Systematic literature review. Eligible studies performed >1 EIA test for C. diff toxin and were published in English. Electronic searches of MEDLINE and EMBASE were performed and bibliographies of review articles and conference abstracts were hand searched. Of 805 citations identified, 32 were reviewed in detail and nine were included in the final review. All studies except one were retrospective chart reviews. Seven studies had data on number of participants (32,526, and the overall reporting of test setting and patient characteristics was poor. The prevalence of C. diff infection ranged from 9.1% to 18.5%. The yield of the first EIA test ranged from 8.4% to 16.6%, dropping to 1.5-4.7% with a second test. The utility of repeat testing was evident in outbreak settings, where the yield of repeat testing was 5%. Repeat C. diff testing for hospitalized patients has low clinical utility and may be considered in outbreak settings or when the pre-test probability of disease is high. Future studies should aim to identify patients with a likelihood of disease and determine the utility of repeat testing compared with empiric treatment.

  19. Evaluation of the C6 Lyme Enzyme Immunoassay for the Diagnosis of Lyme Disease in Children and Adolescents.

    Science.gov (United States)

    Lipsett, Susan C; Branda, John A; McAdam, Alexander J; Vernacchio, Louis; Gordon, Caroline D; Gordon, Catherine R; Nigrovic, Lise E

    2016-10-01

    The commercially-available C6 Lyme enzyme immunoassay (EIA) has been approved to replace the standard whole-cell sonicate EIA as a first-tier test for the diagnosis of Lyme disease and has been suggested as a stand-alone diagnostic. However, the C6 EIA has not been extensively studied in pediatric patients undergoing evaluation for Lyme disease. We collected discarded serum samples from children and adolescents (aged ≤21 years) undergoing conventional 2-tiered testing for Lyme disease at a single hospital-based clinical laboratory located in an area endemic for Lyme disease. We performed a C6 EIA on all collected specimens, followed by a supplemental immunoblot if the C6 EIA result was positive but the whole-cell sonicate EIA result was negative. We defined a case of Lyme disease as either a clinician-diagnosed erythema migrans lesion or a positive standard 2-tiered serologic result in a patient with symptoms compatible with Lyme disease. We then compared the performance of the C6 EIA alone and as a first-tier test followed by immunoblot, with that of standard 2-tiered serology for the diagnosis of Lyme disease. Of the 944 specimens collected, 114 (12%) were from patients with Lyme disease. The C6 EIA alone had sensitivity similar to that of standard 2-tiered testing (79.8% vs 81.6% for standard 2-tiered testing; P = .71) with slightly lower specificity (94.2% vs 98.8% 2; P Lyme disease, the C6 EIA could guide initial clinical decision making, although a supplemental immunoblot should still be performed. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  20. Low sensitivity of fecal toxin A/B enzyme immunoassay for diagnosis of Clostridium difficile infection in immunocompromised patients.

    Science.gov (United States)

    Erb, S; Frei, R; Strandén, A M; Dangel, M; Tschudin-Sutter, S; Widmer, A F

    2015-11-01

    The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12,481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50-5.90, p 0.002), leukocytopenia <1000/μL (OR 2.52, 95% CI 1.22-5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39-3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  1. Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.

    Science.gov (United States)

    Deguchi, Matsuo; Kagita, Masanori; Yoshioka, Nori; Tsukamoto, Hiroko; Takao, Miyuki; Tahara, Kazuko; Maeda, Ikuhiro; Hidaka, Yoh; Yamauchi, Satoshi; Kaneko, Atsushi; Miyakoshi, Hideo; Isomura, Mitsuo

    2017-10-06

    Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients. © 2017 Wiley Periodicals, Inc.

  2. Hepatitis virus genotyping by Polymerase Chain Reaction and DNA Enzyme immunoassay among Saudi patients in the Western Province, Saudi Arabia

    International Nuclear Information System (INIS)

    Osoba, A.O.; Ibrahim, M.; Abdelaal, M.A.; Al-Mowallad, A.; Al-Shareef, B.; Hussein, B.A.

    2000-01-01

    The distribution of hepatitis C virus (HCV) genotypes in the Western Province of Saudi Arabia is unknown. The purpose of our study was to determine the prevalent HCV genotypes among HCV seropositive Saudi patients in the Western Province and to study the relationship between types/subtypes, clinical status and liver histology. Serum samples were collected from 140 consecutive patients attending the Hematology Clinic with varying grades of liver diseases, high almandine transferees (ALT) for > 6 months, positive HCV, qualitative PCR and who had liver biopsy. HCV genotyping was determined on patients who had tested positive by both HCV enzyme immunoassay (EIA) and the recombinant immunoblot assay (RIBA). Of the 140 patients, 97 (69.2%) had genotype 4, 18 (12.8%) had genotype 1a, and 16 (11.4%) had genotype 1b. Genotype 2b and 5 were found in two patients (1.4%) each, while 5 patients (3.6%) had mixed infections with genotype 4 and 5. Of the 97 patients infected with genotype 4, 84 (86.6%) had chronic active hepatitis (CAH), two (2.1%) had CAH with active cirrhosis, 9(9.3%) had cirrhosis and two (2.1%) had normal liver histology (NLH). The most prevalent HCV genotype in the Western Province of Saudi Arabia was genotype 4 (69.2%). Genotype 1b was encountered in 16 (11.4%) patients. For the first time, genotype 5 was identified in the Western Province of Saudi Arabia. Genotype 1b and 4 were associated with different histological grades of liver disease. (author)

  3. Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

    Science.gov (United States)

    Cortina, María E; Novak, Analía; Melli, Luciano J; Elena, Sebastián; Corbera, Natalia; Romero, Juan E; Nicola, Ana M; Ugalde, Juan E; Comerci, Diego J; Ciocchini, Andrés E

    2017-09-01

    Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. A Comparison of Anti-Nuclear Antibody Quantification Using Automated Enzyme Immunoassays and Immunofluorescence Assays

    DEFF Research Database (Denmark)

    Baronaite, Renata; Engelhart, Merete; Mørk Hansen, Troels

    2014-01-01

    using IFA and automated EIA techniques. The IFA results generated by two independent laboratories were compared with the EIA results from antibodies against double-stranded DNA (dsDNA), from ANA screening, and from tests of the seven included subantigens. The final IFA and EIA results for 386 unique......, with Cohen's kappa value of 0.30 (95% confidence interval (CI) = 0.14-0.46), which decreased to 0.23 (95% CI = 0.06-0.40) when the results for dsDNA were omitted. The EIA method was less reliable for assessing nuclear and speckled reactivity patterns, whereas the IFA method presented difficulties detecting...... dsDNA and Ro activity. The automated EIA method was performed in a similar way to the conventional IFA method using HEp-2 cells; thus, automated EIA may be used as a screening test....

  5. Novel photoluminescence enzyme immunoassay based on supramolecular host-guest recognition using L-arginine/6-aza-2-thiothymine-stabilized gold nanocluster.

    Science.gov (United States)

    Wang, Youmei; Lu, Minghua; Tang, Dianping

    2018-06-30

    A new photoluminescence (PL) enzyme immunoassay was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) via an innovative enzyme substrate, 6-aza-2-thiothymine-stabilized gold nanocluster (AAT-AuNC) with L-arginine. The enzyme substrate with strong PL intensity was formed through supramolecular host-guest assembly between guanidine group of L-arginine and AAT capped on the surface of AuNC. Upon arginase introduction, the captured L-arginine was hydrolyzed into ornithine and urea, thus resulting in the decreasing PL intensity. Based on this principle, a novel competitive-type immunoreaction was first carried out on AFB 1 -bovine serum albumin (AFB 1 -BSA) conjugate-coated microplate, using arginase-labeled anti-AFB 1 antibody as the competitor. Under the optimum conditions, the PL intensity increased with the increment of target AFB 1 , and allowed the detection of the analyte at concentrations as low as 3.2 pg mL -1 (ppt). Moreover, L-arginine-AAT-AuNC-based PL enzyme immunoassay afforded good reproducibility and acceptable specificity. In addition, the accuracy of this methodology, referring to commercial AFB 1 ELISA kit, was evaluated to analyze naturally contaminated or spiked peanut samples, giving well-matched results between two methods, thus representing a useful scheme for practical application in quantitative monitoring of mycotoxins in foodstuff. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Evaluation of serum galactomannan enzyme immunoassay at two different cut-offs for the diagnosis of invasive aspergillosis in patients with febrile neutropenia

    Directory of Open Access Journals (Sweden)

    Ritin Mohindra

    2017-01-01

    Full Text Available Background: Invasive aspergillosis (IA is an increasingly common and fatal opportunistic fungal infection in patients with haematological diseases. Early diagnosis is difficult as mycological culture techniques have low sensitivity and the radiological tools have low specificity. Galactomannan enzyme immunoassay (GEI detects galactomannan in the human serum with a reported sensitivity and specificity between 30% and 100%. Aims: The aim of this study was to analyse the role of GEI in diagnosis of IA in patients with febrile neutropenia and to evaluate the role of GEI in the diagnosis of IA as per the revised (2008 European Organization for Research and Treatment of Cancer–Mycoses Study Group (EORTC–MSG criteria at two different optical density (OD cut-offs of 0.5 and 1.0. Setting: This prospective study was conducted in Safdarjung Hospital, New Delhi, India. Methods: GEI testing was performed in adult patients of febrile neutropenia with evidence of IA. Results at two different OD indices (ODIs of 0.5 and 1.0 were analysed. The evaluation of the diagnostic parameter, that is, GEI was measured in terms of sensitivity, specificity and positive and negative predictive value and was validated with the revised (2008 EORTC–MSG diagnostic criteria of IA. Results: One hundred and eleven patients had evidence of IA, of which 79 patients were GEI positive when cut-off ODI was 0.5, whereas with cut-off ODI 1.0, 55 patients were GEI positive. Conclusion: ODI of 1.0 should be considered as positive while in patients with OD between 0.5 and 1.0, repeat sampling from the patient is recommended.

  7. Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella sp and Yersinia enterocolitica O : 9 in cattle and pigs

    DEFF Research Database (Denmark)

    Nielsen, K.; Smith, P.; Yu, W.

    2006-01-01

    A rapid, inexpensive and rugged serological test that distinguishes cattle and swine infected with Brucella sp. or Yersinia enterocolitica O:9 is described. The test protocol, which is an indirect enzyme immunoassay uses a high concentration of divalent cation chelating agents to minimize binding...... with Brucella sp. Sera from 58 cattle and 38 swine exposed to Y. enterocolitica O:9 were negative while only 20 sera from 121 'false positive' reactors of unspecified origin gave low level positive reactions, eliminating 84% of the false positive reactions. Crown...

  8. Clinical Comparison of the Treponema pallidum CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing

    OpenAIRE

    Halling, V. W.; Jones, M. F.; Bestrom, J. E.; Wold, A. D.; Rosenblatt, J. E.; Smith, T. F.; Cockerill, F. R.

    1999-01-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTI...

  9. The significance for epidemiological studies anti-measles antibody detection examined by enzyme immunoassay (EIA) and plaque reduction neutralization test (PRNT).

    Science.gov (United States)

    Siennicka, Joanna; Częścik, Agnieszka; Trzcińska, Agnieszka

    2014-01-01

    The paper discusses the role of anti-measles antibodies for protection and significance for epidemiological studies determination of antibodies by different serological methods. The comparison of anti-measles virus antibodies levels measured by enzyme immunoassay (EIA) and Plaque Reduction Neutralization Test (PRNT) was described. It was found that the 200 mIU/ml of anti-measles activity measured by PRNT (level protection against symp- tomatic disease) is equivalent of 636 mIU/ml measured by EIA (Enzygnost®Anti-Measles Virus/IgG, Simens).

  10. [DIAGNOSTIC VALUE OF COMBINED USE OF COMBINED METHOD OF ENZYME IMMUNOASSAY AND POLYMERASE CHAIN REACTION TO DETECT OF INTRAUTERINE FETAL INFECTION BY PARVOVIRUS B19].

    Science.gov (United States)

    Bondarenko, N P; Lakatosh, V P; Lakatosh, P V; Malanchuk, O B; Poladich, I V

    2015-01-01

    The combined method of diagnosis parvovirus infection during pregnancy by maternal serum enzyme immunoassay and deoxyribonucleic acid isolation parvovirus B19 polymerase chain reaction in amnniotic fluid and fetal cord blood newborns, can diagnose vertical transmission and anticipate a negative effect on the fetus parvovirus. Lack of maternal IgM antibodies in serum due to parvovirus seroconversion during pregnancy does not exclude the persistence of the virus in the fetus. To analyze the diagnostic value of the method for determining the LHP parvovirus B19 DNA in the amniotic fluid, umbilical cord blood of newborns to determine vertical transmission of parvovirus infection when infected mothers B19 during pregnancy.

  11. Comparison among performances of a ligase chain reaction-based assay and two enzyme immunoassays in detecting Chlamydia trachomatis in urine specimens from men with nongonococcal urethritis.

    Science.gov (United States)

    Deguchi, T; Yasuda, M; Uno, M; Tada, K; Iwata, H; Komeda, H; Maeda, S; Latila, V; Saito, I; Kawada, Y

    1996-01-01

    We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis. PMID:8784574

  12. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

    Science.gov (United States)

    Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

    2015-01-01

    The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

  13. Measurements in international units of antibody to hepatitis B surface antigen(anti-HBs) after immunization with a yeast-derived, subtype adr hepatitis B vaccine are considerably different between chemiluminescent immunoassay (CLIA) and chemiluminescent enzyme immunoassay (CLEIA).

    Science.gov (United States)

    Ogata, Norio

    2006-04-01

    The worldwide consensus of the minimum protective anti-HBs level against HBV infection is 10 mIU/mL on assays standardized by the World Health Organization (WHO) reference preparations. To investigate whether this value could be applied to recipients of yeast-derived recombinant HB vaccine containing the major surface protein of subtype adr (Bimmugen, Astellas Pharmaceutical, Tokyo), we compared anti-HBs measurements between chemiluminescent immunoassay (CLIA) (Architect Ausab, Abbott Japan, Tokyo) and chemiluminescent enzyme immunoassay (CLEIA) (Lumipulse Forte, Fujirebio, Tokyo) in given serum samples obtained from the vaccinees. The vaccine and the two assay methods are currently in a wide use in Japan. The study included 300 medical students who completed a standard vaccination course (0, 1 and 6 months). Serum samples obtained 1 month or 13 months after completing the vaccination were simultaneously tested for anti-HBs by CLIA and CLEIA. In 147 samples with quantifiable values on both CLIA and CLEIA (10 to 1000 mIU/mL) the geometric mean titer on CLEIA (225.0 mIU/mL) was significantly higher than that on CLIA (94.5 mIU/mL) (p < 0.0001). Of 26 subjects with CLIA measurements below 10 mIU/mL, 15 samples (57.7%) showed CLEIA measurements more than 10 mIU/mL. Thus, in the subtype adr-vaccinees CLEIA demonstrated considerably high serum anti-HBs measurements compared to CLIA and discordance in determining critical anti-HBs level of 10 mIU/mL was observed in more than half the samples. This suggests that the minimum HBV-protective anti HBs titer of 10 mIU/mL is difficult to be introduced to Japan where subtype adr-HB vaccines or -HBV infection are prevalent, unless characteristics of assay methods are carefully evaluated.

  14. Specificity of immunoassays. Pt. 2

    International Nuclear Information System (INIS)

    Pratt, J.J.; Woldring, M.G.; Boonman, R.; Kittikool, J.

    1979-01-01

    Practical aspects of the measurement of the specificity of immunoassay are reviewed. Antibody heterogeneity in an antiserum makes a pragmatic rather than a theoretical approach necessary. A new method for the measurement of immunoassay specificity is described. This method is based on the errors caused by the cross-reacting antigens and is directly relevant to the validity of results obtained by immunoassay methods. The effect of selectively blocking the least specific antibodies in antisera raised against steroid haptens is tested. The practical consequences of these considerations are tested using steroid radioimmunoassay and enzyme-immunoassay. (orig.) [de

  15. Comparasion of polyacrylamide gel electrophoresis (PAGE, immuno-electron microscopy (IEM and enzyme immunoassay (EIA for the rapid diagnosis of rotavirus infection in children

    Directory of Open Access Journals (Sweden)

    H. G. Pereira

    1983-12-01

    Full Text Available Detection of rotavirus RNA by polyacrylamide gel electrophoresis (PAGE proved to be a highly sensitive and rapid diagnostic test. A comparison of this assay with immuno-electron microscopy (IEM and enzyme immunoassay (EIA in 245 faeces from children with gastroenteritis revealed complete agreement between the three assays in 238 (97.14% samples. Among 75 samples positive in at least one of the three assays, negative results were observed in 5 (6.48% by PAGE, in 6 (6.76% by EIA and in none by IEM. Silver staining greatly increased the sensitivity of the PAGE assay. We conclude that although IEM remains the most sensitive and rapid rotavirus diagnostic assay, the PAGE technique has many advantages in its favour, including the non-requirement of expensive equipment, the use of only chemically defined reagents and the capacity to distinguish virus subgroup and variants and to detect non-crossreactive rotaviruses which are missed in serological assays.A evidenciação da presença de ácido ribonucleico (ARN viral por eletroforese em gel de policrilamida (EGPA foi comprovada como um método altamente sensível e rápido para o diagnóstico de infecções por rotavirus. Uma comparação desta prova com a imunomicroscopia eletrônica (IEM e com o ensaio imunoenzimático (EIE no exame de 245 fezes de crianças com gastroenterite revelou completa concordancia entre os três ensaios em 238 (97.14% amostras. Entre 75 amostras positivas pelo menos em um dos três ensaios, resultados negativos foram observados em 5 (6.48% por EGPA, em 6 (6.76% por EIE e em nenhum por IEM. Coloração pela prata aumentou consideravelmente a sensibilidade do ensaio por EGPA. Concluímos que embora a IEM ainda seja a prova mais sensível e rápida para o diagnóstico de infecções por rotavirus, o ensaio por EGPA tem muitas vantagens em seu favor, sendo as principais as de não necessitar equipamentos caros, de empregar exclusivamente reagentes quimicamente definidos, de

  16. Application of a newly developed high-sensitivity HBsAg chemiluminescent enzyme immunoassay for hepatitis B patients with HBsAg seroclearance.

    Science.gov (United States)

    Shinkai, Noboru; Matsuura, Kentaro; Sugauchi, Fuminaka; Watanabe, Tsunamasa; Murakami, Shuko; Iio, Etsuko; Ogawa, Shintaro; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2013-11-01

    We modified and automated a highly sensitive chemiluminescent enzyme immunoassay (CLEIA) for surface antigen (HBsAg) detection using a combination of monoclonal antibodies, each for a specific epitope of HBsAg, and by improving an earlier conjugation technique. Of 471 hepatitis B virus (HBV) carriers seen in our hospital between 2009 and 2012, 26 were HBsAg seronegative as determined by the Abbott Architect assay. The Lumipulse HBsAg-HQ assay was used to recheck those 26 patients who demonstrated seroclearance by the Abbott Architect assay. The performance of the Lumipulse HBsAg-HQ assay was compared with that of a quantitative HBsAg detection system (Abbott Architect) and the Roche Cobas TaqMan HBV DNA assay (CTM) (lower limit of detection, 2.1 log copies/ml) using blood serum samples from patients who were determined to be HBsAg seronegative by the Abbott Architect assay. Ten patients had spontaneous HBsAg loss. Of 8 patients treated with nucleotide analogues (NAs), two were HBsAg seronegative after stopping lamivudine therapy and 6 were HBsAg seronegative during entecavir therapy. Eight acute hepatitis B (AH) patients became HBsAg seronegative. Of the 26 patients, 16 were HBsAg positive by the Lumipulse HBsAg-HQ assay but negative by the Abbott Architect assay. The differences between the two assays in terms of detectable HBsAg persisted over the long term in the spontaneous loss group (median, 10 months), the NA-treated group (2.5 months), and the AH group (0.5 months). In 9 patients, the Lumipulse HBsAg-HQ assay detected HBsAg when HBV DNA was negative by the CTM assay. HBsAg was also detected by the Lumipulse HBsAg-HQ assay in 4 patients with an anti-HBs concentration of >10 mIU/ml, 3 of whom had no HBsAg escape mutations. The automatic, highly sensitive HBsAg CLEIA Lumipulse HBsAg-HQ is a convenient and precise assay for HBV monitoring.

  17. Central nervous system blastomycosis diagnosed using the MVista® Blastomyces quantitative antigen enzyme immunoassay test on cerebrospinal fluid: A case report and review of the literature.

    Science.gov (United States)

    Walkty, Andrew; Keynan, Yoav; Karlowsky, James; Dhaliwal, Perry; Embil, John

    2018-02-01

    Blastomyces dermatitidis is a thermally dimorphic fungus that is capable of causing pulmonary and extra-pulmonary disease, including infections of the central nervous system (CNS). Diagnosis of CNS blastomycosis with non-invasive testing can be difficult, and a surgical biopsy may ultimately be required for microbiological and/or histopathological confirmation. A case of B. dermatitidis meningitis is presented where the diagnosis was made by testing cerebrospinal fluid (CSF) using the MVista® Blastomyces Quantitative Antigen Enzyme Immunoassay test. The utility of performing this test on CSF for diagnosis of CNS mass lesions/abscesses caused by B. dermatitidis in the absence of associated meningitis remains unclear. Cross reaction of the Blastomyces antigen test with other dimorphic fungi is a concern, necessitating that positive test results are interpreted in the context of the patient's exposure and travel history. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24)

    International Nuclear Information System (INIS)

    Neurath, A.R.; Strick, N.; Lee, Y.S.; Nilsen, T.; Baker, L.; Sproul, P.; Rubinstein, P.; Taylor, P.; Stevens, C.E.; Gold, J.W.M.

    1985-01-01

    Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and 125 I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA) - or enzyme-linked immunoassay (ELISA) - inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed. (Auth.)

  19. Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24)

    Energy Technology Data Exchange (ETDEWEB)

    Neurath, A R; Strick, N; Lee, Y S; Nilsen, T; Baker, L; Sproul, P; Rubinstein, P; Taylor, P; Stevens, C E; Gold, J W.M.

    1985-10-01

    Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and /sup 125/I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA) - or enzyme-linked immunoassay (ELISA) - inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed. 17 refs.; 2 figs.; 1 table.

  20. Galactomannan enzyme immunoassay and quantitative Real Time PCR as tools to evaluate the exposure and response in a rat model of aspergillosis after posaconazole prophylaxis.

    Science.gov (United States)

    Cendejas-Bueno, Emilio; Forastiero, Agustina; Ruiz, Isabel; Mellado, Emilia; Buitrago, María José; Gavaldà, Joan; Gomez-Lopez, Alicia

    2016-11-01

    A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole. Two different strains of Aspergillus fumigatus with different in vitro posaconazole susceptibility were used. Serum concentrations demonstrated similar posaconazole exposure for all treated animals. However, response to posaconazole relied on the in vitro susceptibility of the infecting strain. After prophylaxis, galactomannan index and fungal burden only decreased in those animals infected with the most susceptible strain. This study demonstrated that both biomarkers may be useful tools for predicting efficacy of antifungal compounds in prophylaxis. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  1. Sandwich-type enzyme immunoassay for big endothelin-I in plasma: concentrations in healthy human subjects unaffected by sex or posture.

    Science.gov (United States)

    Aubin, P; Le Brun, G; Moldovan, F; Villette, J M; Créminon, C; Dumas, J; Homyrda, L; Soliman, H; Azizi, M; Fiet, J

    1997-01-01

    A sandwich-type enzyme immunoassay has been developed for measuring human big endothelin-1 (big ET-1) in human plasma and supernatant fluids from human cell cultures. Big ET-1 is the precursor of endothelin 1 (ET-1), the most potent vasoconstrictor known. A rabbit antibody raised against the big ET-1 COOH-terminus fragment was used as an immobilized antibody (anti-P16). The Fab' fragment of a monoclonal antibody (1B3) raised against the ET-1 loop fragment was used as the enzyme-labeled antibody, after being coupled to acetylcholinesterase. The lowest detectable value in the assay was 1.2 pg/mL (0.12 pg/well). The assay was highly specific for big ET-1, demonstrating no cross-reactivity with ET-1, big endothelin-2 (big ET-2), and big endothelin-3 (big ET-3). We used this assay to evaluate the effect of two different postural positions (supine and standing) on plasma big ET-1 concentrations in 11 male and 11 female healthy subjects. Data analysis revealed that neither sex nor body position influenced plasma big ET-1 concentrations. This assay should thus permit the detection of possible variations in plasma concentrations of big ET-1 in certain pathologies and, in association with ET-1 assay, make possible in vitro study of endothelin-converting enzyme activity in cell models. Such studies could clarify the physiological and clinical roles of this family of peptides.

  2. Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources.

    Science.gov (United States)

    Beutin, L; Steinrück, H; Krause, G; Steege, K; Haby, S; Hultsch, G; Appel, B

    2007-03-01

    To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

  3. Development of an enzyme immunoassay for the antibiotic cefquinome and its application for residue determination in cow's milk after therapeutical mastitis treatment.

    Science.gov (United States)

    Thal, Johannes; Steffen, Monika; Meier, Bianca; Schneider, Elisabeth; Adriany, Ansgar; Usleber, Ewald

    2011-01-01

    The aim of this study was to develop and evaluate an enzyme immunoassay (EIA) for the cephalosporin antibiotic in milk, in combination with a new microbiological test system (brilliant black reduction test, BRT-P). Polyclonal antibodies against cefquinome were produced in rabbits, using cefquinome-keyhole limpet hemocyanine as the immunogen. These antibodies and a cefquinome-glucose oxidase conjugate were used in a competitive indirect EIA. The detection limit for cefquinome in milk was 1.5 ng ml(-1), recoveries were 80-128% at 4-40 ng ml(-1). Cross-reactivities with other cephalosporins/penicillins were all <1%. The EIA was used to determine cefquinome in incurred raw milk, the BRT-P (detection limit ≈ 20 ng ml(-1)) and a receptor assay (ßeta-s.t.a.r., detection limit ≈ 15 ng ml(-1)) were used in parallel. Five lactating cows, suffering from clinical mastitis, were treated with cefquinome by simultaneous intramammary and intramuscular injection. Cefquinome residues (maximum 10-27 μg ml(-1)) were most exclusively found in the udder quarter which was treated intramammary, residue levels in the other three quarters were low (<20 ng ml(-1)). Even in milk from intramammary-dosed quarters, residue levels fell below European Union maximum residue level (MRL, 20 μg kg(-1)) 2 days before the end of the withdrawal period. EIA, BRT-P, and ßeta-s.t.a.r. results showed acceptable agreement for milk samples, but the newly developed EIA is superior in aspects of sensitivity. In conclusion, this is the first one description of immunoassay and microbiological tests capable to determine cefquinome in milk at the MRL in incurred sample material.

  4. Pregnancy does not affect HIV incidence test results obtained using the BED capture enzyme immunoassay or an antibody avidity assay.

    Directory of Open Access Journals (Sweden)

    Oliver Laeyendecker

    2010-10-01

    Full Text Available Accurate incidence estimates are needed for surveillance of the HIV epidemic. HIV surveillance occurs at maternal-child health clinics, but it is not known if pregnancy affects HIV incidence testing.We used the BED capture immunoassay (BED and an antibody avidity assay to test longitudinal samples from 51 HIV-infected Ugandan women infected with subtype A, C, D and intersubtype recombinant HIV who were enrolled in the HIVNET 012 trial (37 baseline samples collected near the time of delivery and 135 follow-up samples collected 3, 4 or 5 years later. Nineteen of 51 women were also pregnant at the time of one or more of the follow-up visits. The BED assay was performed according to the manufacturer's instructions. The avidity assay was performed using a Genetic Systems HIV-1/HIV-2 + O EIA using 0.1M diethylamine as the chaotropic agent.During the HIVNET 012 follow-up study, there was no difference in normalized optical density values (OD-n obtained with the BED assay or in the avidity test results (% when women were pregnant (n = 20 results compared to those obtained when women were not pregnant (n = 115; for BED: p = 0.9, generalized estimating equations model; for avidity: p = 0.7, Wilcoxon rank sum. In addition, BED and avidity results were almost exactly the same in longitudinal samples from the 18 women who were pregnant at only one study visit during the follow-up study (p = 0.6, paired t-test.These results from 51 Ugandan women suggest that any changes in the antibody response to HIV infection that occur during pregnancy are not sufficient to alter results obtained with the BED and avidity assays. Confirmation with larger studies and with other HIV subtypes is needed.

  5. Nanodiamonds as pH-switchable oxidation and reduction catalysts with enzyme-like activities for immunoassay and antioxidant applications.

    Science.gov (United States)

    Chen, T M; Tian, X M; Huang, L; Xiao, J; Yang, G W

    2017-10-19

    Nanodiamonds (NDs) have recently become a focus of interest from the viewpoints of both science and technology. Their intriguing properties make them suitable as biologically active substrates, in biosensor applications as well as diagnostic and therapeutic biomedical imaging probes. Here, we demonstrate that NDs, as oxidation and reduction catalysts, possess intrinsic enzyme mimetic properties of oxidase, peroxidase and catalase, and these behaviors can be switched by modulating the pH value. NDs not only catalyze the reduction of oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) at acidic pH, but also catalyze the dismutation decomposition of H 2 O 2 to produce O 2 at alkaline pH. It was proposed that the molecular mechanism of their peroxidase-like activity is electron-transfer acceleration, the source of which is likely derived from oxygen containing functional groups on their surface. Based on the color reaction, a nanodiamond-based enzyme linked immunosorbent assay (ELISA) was established for the detection of immunoglobulin G (IgG). Surprisingly, NDs display an excellent antioxidant activity due to the protective effect against H 2 O 2 -induced cellular oxidative damage. These findings make NDs a promising enzyme mimetic candidate and expand their applications in biocatalysis, bioassays and nano-biomedicine.

  6. Assessment of radio biochemical problems for optimization of radio - immunoassay technique of insulin in blood

    Energy Technology Data Exchange (ETDEWEB)

    El-Nabarawy, F E

    1979-07-01

    Many biologically active substance such as polypeptides, steroids and others exist in biological fluids att extremely low concentrations. There is no available chemical technique that is capable of measuring these compounds. Under these conditions since they have no unique chemical properties . Two possible solutions to this problem are available. The first is to use the biological actionof the substance as an assay. However, biological assays without prior extraction and concentration of ten lack sufficient sensitivity to detect them in plasma.

  7. Production of monoclonal antibodies for use in immunoassays based on the magnetizable solid phase separation technique

    International Nuclear Information System (INIS)

    Charoensiriwatana, W.; Janejai, N.; Krasao, P.

    1996-01-01

    Monoclonal antibodies to TSH were produced by using mouse-ascites techniques. Various methods for purifying the antibody from the ascetic fluid have been tried in order to obtain an appropriate TSH kit production protocol. The purified antibodies were then immobilized on magnetizable cellulose for developing an IRMA for TSH. A detailed study of the assay system, including the stability of the magnetic adsorbent was made, which showed that the SCIPAc magnetizable cellulose is suitable for the production of TSH - Blood spot IRMA kits for use in the Neonatal hypothyroid screening programme to be launched in Thailand in the near future. (author). 4 refs, 12 figs, 2 tabs

  8. Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

    Science.gov (United States)

    Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

    2007-02-01

    Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

  9. Evaluation and Comparison of Enzyme Immunoassay (Eia and Acid Fast Staining with Confirmation by Immunofluorescent Antibody Assay for Detection of Cryptosporidium Species in Infants and Young Children.

    Directory of Open Access Journals (Sweden)

    D Dorostcar Moghaddam

    2005-01-01

    Full Text Available Introduction: Cryptosporidiosis is prevalent world wide, causing a variety of problems ranging from acute, self-limiting diarrhea to fatal cases in immunocompromised persons, particulary those with acquired immunodeficiency (AIDS. Diagnosis of Cryptosporidium is made by identification of oocysts in stool specimens. The detection is most commonly made by the acid-fast staining method followed by microscopic examination which has low specificity and sensitivity. Material and Methods: In the present study, we evaluated diagnostic utility of a commercially available enzyme immunoassay (EIA, which detects Cryptosporidium-Specific antigen (CSA in 204 unprocessed stool specimens obtained from patients less than 3 years of age. Results: When compared with the routine screening procedure applied in this field study (screening by acid-fast staining and microscopy after concentration of positive results by IFA, both sensitivity and specificity were 98%. Of the 139 specimens negative by microscopy, 13 (9.3% were positive by EIA, 11 of which were confirmed by inhibition with antibody to Cryptosporidia-specific antigen. Conclusion: The EIA is an important tool for identifying Cryptosporidium in fecal specimens in field studies since it is sensitive, specific, simple to use and unaffected by the presence of a preservative.

  10. Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

    Science.gov (United States)

    Pribbenow, Susanne; Wachter, Bettina; Ludwig, Carsten; Weigold, Annika; Dehnhard, Martin

    2016-03-01

    In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

    International Nuclear Information System (INIS)

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

    2015-01-01

    Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B) 2 complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for

  12. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jin-Bao [School of Pharmacy, Weifang Medical University, Weifang 261053 (China); Tang, Ying [Affiliated Hospital of Weifang Medical University, Weifang 261041 (China); Yang, Hong-Ming, E-mail: yanghongming2006@sohu.com [School of Pharmacy, Weifang Medical University, Weifang 261053 (China)

    2015-02-15

    Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B){sub 2} complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable

  13. Plasma cortisol and 11-ketotestosterone enzyme immunoassay (EIA) kit validation for three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus.

    Science.gov (United States)

    Mills, S C; Mourier, J; Galzin, R

    2010-08-01

    Commercially available enzyme immunoassay (EIA) kits were validated for measuring steroid hormone concentrations in blood plasma from three fish species: the orange clownfish Amphiprion percula, the orangefin anemonefish Amphiprion chrysopterus and the blacktip reef shark Carcharhinus melanopterus. A minimum of 5 microl plasma was required to estimate hormone concentrations with both kits. These EIA kits are a simple method requiring minimal equipment, for measuring hormone profiles under field conditions.

  14. Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species.

    Science.gov (United States)

    Bienboire-Frosini, Cecile; Chabaud, Camille; Cozzi, Alessandro; Codecasa, Elisa; Pageat, Patrick

    2017-01-01

    The neurohormone oxytocin (OT) has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA) for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat). The Oxytocin EIA kit (EnzoLifeSciences) was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml). To validate the method, the following performance characteristics were evaluated using Validation Samples (VS) at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was verified. Although

  15. Validation of a Commercially Available Enzyme ImmunoAssay for the Determination of Oxytocin in Plasma Samples from Seven Domestic Animal Species

    Directory of Open Access Journals (Sweden)

    Cecile Bienboire-Frosini

    2017-09-01

    Full Text Available The neurohormone oxytocin (OT has a broad range of behavioral effects in mammals. It modulates a multitude of social behaviors, e.g., affiliative and sexual interactions. Consequently, the OT role in various animal species is increasingly explored. However, several issues have been raised regarding the peripheral OT measurement. Indeed, various methods have been described, leading to assay discrepancies and inconsistent results. This highlights the need for a recognized and reliable method to measure peripheral OT. Our aim was to validate a method combining a pre-extraction step, previously demonstrated as essential by several authors, and a commercially available enzyme immunoassay (EIA for OT measurement, using plasma from seven domestic species (cat, dog, horse, cow, pig, sheep, and goat. The Oxytocin EIA kit (EnzoLifeSciences was used to assay the solid-phase extracted samples following the manufacturer's instructions with slight modifications. For all species except dogs and cats, concentration factors were applied to work above the kit's sensitivity (15 pg/ml. To validate the method, the following performance characteristics were evaluated using Validation Samples (VS at various concentrations in each species: extraction efficiency via spiking tests and intra- and inter-assay precision, allowing for the calculation of total errors. Parallelism studies to assess matrix effects could not be performed because of too low basal concentrations. Quantification ranges and associated precision profiles were established to account for the various OT plasma concentrations in each species. According to guidelines for bioanalytical validation of immunoassays, the measurements were sufficiently precise and accurate in each species to achieve a total error ≤30% in each VS sample. In each species, the inter-assay precision after 3 runs was acceptable, except in low concentration samples. The linearity under dilution of dogs and cats' samples was

  16. Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo-immunoassays for Chemical Contaminants in Food and Environmental Samples.

    Science.gov (United States)

    Chen, Chaochao; Luo, Jiaxun; Li, Chenglong; Ma, Mingfang; Yu, Wenbo; Shen, Jianzhong; Wang, Zhanhui

    2018-03-21

    The chemical contaminants in food and the environment are quite harmful to food safety and human health. Rapid, accurate, and cheap detection can effectively control the potential risks derived from these chemical contaminants. Among all detection methods, the immunoassay based on the specific interaction of antibody-analyte is one of the most widely used techniques in the field. However, biological antibodies employed in the immunoassay usually cannot tolerate extreme conditions, resulting in an unstable state in both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class of polymers with specific molecular recognition abilities, which are highly robust, showing excellent operational stability under a wide variety of conditions. Recently, MIPs have been used in biomimetic immunoassays for chemical contaminants as an antibody substitute in food and the environment. Here, we reviewed these applications of MIPs incorporated in different analytical platforms, such as enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, microfluidic paper-based immunoassay, and homogeneous immunoassay, and discussed current challenges and future trends in the use of MIPs in biomimetic immunoassays.

  17. Chemiluminescence immunoassay for chloramphenicol

    International Nuclear Information System (INIS)

    Lin Si; Xu Wenge; Liu Yibing

    2007-06-01

    A simple, solid-phase chemiluminescence immunoassay (CLIA) for the measurement of Chloramphenicol(CAP) in foodstuffs is described. A rabbit anti-CAP IgG is passively adsorbed onto the walls of polypropylene plates. The labeled conjugant is horseradish peroxidase(HRP) conjugate of CAP. Luminol solution is used as the substrate of HRP. The light yield is inversely proportional to the concentration of CAP. The method has a similar sensitivity (0.05 ng/mL), specificity, precision, and accuracy to a conventional enzyme immunoassay (EIA). The intra-assay and inter-assay CVs of ten samples were <8 and <20%, respectively, and the analytical recovery of the method was 87% 100%. The experimental correlation coefficient of dilution was found to be 0.999 using milk supernatant as buffer. The assay range for the method was 0.1-10 ng/mL, and it displayed good linearity. (authors)

  18. Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species.

    Science.gov (United States)

    Bojanić, Krunoslav; Midwinter, Anne Camilla; Marshall, Jonathan Craig; Rogers, Lynn Elizabeth; Biggs, Patrick Jon; Acke, Els

    2016-08-01

    Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Utilization of a DNA enzyme immunoassay for the detection of proviral DNA of human immunodeficiency virus type 1 by polymerase chain reaction.

    Science.gov (United States)

    Zella, D; Cavicchini, A; Cattaneo, E; Cimarelli, A; Bertazzoni, U

    1995-02-01

    The detection of proviral DNA by Polymerase Chain Reaction (PCR) is regarded as an important tool in the diagnosis of HIV-1 infection, specially among adults at risk of AIDS and children born to seropositive mothers. However, application of PCR in routine testing is hampered by the need to use radioactive probes. In this study, a non-radioactive test based on a microtiter plate (DNA Enzyme ImmunoAssay, DEIA) was used for the detection of proviral sequences of HIV-1 in peripheral blood cells of different patients. The results of the PCR-DEIA assay were compared to those obtained by liquid hybridization (PCR-LH), virus isolation (VI) and Western blot (WB). The study population included 92 patients belonging to three different groups: seropositive subjects with a well-defined clinical status and WB profile; adults at risk of infection with negative or indeterminate WB; children born to seropositive mothers with still unestablished HIV-1 infection. In the seropositive subjects, both PCR-LH and PCR-DEIA confirmed infection and gave the same results as WB. In adults at risk of infection, PCR with both methods anticipated the seroconversion in one patient with indeterminate WB and confirmed the absence of infection among seronegative and other indeterminate patients. In children born to seropositive mothers, both PCR systems as well as VI permitted an early diagnosis of infection, as confirmed by the clinical follow-up. This study has shown that in subjects at risk of AIDS and in children born to seropositive mothers, the non-isotopic DEIA method presents the same sensitivity and specificity for the detection of HIV-1 infection as the radioactive procedure. The DEIA method appears to be particularly useful for the detection of PCR products in routine diagnostic analyses.

  20. Assessment of pregnancy status of Asian elephants (Elephas maximus) by measurement of progestagen and glucocorticoid and their metabolite concentrations in serum and feces, using enzyme immunoassay (EIA).

    Science.gov (United States)

    Kajaysri, Jatuporn; Nokkaew, Weerapun

    2014-03-01

    The study was to find patterns of progestagen (progesterone and its metabolite) and glucocorticoid and their metabolite concentrations in serum and feces of pregnant Asian elephants (Elephas maximus). The 5 female Asian domestic elephants were naturally mated until pregnancy. After that, blood and feces samples were collected monthly during pregnancy for progestagen, glucocorticoid and their metabolites analysis by enzyme immunoassay (EIA). The results showed the serum progestagen concentration during gestation was 2.11 ± 0.60 to 18.44 ± 2.28 ng/ml. Overall, serum progestagen concentration rose from the 1st month to reach peak in the 11th month, after which it declined to its lowest level in the 22nd month of pregnancy. Fecal progestagen concentration varied from 1.18 ± 0.54 to 3.35 ± 0.45 µg/g during pregnancy. In general, fecal progestagen concentration increased from the 1st month to its highest level in the 12th month. After this, it declined reaching its lowest point in the 22nd month of pregnancy. Glucocorticoid hormones and their metabolite concentrations both in serum and feces fluctuated from low to medium throughout almost the entire pregnancy period and then rapidly increased around the last week before calving. Our study suggests that this profile of progestagen and glucocorticoid hormones and their metabolite concentration levels in serum and feces can be used to assess the pregnancy status of Asian elephants. If serum and fecal progestagen concentrations were found in very low levels and glucocorticoid and their metabolite concentrations were found in very high levels, it was indicated that the cow elephant would calve within 7 days.

  1. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection.

    Science.gov (United States)

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-06-01

    Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

  2. Development of a single-chain variable fragment-alkaline phosphatase fusion protein and a sensitive direct competitive chemiluminescent enzyme immunoassay for detection of ractopamine in pork

    Energy Technology Data Exchange (ETDEWEB)

    Dong Jiexian; Li Zhenfeng; Lei Hongtao; Sun Yuanming [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Ducancel, Frederic [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Xu Zhenlin [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Boulain, Jean-Claude [CEA, iBiTec-S, Service de Pharmacologie et d' Immnoanalyse (SPI), CEA Saclay, F-91191 Gif sur Yvette (France); Yang Jinyi; Shen Yudong [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China); Wang Hong, E-mail: gzwhongd@63.com [Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642 (China)

    2012-07-29

    Graphical abstract: Detection model of dc-CLEIA based on anti-RAC scFv-AP fusion protein. Highlights: Black-Right-Pointing-Pointer The scFv-AP fusion protein against ractopamine (RAC) was produced. Black-Right-Pointing-Pointer A dc-CLEIA for RAC was developed based on the purified scFv-AP fusion protein. Black-Right-Pointing-Pointer The sensitivity of dc-CLEIA was 10 times as sensitive as dc-ELISA for RAC. Black-Right-Pointing-Pointer Recovery tests from pork samples were studied. Black-Right-Pointing-Pointer Good accuracy was obtained. - Abstract: A rapid, sensitive chemiluminescent enzyme immunoassay (CLEIA) for ractopamine (RAC) based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein was developed. The scFv gene was prepared by cloning the heavy- and light-chain variable region genes (V{sub H} and V{sub L}) from hybridoma cell line AC2, which secretes antibodies against RAC, and assembling V{sub H} and V{sub L} genes with a linker by means of splicing overlap extension polymerase chain reaction. The resulting scFv gene was inserted into the expression vector pLIP6/GN containing AP to produce the fusion protein in Escherichia coli strain BL21. The purified scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) protocol for detection of RAC. The average concentration required for 50% inhibition of binding and the limit of detection of the assay were 0.25 {+-} 0.03 and 0.02 {+-} 0.004 ng mL{sup -1}, respectively, and the linear response range extended from 0.05 to 1.45 ng mL{sup -1}. The assay was 10 times as sensitive as the corresponding enzyme-linked immunosorbent assay based on the same fusion protein. Cross-reactivity studies showed that the fusion protein did not cross react with RAC analogs. DcCLEIA was used to analyze RAC spiked pork samples, and the validation was confirmed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The results showed a good correlation between

  3. Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

    Science.gov (United States)

    Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

    1999-10-01

    Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests

  4. Challenges, pitfalls and surprises: development and validation of a monoclonal antibody for enzyme immunoassay of the steroid 1α-hydroxycorticosterone in elasmobranch species.

    Science.gov (United States)

    Wheaton, Catharine J; Mylniczenko, Natalie D; Rimoldi, John M; Gadepalli, Rama S V S; Hart, R; O'Hara, Bobbi R; Evans, Andrew N

    2018-01-31

    Sharks and rays are popular species used in wildlife ecotourism and aquariums to educate the public on the behavior, ecology and conservation challenges of elasmobranchs. To understand long-term physiological health and welfare under varying social and husbandry conditions, we developed and validated an enzyme immunoassay (EIA) to measure stress/ionoregulatory hormones in managed and semi-free range southern rays (Hypanus americanus). Banked serum and interrenal samples from 27 female rays managed at Disney's The Seas with Nemo and Friends® and Castaway Cay were used to evaluate measurement of 1α-hydroxycorticosterone (1αOHB) relative to corticosterone (B). Although commercial EIAs are available for B, those tested exhibit only low relative cross-reactivity to 1αOHB (3-5%). To improve measurement of 1αOHB, we developed a monoclonal antibody using a synthesized 1αOHB-derivative for evaluation using high-performance liquid chromatography (HPLC) and EIA. Relative displacements of cross-reactant compounds showed that the antibody had good sensitivity for the target antigen 1αOHB, and low sensitivity to related steroids (desoxycorticosterone and B), but greater sensitivity to 11-dehydrocorticosterone. Tests of competitive vs. noncompetitive EIA formats, reagent titration, and incubation times of the antibody and conjugate were used to optimize sensitivity, repeatability and precision of measured 1αOHB in standards and samples (4 ng/ml, 90% binding). Tests of sample pre-treatment (pH adjustment) and extraction with varying solvent polarity were used to optimize measurement of 1αOHB in <1 ml (serum) or 1 g (interrenal) samples. HPLC analysis revealed the 1αOHB EIA to be superior for measurement of 1αOHB compared to use of a B EIA with or without HPLC fractioning. Results may prove useful for extrapolation to guide best practices for 1αOHB measurement in other elasmobranch species. Improved measurement of stress/ionoregulatory hormones in sharks and rays

  5. A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody

    International Nuclear Information System (INIS)

    Yoshida, Shinichi

    1986-01-01

    Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng per tube, and linearity was obtained between 0.01 to 30 ng per tube. The radioimmunoassay used a 125 I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. (Auth.)

  6. Comparison of a novel chemiluminescence enzyme immunoassay (CLEIA) with enzyme-linked immunosorbent assay (ELISA) for the determination of MPO-ANCA in patients with ANCA-associated vasculitis.

    Science.gov (United States)

    Hirose, Orie; Itabashi, Mitsuyo; Takei, Takashi; Nitta, Kosaku

    2015-03-01

    Myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA) represents the serological hallmark of ANCA-associated vasculitis (AAV). We evaluated the analytical and diagnostic accuracy of chemiluminescence enzyme immunoassay (CLEIA) versus enzyme-linked immunosorbent assay (ELISA) for the detection of MPO-ANCA. A total of 242 sera obtained from 51 patients with AAV and 103 patients without AAV were tested for MPO-ANCA by ELISA (NephroScholor MPOANC II) and CLEIA (the STACIA MEBLux test). Disease activity in the patients with AAV was determined based on the Birmingham Vasculitis Activity Score. We analyzed the correlations between the MPO-ANCA titers determined by the CLEIA and those determined by the ELISA, and also between the MPO-ANCA titers and the disease activity. The MPO-ANCA titers determined by the CLEIA (x) were strongly correlated with those determined by the ELISA (y). The correlation could be expressed by the following equation in this study: y = 1.8x + 7.7 (r = 0.96; p ELISA yielded positive test results in 57 of the 242 sera (23.6%). The CLEIA yielded false-positive test results in 4 of the 120 sera obtained from the non-AAV patients (3.3%), whereas the ELISA yielded a false-positive result in only 1 of the 120 sera obtained from the non-AAV patients (0.8%). The sensitivity and specificity of the CLEIA for the diagnosis of AAV were 100% and 96.7%, respectively, while those of the ELISA were 94.3% and 99.2%, respectively. The sensitivity and specificity of the CLEIA for the prediction of active disease were 100% and 64.4%, respectively, while those of the ELISA were 94.3% and 73.6%, respectively. The false positivity rate of the CLEIA for MPO-ANCA tended to be high as compared with that of the ELISA. Also, according to the correlation coefficient between the results of the CLEIA and the ELISA calculated in this study, it is necessary to pay attention to the differences in the sensitivity and specificity between CLEIA and ELISA.

  7. Comparison of gas chromatography/mass spectrometry and immunoassay techniques on concentrations of atrazine in storm runoff

    Science.gov (United States)

    Lydy, Michael J.; Carter, D.S.; Crawford, Charles G.

    1996-01-01

    Gas chromatography/mass spectrometry (GC/MS) and enzyme-linked immunosorbent assay (ELISA) techniques were used to measure concentrations of dissolved atrazine in 149 surface-water samples. Samples were collected during May 1992–September 1993 near the mouth of the White River (Indiana) and in two small tributaries of the river. GC/MS was performed on a Hewlett-Packard 5971 A, with electron impact ionization and selected ion monitoring of filtered water samples extracted by C-18 solid phase extraction; ELISA was performed with a magnetic-particle-based assay with photometric analysis. ELISA results compared reasonably well to GC/MS measurements at concentrations below the Maximum Contaminant Level for drinking water set by the U.S. Environmental Protection Agency (3.0 μg/L), but a systematic negative bias was observed at higher concentrations. When higher concentration samples were diluted into the linear range of calibration, the relation improved. A slight positive bias was seen in all of the ELISA data compared to the GC/MS results, and the bias could be partially explained by correcting the ELISA data for cross reactivity with other triazine herbicides. The highest concentrations of atrazine were found during the first major runoff event after the atrazine was applied. Concentrations decreased throughout the rest of the sampling period even though large runoff events occurred during this time, indicating that most atrazine loading to surface waters in the study area occurs within a few weeks after application.

  8. An Investigation of Students' Views about Enzymes by Fortune Lines Technique

    Science.gov (United States)

    Özarslan, Murat; Çetin, Gülcan

    2014-01-01

    This study aimed to investigate ninth grade students' views about enzymes using fortune lines technique and to obtain the students' views about fortune lines technique. Participants were 38 ninth grade students in a Technique Vocational Girls High School in the city of Kocaeli, Turkey. After instruction of subject of enzymes, the participants were…

  9. Industrial Applications of Enzymes: Recent Advances, Techniques, and Outlooks

    Directory of Open Access Journals (Sweden)

    Jordan Chapman

    2018-06-01

    Full Text Available Enzymes as industrial biocatalysts offer numerous advantages over traditional chemical processes with respect to sustainability and process efficiency. Enzyme catalysis has been scaled up for commercial processes in the pharmaceutical, food and beverage industries, although further enhancements in stability and biocatalyst functionality are required for optimal biocatalytic processes in the energy sector for biofuel production and in natural gas conversion. The technical barriers associated with the implementation of immobilized enzymes suggest that a multidisciplinary approach is necessary for the development of immobilized biocatalysts applicable in such industrial-scale processes. Specifically, the overlap of technical expertise in enzyme immobilization, protein and process engineering will define the next generation of immobilized biocatalysts and the successful scale-up of their induced processes. This review discusses how biocatalysis has been successfully deployed, how enzyme immobilization can improve industrial processes, as well as focuses on the analysis tools critical for the multi-scale implementation of enzyme immobilization for increased product yield at maximum market profitability and minimum logistical burden on the environment and user.

  10. Rapid detection of fungal alpha-amylase in the work environment with a lateral flow immunoassay

    NARCIS (Netherlands)

    Bogdanovic, J.; Koets, M.; Sander, I.; Wouters, I.; Meijster, T.; Heederik, D.J.J.; Amerongen, van A.; Doekes, G.

    2006-01-01

    Background Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with

  11. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  12. Enzyme immunoassay and proteomic characterization of troponin I as a marker of mammalian muscle compounds in raw meat and some meat products.

    Science.gov (United States)

    Zvereva, Elena A; Kovalev, Leonid I; Ivanov, Alexei V; Kovaleva, Marina A; Zherdev, Anatoly V; Shishkin, Sergey S; Lisitsyn, Andrey B; Chernukha, Irina M; Dzantiev, Boris B

    2015-07-01

    The skeletal muscle protein troponin I (TnI) has been characterized as a potential thermally stable and species-specific biomarker of mammalian muscle tissues in raw meat and meat products. This study proposed a technique for the quantification of TnI comprising protein extraction and sandwich enzyme-linked immunosorbent assay (ELISA). The technique is characterized by a TnI detection limit of 4.8 ng/ml with quantifiable concentrations ranging from 8.7 to 52 ng/ml. The method was shown to be suitable for detection of TnI in mammalian (beef, pork, lamb, and horse) meat but not in poultry (chicken, turkey, and duck) meat. In particular, the TnI content in beef was 0.40 3 ± 0.058 mg/g of wet tissue. The TnI estimations obtained for the pork and beef samples using ELISA were comparable to the proteomic analysis results. Thus, the quantitative study of TnI can be a convenient way to assess the mammalian muscle tissue content of various meat products. Copyright © 2015. Published by Elsevier Ltd.

  13. A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

    Science.gov (United States)

    Lai, Wenqiang; Zeng, Qiao; Tang, Juan; Zhang, Maosheng; Tang, Dianping

    2018-01-10

    The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B 1 (AFB 1 ). It is based on the just-in-time generation of an MnO 2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO 4 ) is converted into manganese dioxide (MnO 2 ) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO 4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO 2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO 4 . Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB 1 and magnetic beads carrying bovine serum albumin conjugated to AFB 1 are used for the determination of AFB 1 . In presence of AFB 1 , it will compete with the BSA-conjugated AFB 1 (on the magnetic beads) for the labeled antibody against AFB 1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB 1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB 1 concentrations in the range from 0.1 to 100 ng mL -1 , with a 0.1 ng mL -1 detection limit (at the 3S blank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract

  14. Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

    International Nuclear Information System (INIS)

    Dickinson, D.B.

    1975-01-01

    Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

  15. A sensitive progesterone enzyme immunoassay for cow, goat and llama plasma using a monoclonal antibody and Danazol (17-α-2,4-pregnadien-20-yno (2,3-D) isoxazol-17-ol) as a displacing agent

    International Nuclear Information System (INIS)

    Aba, M.A.; Carlsson, M.A.; Karlsson, A.; Forsberg, M.

    2001-01-01

    A sensitive progesterone enzyme immunoassay was developed for cow, goat and llama plasma using a monoclonal antibody and Danazol (17-α-2,4-pregnadien-20-yno (2,3-d) isoxazol-17-ol) as a displacing agent. The microtitration plates were first coated with progesterone 3 (o-carboxy-methyl) oxine: BSA conjugate. The immune reaction was performed by incubating overnight a mixture of 50 μL of plasma and 100 μL of first antibody. After washing, 100 μL of the second antibody (horse radish peroxidase conjugated anti-mouse IgG) were added. The plates were incubated for 1 hour and washed. Immediately the substrate solution was added and finally the reaction stopped and optical density measured. This assay allows accurate determination of progesterone in plasma from several species with good specificity, precision and accuracy, and is suitable for the rapid assessment of luteal function and reproductive status in both clinical and research situations. (author)

  16. Procedures for Sensitive Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Givol, D. [Department of Chemical Immunology, Weizmann Institute of Science, Rehovot (Israel)

    1970-02-15

    Sensitive immunoassay methods should be applied to small molecules of biological importance, which are non-immunogenic by themselves, such as small peptide hormones (e.g. bradykinin), plant hormones (e.g. indoleacetic acid), nucleotides and other small molecules. Methods of binding these small molecules, as haptens, to immunogenic carriers by various cross-linking agents are described (dicyclohexylcarbodiimide, tolylene-diisocyanate and glutaraldehyde), and the considerations involved in relation to the methods of binding and the specificity of the antibodies formed are discussed. Some uses of antibody bound to bromoacetyl cellulose as an immuno adsorbent convenient for assay of immunoglobulins are described. Finally, the sensitive immunoassay method of chemically modified phage is described. This includes methods of binding small molecules (such as the dinitrophenyl group, penicillin, indoleacetic acid) or proteins (such as insulin, immunoglobulins) to phages. Methods of direct chemical conjugation, or an indirect binding via anti-phage Fab, are described. The phage inactivation method by direct plating and its modifications (such as decision technique and complex inactivation) are compared with the more simple end-point titration method. The inhibition of phage inactivation has some advantages as it does not require radioactive material, or expensive radioactive counters, and avoids the need for separation between bound and unbound antigen. Hence, if developed, it could be used as an alternative to radioimmunoassay. (author)

  17. Rotifer digestive enzymes: direct detection using the ELF technique

    Czech Academy of Sciences Publication Activity Database

    Štrojsová, M.; Vrba, Jaroslav

    2007-01-01

    Roč. 593, č. 1 (2007), s. 159-165 ISSN 0018-8158. [International Symposium on Rotifers /11./. Mexiko City, 08.03.2006-13.03.2006] R&D Projects: GA AV ČR(CZ) IAA6017202; GA AV ČR(CZ) IAA600170602; GA AV ČR(CZ) 1QS600170504 Institutional research plan: CEZ:AV0Z60170517 Keywords : rotifers * enzyme localization * phosphatase * lipase * .beta.-N-acetylhexosaminidase Subject RIV: DA - Hydrology ; Limnology Impact factor: 1.201, year: 2007

  18. Validity and reliability of enzyme immunoassays using Leishmania major or L. infantum antigens for the diagnosis of canine visceral leishmaniasis in Brazil.

    Directory of Open Access Journals (Sweden)

    Mauro Maciel de Arruda

    Full Text Available BACKGROUND: American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. METHODS: A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. RESULTS: The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. INTERPRETATION: ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.

  19. Determination of Glutamic Acid Decarboxylase (GAD65 in Pancreatic Islets and Its In Vitro and In Vivo Degradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

    Directory of Open Access Journals (Sweden)

    Michael Schlosser

    2008-01-01

    Full Text Available Glutamic acid decarboxylase GAD65 autoantibodies (GADA are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human, 43.7 (LEW.1A rat and 37.4 (BB/OK rat ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65.

  20. Immunoassays in Biotechnology

    Science.gov (United States)

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  1. Determination of the reproductive behavior of dairy cows, post-partum, by means of radio immunoassay (RIA) techniques

    International Nuclear Information System (INIS)

    Villalba, Patricio; Guerron, Edwin

    1992-01-01

    The main objective of this trial was to determine the post-partum ovaric activity in dairy cattle recently introduced in the Ecuadorian subtropic. On this trial, progesterone levels in milk of 114 post-partum cows from 6 different farms were determined. The progesterone level in milk was analyzed by RIA and the milk sampling was done according to established protocol. Additional data about management, nutritional condition and health of the animals were also taken in each farm. In the Santo Domingo valley, the ovaries of post-partum cows, with a good nutritious diet, start functioning after 54.21 days with a range of 10 to 161. The duration of the first oestral cycle was 24.07 with the range of 6 to 29 days. Fifty six cows (49.12 percent) remained pregnant up to the end of the trial and the average of interval calving was 409. 95 - 115. 69 days. In the area, there is high incidence of ovaric hypofunctions ( 30.70 percent) and return of anoestrus ( 14.03 percent). heats of the cows were observed ( 62.90 percent) by the farmers, but they were determined by progesterone levels, through RIA technique

  2. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  3. Cost-effectiveness of a modified two-step algorithm using a combined glutamate dehydrogenase/toxin enzyme immunoassay and real-time PCR for the diagnosis of Clostridium difficile infection.

    Science.gov (United States)

    Vasoo, Shawn; Stevens, Jane; Portillo, Lena; Barza, Ruby; Schejbal, Debra; Wu, May May; Chancey, Christina; Singh, Kamaljit

    2014-02-01

    The analytical performance and cost-effectiveness of the Wampole Toxin A/B EIA, the C. Diff. Quik Chek Complete (CdQCC) (a combined glutamate dehydrogenase antigen/toxin enzyme immunoassay), two RT-PCR assays (Progastro Cd and BD GeneOhm) and a modified two-step algorithm using the CdQCC reflexed to RT-PCR for indeterminate results were compared. The sensitivity of the Wampole Toxin A/B EIA, CdQCC (GDH antigen), BD GeneOhm and Progastro Cd RT-PCR were 85.4%, 95.8%, 100% and 93.8%, respectively. The algorithm provided rapid results for 86% of specimens and the remaining indeterminate results were resolved by RT-PCR, offering the best balance of sensitivity and cost savings per test (algorithm ∼US$13.50/test versus upfront RT-PCR ∼US$26.00/test). Copyright © 2012. Published by Elsevier B.V.

  4. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

    Science.gov (United States)

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

  5. Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9-12 Years Children.

    Science.gov (United States)

    Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

    2015-01-01

    The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9-11 years were determined by two methods of CPBA and HPLC. Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes.

  6. Determination of relative assay response factors for toxic chlorinated and brominated dioxins/furans using an enzyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX).

    Science.gov (United States)

    Samara, Fatin; Gullett, Brian K; Harrison, Robert O; Chu, Andrew; Clark, George C

    2009-04-01

    Determination of toxic activity requires knowledge of both the concentration and toxicity to evaluate the risk for adverse human health and environmental effects. A chemically-activated luciferase gene expression cell bioassay system (CALUX) and an antibody-based method enzyme immunoassay (EIA) were used to detect the dioxin-like response of several polybrominated, polychlorinated, and polybrominated/chlorinated dibenzo-p-dioxins/furans (PBDDs/Fs, PCDDs/Fs, and PBCDDs/Fs, respectively). It has been suggested that the biological activity of the brominated and mixed bromo/chloro compounds is similar to their chlorinated analogues (measured by binding to the Ah receptor). PBDD/F, PCDD/F, and PBCDD/F laboratory standards exhibited biological activity ranging over three orders of magnitude. The highest relative potency (REP) values from CALUX analysis, when compared to 2,3,7,8-TCDD, were 2,3,7,8-TBDD at 0.99 (+/-0.07), 1,2,3,7,8-PeCDD at 0.69, and 2-Br-3,7,8-TriCDD at 0.72 (+/-0.02). Cross-reactivities were calculated using EIA for several PBDDs/Fs and PBCDDs. The highest percent cross-reactivity was found for 2,3,7,8-TBDD at 138 (+/-34%), and 2,3,7-TriBDD at 84 (+/-36%).

  7. [Detecting the markers of HIV infection with the new enzyme immunoassay diagnostic kit "DS-EIA-HIV-AB-AG-SPECTRUM" at the laboratories of AIDS prevention and control centers in the Volga Federal District].

    Science.gov (United States)

    Ivanova, N I; Peksheva, O Iu

    2009-03-01

    A possibility of simultaneously detecting specific antibodies to HIV-1 and HIV-2 by enzyme immunoassay (EIA) at lower concentrations than those by immunoblotting (IB), and well as an additional possibility of earlier diagnosis of HIV infection, by identifying the HIV-1 antigen p24 lay the foundation of the "DS-EIA-HIV-AB-AG-SPECTRUM" test system made by OOO "Research-and-Production Association "Diagnosticheskiye Sistemy" (Diagnostic Systems). These peculiarities were compared with those of IB at a number of laboratories of AIDS prevention and control centers in the Volga Federal District, by using native serum/plasma samples and a specially designed control panel. The analysis of the conducted studies to identify HIV-1 and HIV-2 antibodies and HIV-1 antigen p24 in 65 plasma/serum samples in the "DS-EIA-HIV-AB-AG-SPECTRUM" and "LIA-HIV-1/2" (OOO "Niarmedik plus") test systems while confirming the positive result indicated agreement in 57 (87.7%) cases. The diagnostic possibilities of the "DS-EIA-HIV-AB-AG-SPECTRUM" test system versus the "New Lav-Blot I" one to make a laboratory diagnosis of HIV infection were studied. Irrefragable answers as to the availability of HIV-1 markers in the study serum samples on the enciphered panel were provided by IB in 73.3% of cases and EIA in 92%.

  8. Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

    Science.gov (United States)

    Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

    2015-08-25

    Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Radioimmunoassay and other related techniques

    International Nuclear Information System (INIS)

    Zarkawi, Moutaz

    1993-04-01

    The article reviews principles, requirements and reliability criteria of radioimmunoassay (RIA). Since basic reactions involved in RIA and related techniques are derived from reactions which take place in the immune system (IS) of humans and animals, the IS and the way it works will be described. In addition to RIA which involves the use of isotopes as tracers (labels), other non-radioisotopic and recent immunoassay techniques i.e. enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA) and fluoroimmunoassay (FIA) will be dealt with. Some important and related terms will be defined and explained. (author). 59 refs., 4 figs

  10. Immunoassay for thymopoietin

    International Nuclear Information System (INIS)

    Goldstein, G.

    1979-01-01

    The patent describes the development of a radio-immunoassay for thymopoietin in biological samples. The method of raising antibodies to this polypeptide hormone is described. This is achieved by injecting a host animal with an antigen consisting of thymopoietin covalently bonded by glutaraldehyde to a carrier protein such as bovine serum albumin and equine globulin. Different methods of radiolabelling thymopoietin with 125 I for use as the tracer antigen are described. The Bolton-Hunter procedure was preferred to the chloramine-T method since direct iodination of the tyrosyl moieties of thymopoietin resulted in some loss of immunoreactivity. Systems for separating the antigen-antibody complex and unbound antigen are compared. Binding-inhibition curves for unlabelled thymopoietin in the assay employing polyethylene glycol separation showed a sensitivity of 5 ng thymopoietin/ml. However, using the double antibody or dextran coated charcoal separation techniques, the sensitivity of thymopoietin was 0.1 ng/ml. Thus these latter two procedures are thus especially suitable for measuring thymopoietin levels in serum or plasma samples. The assay was shown to be specific for thymopoietin, no significant displacement being produced by control polypeptides. (U.K.)

  11. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test

    International Nuclear Information System (INIS)

    Casali, P.; Bossus, A.; Carpentier, N.A.; Lambert, P.H.

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample was incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes were detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test did not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG was 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the 125 I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, 125 I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application. (author)

  12. A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

    Science.gov (United States)

    Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

    2012-01-01

    ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

  13. A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.

    Science.gov (United States)

    Grace, M; Fletcher, L; Powers, S K; Hughes, M; Coombes, J

    1996-12-01

    Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.

  14. Comparison of premier CAMPY enzyme immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY tests with culture for laboratory diagnosis of Campylobacter enteric infections.

    Science.gov (United States)

    Granato, Paul A; Chen, Li; Holiday, Iris; Rawling, Russell A; Novak-Weekley, Susan M; Quinlan, Tammy; Musser, Kimberlee A

    2010-11-01

    Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the "gold standard" for

  15. Comparison of Premier CAMPY Enzyme Immunoassay (EIA), ProSpecT Campylobacter EIA, and ImmunoCard STAT! CAMPY Tests with Culture for Laboratory Diagnosis of Campylobacter Enteric Infections ▿ †

    Science.gov (United States)

    Granato, Paul A.; Chen, Li; Holiday, Iris; Rawling, Russell A.; Novak-Weekley, Susan M.; Quinlan, Tammy; Musser, Kimberlee A.

    2010-01-01

    Campylobacter enteritis is a food-borne or waterborne illness caused almost exclusively by Campylobacter jejuni and, to a lesser extent, by Campylobacter coli. These organisms produce indistinguishable clinical diseases and together represent the second most common cause of bacterial diarrhea in the United States and the leading cause of enteric infection throughout the world. The conventional approach to the laboratory diagnosis of Campylobacter enteritis is based on the recovery of the organism from a stool specimen, which requires the use of a specialized medium incubated at 42°C for several days in an artificially created microaerophilic environment. Recently, several commercially available enzyme immunoassays (EIAs) have been developed for the direct detection of C. jejuni and C. coli in stool specimens. This study compared conventional culture with three EIA methods, the Premier CAMPY EIA (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter EIA (Remel, Lenexa, KS), and the ImmunoCard STAT! CAMPY test (Meridian Bioscience, Cincinnati, OH), for the detection of C. jejuni and C. coli in 485 patient stool samples. Discordant results were arbitrated by using an in-house, real-time PCR assay that was developed and validated by a public health reference laboratory. Following analyses of the discrepant specimens by PCR, the sensitivity and specificity of both the Premier CAMPY and ProSpecT Campylobacter EIAs were 99.3% and 98%, respectively, while the ImmunoCard STAT! CAMPY test had a sensitivity of 98.5% and a specificity of 98.2%. By use of the PCR test as the reference standard, culture detected 127 of 135 Campylobacter-positive stool specimens, yielding a sensitivity of 94.1%. These results showed that the three EIAs evaluated in this study provide a rapid and reliable alternative for the laboratory diagnosis of enteric infections with C. jejuni and C. coli and that conventional culture may no longer be recognized as the “gold standard” for

  16. Studies on direct and indirect electrochemical immunoassays

    OpenAIRE

    Buckley, Eileen

    1989-01-01

    Two approaches to electrochemical immunoassay are reported. The first approach was an indirect method, involving an electroactive, enzyme-catalysed, substrate to product reaction. Conditions were optimised for the amperometric detection of para-aminophenol, the electroactive product of the alkaline phosphatase catalysed hydrolysis of a new substrate, p-aminophenylphosphate, after separation by HPLC. The second approach involved the direct electrochemical detection of an immunoglo...

  17. Hydrogel nanoparticle based immunoassay

    Science.gov (United States)

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

    2015-04-21

    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  18. Recent advancements in the immunoassay domain

    International Nuclear Information System (INIS)

    Pradelles, Ph.

    1997-01-01

    The two types of immunoassay techniques, the competition analysis and the immuno-metric analysis (sandwich type), are described; the tracers used with theses methods have high specific radioactivity levels in order to be traced at extremely low content. Non radioactive tracers have been also developed, such as enzymatic, fluorescent, luminescent tracers, which are simpler and may be used at home. The Cea has recently developed some innovative immunoassay formats, such as acetylcholinesterase as a new enzymatic tracer, and immuno-metric dosage for very small molecules such as haptenes

  19. The right environment for the immunoassay

    International Nuclear Information System (INIS)

    Emon, J.M. Van; Gerlach, C.L.

    1995-01-01

    For the US Environmental Protection Agency (EPA), the first in-house research effort began in 1987, when results of an early immunoassay field study verified the technology's potential for environmental applications. Looking at the fundamental features of immunochemical reactions from the clinical laboratories, analytical chemists realized the potential value of these methods for hazardous waste site characterization and pesticide monitoring. Immunoassays rely on the interaction between an antibody and a target analyte. For environmental purposes, enzyme immunoassays are generally used. After the target analyte binds to the antibody, an enzymatic reaction yields a colorimetric change. This change, read visually or by a spectrophotometer, indicates the concentration of the target analyte. Promising results with assays for compounds (such as paraquat and pentachlorophenol) and compound groups (such as total petroleum hydrocarbons and polychlorinated biphenyls) spurred interest among various entrepreneurs. The first target market for immunoassays was environmental engineers and field crews who needed quick answers on-site to determine the direction of further remediation efforts

  20. Bead-based immunoassays

    NARCIS (Netherlands)

    Wal, van der F.J.; Bergervoet, J.H.W.; Achterberg, R.P.; Haasnoot, W.

    2014-01-01

    Since the first immunoassay with (radioactive) labeled antibodies in the middle of the 20th century [1], many different formats on various platforms have been developed, using antibodies for capture and/or detection. If antibodies are used to capture compounds, a support, such as the wall of a

  1. The immunoassay handbook

    National Research Council Canada - National Science Library

    Wild, David (David G.)

    2001-01-01

    ... importantly, enabling them to keep current on the basic theory behind immunoassay. Since the publication of the previous edition in 1994, the field has continued to evolve rapidly, and the need for a fully updated version of this book is now paramount. The second edition has been comprehensively updated and new chapters have been added to each section" [publisher's web site].

  2. Synthesis of an oxytetracyline-tolidin-BSA immunogen and antibodies production of anti-oxytetracyline developed for oxytetracyline residue detection with enzyme-linked immunosorbent assays technique

    Directory of Open Access Journals (Sweden)

    Widiastuti R

    2013-06-01

    Full Text Available An oxytetracycline-tolidin-bovine serum albumin (OTC-tolidin-BSA-conjugate was synthezed as immunogen for producing specific antibodies in immunized rabbits that would be used as reagent for development of OTC residue detection with enzym-linked immunoassays technique. The immunogen was prepared through diazotization tolidin and subsequently reacted with OTC. The red purple immunogen of OTC-tolidin-BSA absorbed at wave lengths of 277 nm and 488 nm under UV screening absorbances and confirmation with the high performance liquid chromatography (HPLC showed the absence of peak at retention time of 3.46 minutes. Characaterized result with SDS-PAGE showed the molecular weight of the OTC-tolidin-BSA at 69.79 kDA. Subsequently, the immunogen was immunized into New Zealand rabbits in order to produce the polyclonal antibodies. The antibodies were purified using a protein A sepharose column. The OD optimum responses of 0.92 to 1.20 were obtained from the second fractionation at dilution of 1/1000 by titrating the antibodies and OTC-tolidin-BSA coating antigen at concentration of 10 µg/mL on several bleeding times.

  3. Determination of phospholipid transfer proteins in rat tissues by immunoassays

    International Nuclear Information System (INIS)

    Teerlink, T.

    1983-01-01

    Several quantitative immunoassays have been developed for two phospholipid transfer proteins from rat liver, i.e. the phosphatidylcholine transfer protein and the non-specific lipid transfer protein. The development of a double-antibody radioimmunoassay for the phosphatidylcholine transfer protein is described. The transfer protein was labelled with iodine-125 by the mild glucose oxidase-lactoperoxidase method. Although less than one tyrosine residue per molecule of transfer protein was labelled, only 20% of the labelled transfer protein was immunoprecipitable. This value could be increased to 80% by purifying the labelled protein by affinity chromatography on a column of anti-phosphatidylcholine transfer protein-IgG coupled to Sepharose 4B. The radioimmunoassay was used to determine the levels of phosphatidylcholine transfer protein in homogenates and 105 000 xg supernatants from various rat tissues as well as several Morris hepatomas. An enzyme immunoassay for the non-specific lipid transfer protein is also described. The antiserum that was raised especially by the author was cross-reactive with the non-specific lipid transfer protein present in 105 000 xg supernatants from human, mouse and bovine liver. The non-specific lipid transfer protein lost its immunoreactivity upon labelling with iodine-125 using different labelling techniques. Therefore, a regular radioimmunoassay could not be developed. The results of these different assays were compared. (Auth.)

  4. Role of signal-to-cut-off ratios of anti-hepatitis C virus antibody by enzyme immunoassays along with ID-NAT for screening of whole blood donors in India

    Directory of Open Access Journals (Sweden)

    Satyam Arora

    2016-01-01

    Full Text Available Background: The use of elevated signal-to-cut off ratios (S/CO as an alternate to further supplemental testing (i.e., RIBA has been included in the guidelines provided by the Centres for Disease Control and Prevention for HCV diagnostic purposes since 2003. With availability of screening by NAT and non availability of RIBA, further confirmation of HCV infection has been possible at the molecular level (RNA. Aims: To study the role of S/CO ratios of anti hepatitis C virus antibody detection by enzyme immunoassays (EIA along with ID-NAT for screening of whole blood donors. Methods: In this study we reviewed the donor screening status for anti HCV from January 2013 to May 2014. All the donations were screened for anti HCV with fourth generation ELISA (BioRad Monolisa Ag-Ab Ultra as well as with ID NAT (Procleix Ultrio. The S/CO ratio of all the anti-HCV reactive samples were analysed for their presence of HCV RNA. Results: On screening 21,115 donors for HCV, 83 donors (0.39% were found reactive on pilot tube and repeat plasma bag testing (S/Co ratio ≥1 by ELISA. 41 donors were HCV RNA reactive with ID-NAT. 4 samples out of 41 were NAT yields and 37 were concordant reactive with ELISA. The S/Co ratio of anti-HCV reactive samples ranged from 0.9-11.1 [mean = 5.1; SD ΁ 2.9] whereas S/Co ratio of anti HCV and NAT reactive samples (concordant positives ranged from 4.1-11.1 [mean 7.3]. In our analysis we found that S/CO ratio of 4 showed positive predictive value (PPV and sensitivity of 100%. Summary/Conclusions: Our study showed that S/CO of 4 for anti HCV on ELISA would have maximum positive predictive value of having donor with HCV RNA. S/CO ratio of 4 is very close to 3.8 which was the CDC guideline. The presence of anti-HCV does not distinguish between current or past infections but a confirmed anti-HCV-positive result indicates the need for counseling and medical evaluation for HCV infection.

  5. Towards the development of a radioenzyme-immunoassay

    International Nuclear Information System (INIS)

    Schuurs, A.H.W.M.; Waart, M. v. d.

    1976-01-01

    We have tried to develop a very sensitive enzyme-immunoassay. For this purpose, a very sensitive radiochemical enzyme assay was used. HCG was chosen as test model and AChE as labelling enzyme. The test appeared to be much more sensitive than the normal enzymeimmunoassay. And, in comparison with RIA, it was about as sensitive but less time-consuming, and it makes use, in principle, of stable reagents. (orig./GSE) [de

  6. Good performance of an immunoassay based method for nevirapine measurements in human breast milk

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Theilgaard, Zahra Persson; Chiduo, Mercy

    2011-01-01

    from HIV-uninfected women. Clinical samples from HIV-infected women receiving a single-dose of nevirapine were analyzed. Results: Precision and accuracy were evaluated with two concentrations of quality control materials analyzed in three replicates on four different days and was......, requires complicated extraction techniques. The ARK method employs an immunoassay technology and requires a small sample volume (40 μL) and no pre-treatment of the samples. Methods: Commercial enzyme and antibody were used and calibration standards and quality controls were prepared from pooled breast milk...

  7. Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

    NARCIS (Netherlands)

    J. Groen (Jan); P. Koraka (Penelope); J. Velzing (Jans); C. Copra (Cederick); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractThe performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid

  8. The Application of Six Sigma Techniques in the Evaluation of Enzyme Measurement Procedures in China.

    Science.gov (United States)

    Zhang, Chuanbao; Zhao, Haijian; Wang, Jing; Zeng, Jie; Wang, Zhiguo

    2015-01-01

    Recently, Six Sigma techniques have been adopted by clinical laboratories to evaluate laboratory performance. Measurement procedures in laboratories can be categorized as "excellent", "good", and "improvement needed" based on sigma (σ) metrics of σ ≥ 6, 3 ≤ σ 1.2 indicates that the trueness of the procedure needs to be improved; 0.8 ≤ QGI ≤ 1.2 indicates that both the precision and trueness of the procedure need to be improved. Fresh frozen sera containing seven enzymes (ALT, ALP, AMY, AST, CK, GGT, and LDH) were sent to 78 clinical laboratories in China. The biases for measurement procedures in each laboratory (Bias) were calculated based on the target values assigned by 18 laboratories performing IFCC (International Federation of Clinical Chemistry and Laboratory medicine) recommended reference methods. The imprecision of each measurement procedure was represented by coefficient variations (CV) calculated based on internal quality control (IQC) data. The σ and QGI values were calculated as follows: σ = (TEa-Bias)/CV; QGI = Bias/(1.5 x CV). TEa is allowable total error for each enzyme derived from biological variation. Our study indicated that 7.9% (6/76, ALP) to 31.0% (18/58, AMY) of the participating laboratories were scored as "excellent" (σ ≥ 6), 21.1% (16/76, ALP) to 41.3% (31/75, CK) of the laboratories were scored as "good" (3 ≤ σ Six Sigma techniques still suggested that approximately 31.1% to 71.0% of the laboratories need to improve their enzyme measurement procedures, either in terms of precision, trueness, or both.

  9. Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)

    Science.gov (United States)

    Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

    2000-03-01

    Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this

  10. Status of immunoassay as an analytical tool in environmental investigations

    International Nuclear Information System (INIS)

    Van Emon, J.M.

    2000-01-01

    Immunoassay methods were initially applied in clinical situations where their sensitivity and selectivity were utilized for diagnostic purposes. In the 1970s, pesticide chemists realized the potential benefits of immunoassay methods for compounds difficult to analyze by gas chromatography. This transition of the technology has extended to the analysis of soil, water, food and other matrices of environmental and human exposure significance particularly for compounds difficult to analyze by chromatographic methods. The utility of radioimmunoassays and enzyme immunoassays for environmental investigations was recognized in the 1980s by the U.S. Environmental Protection Agency (U.S. EPA) with the initiation of an immunoassay development programme. The U.S. Department of Agriculture (USDA) and the U.S. Food and Drug Administration (PDA) have investigated immunoassays for the detection of residues in food both from an inspection and a contamination prevention perspective. Environmental immunoassays are providing rapid screening information as well as quantitative information to fulfill rigorous data quality objectives for monitoring programmes

  11. Interference in immunoassay

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    Interfering factors are evident in both limited reagent (radioimmunoassay) and excess reagent (immunometric assay) technologies and should be suspected whenever there is a discrepancy between analytical results and clinical findings in the investigation of particular diseases. The overall effect of interference in immunoassay is analytical bias in result, either positive or negative of variable magnitude. The interference maybe caused by a wide spectrum of factors from poor sample collection and handling to physiological factors e.g. lipaemia, heparin treatment, binding protein abnormalities, autoimmunity and drug treatments. The range of interfering factors is extensive and difficult to discuss effectively in a short review

  12. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.

    Science.gov (United States)

    Bandura, Dmitry R; Baranov, Vladimir I; Ornatsky, Olga I; Antonov, Alexei; Kinach, Robert; Lou, Xudong; Pavlov, Serguei; Vorobiev, Sergey; Dick, John E; Tanner, Scott D

    2009-08-15

    A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200-300 micros duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard inductively coupled plasma (ICP) operating conditions ( 900 for m/z = 159, the sensitivity with a standard sample introduction system of >1.4 x 10(8) ion counts per second per mg L(-1) of Tb and an abundance sensitivity of (6 x 10(-4))-(1.4 x 10(-3)) (trailing and leading masses, respectively) are shown. The mass range (m/z = 125-215) and abundance sensitivity are sufficient for elemental immunoassay with up to 60 distinct available elemental tags. When 500) can be used, which provides >2.4 x 10(8) cps per mg L(-1) of Tb, at (1.5 x 10(-3))-(5.0 x 10(-3)) abundance sensitivity. The real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown. A real time single cell 20 antigen expression assay of model cell lines and leukemia patient samples immuno-labeled with lanthanide-tagged antibodies is presented.

  13. Can LC and LC-MS ever replace immunoassays?

    Directory of Open Access Journals (Sweden)

    Timothy G. Cross

    2016-10-01

    Full Text Available Immunoassays have been the technology of choice for the analysis of biomolecules for many decades across a wide range of applications in research, diagnostics and infectious disease monitoring. There are good reasons for the wide adoption of immunoassays but even such a well established and characterised technique has limitations and as such investigators are looking at alternative technologies. One such alternative is liquid chromatography (LC and, more specifically, liquid chromatography coupled with mass spectrometry (LC-MS. This article will review both immunoassay and LC and LC-MS technologies and methodologies and discuss the advantages and limitations of both approaches. In addition, the next developments that will need to occur before there is widespread adoption of LC and LC-MS technology preferentially over immunoassays will be examined.

  14. Development of feed supplementation strategies for improving ruminant productivity on small-holder farms in Latin America through the use of immunoassay techniques. Proceedings of the final research co-ordination meeting of a co-ordinated research programme

    International Nuclear Information System (INIS)

    1996-05-01

    The result of a CRP completed in 1989 and entitled ''Regional Network for Improving the Reproductive Management of Milk, Meat and Fibre Producing Livestock with the Use of Radioimmunoassay Techniques'' clearly indicated that nutritional inadequacies and livestock management deficiencies were the major factors affecting livestock productivity in Latin America. Based on these conclusions a CRP entitled ''Development of Feed Supplementation Strategies for Improving Ruminant Productivity on Small-holder Farms in Latin America through the Use of Immunoassay Techniques'' was initiated late in the same year. The primary aim of the Programme was to improve the productivity of indigenous ruminant livestock species maintained on typical small-holder farms in the region. Central to the approach was to first identify the nutritional and management constraints which affect reproductive and productive efficiency, and subsequently to devise and test corrective measures which would be practical, sustainable and economically viable. Important related goals of the Programme were to enhance the level of expertise and the educational quality within animal production research institutes in the region, to encourage close contact between scientists and institutions in developing and developed countries and to promote scientific information exchange on a regional basis. Refs, figs, tabs

  15. Development of feed supplementation strategies for improving ruminant productivity on small-holder farms in Latin America through the use of immunoassay techniques. Proceedings of the final research co-ordination meeting of a co-ordinated research programme

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-05-01

    The result of a CRP completed in 1989 and entitled ``Regional Network for Improving the Reproductive Management of Milk, Meat and Fibre Producing Livestock with the Use of Radioimmunoassay Techniques`` clearly indicated that nutritional inadequacies and livestock management deficiencies were the major factors affecting livestock productivity in Latin America. Based on these conclusions a CRP entitled ``Development of Feed Supplementation Strategies for Improving Ruminant Productivity on Small-holder Farms in Latin America through the Use of Immunoassay Techniques`` was initiated late in the same year. The primary aim of the Programme was to improve the productivity of indigenous ruminant livestock species maintained on typical small-holder farms in the region. Central to the approach was to first identify the nutritional and management constraints which affect reproductive and productive efficiency, and subsequently to devise and test corrective measures which would be practical, sustainable and economically viable. Important related goals of the Programme were to enhance the level of expertise and the educational quality within animal production research institutes in the region, to encourage close contact between scientists and institutions in developing and developed countries and to promote scientific information exchange on a regional basis. Refs, figs, tabs.

  16. Soil zymography - A novel technique for mapping enzyme activity in the rhizosphere

    Science.gov (United States)

    Spohn, Marie

    2014-05-01

    The effect plant roots on microbial activity in soil at the millimeter scale is poorly understood. One reason for this is that spatially explicit methods for the study of microbial activity in soil are limited. Here we present a quantitative in situ technique for mapping the distribution of exoenzymes in soil along with some results about the effects of roots on exoenzyme activity in soil. In the first study we showed that both acid and alkaline phosphatase activity were up to 5.4-times larger in the rhizosphere of Lupinus albus than in the bulk soil. While acid phosphatase activity (produced by roots and microorganisms) was closely associated with roots, alkaline phosphatase activity (produced only by microorganisms) was more widely distributed, leading to a 2.5-times larger area of activity of alkaline than of acid phosphatase. These results indicate a spatial differentiation of different ecophysiological groups of organic phosphorus mineralizing organisms in the rhizosphere which might alleviate a potential competition for phosphorus between them. In a second study cellulase, chitinase and phosphatase activities were analyzed in the presence of living Lupinus polyphyllus roots and dead/dying roots (in the same soils 10, 20 and 30 days after cutting the L. polyphyllus shoots). The activity of all three enzymes was 9.0 to 13.9-times higher at the living roots compared to the bulk soil. Microhotspots of cellulase, chitinase and phosphatase activity in the soil were found up to 60 mm away from the living roots. 10 days after shoot cutting, the areas of high activities of cellulase and phosphatase activity were extend up to 55 mm away from the next root, while the extension of the area of chitinase activity did not change significantly. At the root, cellulase and chitinase activity increased first at the root tips after shoot cutting and showed maximal activity 20 days after shoot cutting. The number and activity of microhotspots of chitinase activity was maximal 10

  17. Developments of sensitive immunoassays for detection of antibodies against hepatitis B surface antigen

    Energy Technology Data Exchange (ETDEWEB)

    Ionescu-Matiu, I; Sanchez, Y; Dreesman, G R [Baylor Univ., Houston, TX (USA). Coll. of Medicine; Fields, H A [Centers for Disease Control, Public Health Service, Department of Health and Human Services, Phoenix, AZ (USA)

    1983-01-01

    Three micro solid phase immunoassays (a micro-SPRIA and two ELISA techniques) were developed and tested for the detection of anti-HBs antibodies. Two different crosslinkers (glutaraldehyde and N-succinimidyl 3-(2-pyridyldithio) propionate) were used to couple a goat anti-mouse IgG reagent to alkaline phosphatase for use as enzyme-labeled probes in the two ELISA tests. With the latter cross-linker, a defined conjugate with a 1 : 1 antibody-enzyme molar ratio was obtained. The sensitivities of micro-SPRIA and the two types of ELISA were compared to that of the commercial solid phase radioimmunoassay AUSAB test. All three microtests were significantly more sensitive than the AUSAB test. The ELISA using the glutaraldehyde cross-linked conjugate was 3-5 times less sensitive than micro-SPRIA, while the ELISA using the disulfide-linked conjugate was 2.6-4.0 times more sensitive than micro-SPRIA.

  18. Monoclonal antibody-based immunoassays.

    Science.gov (United States)

    Appleby, P; Reischl, U

    1998-01-01

    An immunoassay may be defined as an assay that employs an immunological reagent, usually an antibody, to confer specificity for the ligand being measured. As a corollary to this, the discovery, and subsequent development, of monoclonal antibodies (MAbs) has greatly expanded the application and use of immunoassays. Polyclonal reagents, with their associated problems of specificity and quality control, have now been largely replaced by readily available MAbs of potential immortality and well-defined specificity and affinity. This has resulted, in the last two decades, in a great expansion in the range of immunoassays available and also a significant improvement in their reproducibility and reliability.

  19. False Elevation of the Blood Tacrolimus Concentration, as Assessed by an Affinity Column-mediated Immunoassay (ACMIA), Led to Acute T Cell-mediated Rejection after Kidney Transplantation.

    Science.gov (United States)

    Kono, Momoko; Hasegawa, Jumpei; Ogawa, Hina; Yoshikawa, Kanae; Ishiwatari, Ayumi; Wakai, Sachiko; Tanabe, Kazunari; Shirakawa, Hiroki

    2018-05-01

    Tacrolimus is the most commonly used immunosuppressant. Because of its narrow therapeutic range, it is necessary to frequently monitor its concentration. We report the case of a 25-year-old man who underwent kidney transplantation whose tacrolimus concentrations, as measured by an affinity column-mediated immunoassay, were falsely elevated. As we reduced the dose of tacrolimus, the recipient developed T cell-mediated rejection. Using the same blood samples, an enzyme-multiplied immunoassay technique showed that the patient's levels of tacrolimus were extremely low. A further examination indicated that the false increase in the tacrolimus concentration was likely due to an unknown interfering substance. We administered methylprednisolone and antithymocyte-globulin. The patient's serum creatinine level decreased and remained stable after these treatments.

  20. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    International Nuclear Information System (INIS)

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-01-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application

  1. Micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

    Energy Technology Data Exchange (ETDEWEB)

    Layton, G T [Royal Infirmary, Manchester (UK)

    1980-10-01

    Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10/sup -3/ units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus.

  2. A micro-enzyme-linked immunosorbent assay (ELISA) and radioimmunosorbent technique (RIST) for the detection of immunity to clinical tetanus

    International Nuclear Information System (INIS)

    Layton, G.T.

    1980-01-01

    Enzyme-linked immunosorbent assay (ELISA), and radioimmunosorbent assay (RIST) techniques for the detection of tetanus toxin antibodies are described. Both methods proved to be highly sensitive, and allowed the measurement of 5 x 10 -3 units/ml tetanus antitoxin in human serum or plasma, sensitivity and reproducibility comparing well with other techniques previously described, and being superior to haemagglutination and latex agglutination tests. Results of the two methods correlated well, and reflected the immunization histories obtained. Micro ELISA and micro RIST would seem to be suitable for the detection of immunity, or non-immunity to clinical tetanus. (author)

  3. Development of a monoclonal antibody to urinary degradation products from the C-terminal telopeptide alpha 1 chain of type I collagen. Application in an enzyme Immunoassay and comparison to CrossLaps(TM) ELISA

    DEFF Research Database (Denmark)

    C, Fledelius; I, Kolding; P, Quist

    1997-01-01

    A monoclonal antibody MAbA7 was raised against a synthetic peptide having a sequence (EKAHDGGR) specific for a part of the C-telopeptide alpha 1 chain of type I collagen. MAbA7 was labelled with horseradish peroxide and used in a competitive one-step enzyme-linked immunosorbent assay (ELISA...... concentrations decreased 88% (pantibody and the new assay may be useful for further investigations of the physiological...

  4. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability

    DEFF Research Database (Denmark)

    Assarsson, Erika; Lundberg, Martin; Holmquist, Göran

    2014-01-01

    reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput...... detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes......, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research....

  5. Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

    Science.gov (United States)

    Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

    2016-01-15

    Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Immunoassay of DNA damage

    International Nuclear Information System (INIS)

    Gasparro, F.P.; Santella, R.M.

    1988-01-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

  7. Immunoassay of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Gasparro, F P; Santella, R M

    1988-09-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

  8. Improvement of Aspergillus niger 55, a raw corn meal saccharifying enzyme hyperproducer, through mutation and selective screening techniques

    International Nuclear Information System (INIS)

    Oh, S.H.; O, P.S.

    1991-01-01

    Mutation experiments were performed to select the mutant of Aspergillus niger 55, which had lost almost all the ability to produce transglucosidases but retained that of high productivity of raw meal saccharifying enzyme, by means of successive induction with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), ultraviolet(UV) light, and γ-rays. Also, we used the mutant enrichment techniques, such as liquid culture-filtration procedure and differential heat sensitivity of conidia, in order to increase the possibility of obtaining a mutant. The glucoamylase productivity of mutant PFST-38 was 11 times higher than that of the parent strain. The mutant PFST-38 was morphologically identical to the parent strain, except for the size of conidia, the tendency to form conidia and the length of conidiophore. Asp. niger mutant PFST-38 appeared to be useful for the submerged production of the raw corn meal saccharifying enzyme

  9. Extraction of intracellular protein from Chlorella pyrenoidosa using a combination of ethanol soaking, enzyme digest, ultrasonication and homogenization techniques.

    Science.gov (United States)

    Zhang, Ruilin; Chen, Jian; Zhang, Xuewu

    2018-01-01

    Due to the rigid cell wall of Chlorella species, it is still challenging to effectively extract significant amounts of protein. Mass methods were used for the extraction of intracellular protein from microalgae with biological, mechanical and chemical approaches. In this study, based on comparison of different extraction methods, a new protocol was established to maximize extract amounts of protein, which was involved in ethanol soaking, enzyme digest, ultrasonication and homogenization techniques. Under the optimized conditions, 72.4% of protein was extracted from the microalgae Chlorella pyrenoidosa, which should contribute to the research and development of Chlorella protein in functional food and medicine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Clearing of suspensions of Micrococcus lysodeikticus catalysed by lysozymes from hen, goose, and turkey egg whites, human milk, and phage T4. Assessment of potential as signal generators for homogeneous enzyme immunoassays for urinary steroids.

    Science.gov (United States)

    Cooke, Delwyn G; Blackwell, Leonard F

    2007-01-01

    Lysozymes (3.2.1.17) from goose (Anser anser) egg white, turkey (Melagris gallopavo) egg white, phage T4 and human milk were compared with hen egg white lysozyme in their ability to clear a suspension of Micrococcus lysodeikticus. All of the lysozymes, except hen egg white lysozyme, catalysed the clearing of the Micrococcus lysodeikticus suspension in a biphasic fashion. Compared to hen egg white lysozyme, the total absorbance or transmission change over 5 and 20 minutes was less in all cases, except for human lysozyme. Human lysozyme was, therefore, a potential alternative, more rapid signal generator for the measurement of urinary estrone glucuronide excretion rates because of its structural similarity to hen egg white lysozyme. The apparent K(M) values for hen egg white lysozyme increased with the enzyme concentration.

  11. Development and application of radioimmunoassays and enzyme immunoassays in microbiological and immunological diagnosis. 2. Comparative studies for the detection of toxoplasma antibodies with ELISA, RIA and other serological methods

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, W A; Struy, H; Holzwarth, F [Medizinische Akademie, Magdeburg (German Democratic Republic)

    1982-06-01

    Comparative studies of indirect immunofluorescence test (IT), complement binding reaction (CBR), enzyme- and radioimmunoassay (ELISA, RIA) for the detection of toxoplasma antibodies in sera of 513 patients are reported. The precision dependent on time, showed coefficients of variation from 3% to 12% (IFT 3%, CBR 10%, ELISA 12%, RIA 7%). The correlation of IFT and ELISA as well as RIA was relatively unfavourable (coefficient of correlation IFT/ELISA r = 0.52, IFT/RIA r = 0.54, RIA/ELISA r = 0.60). The ELISA is the most sensitive method for the detection of antibodies. The specificity of the Toxo-ELISA has to be improved by application of suitable fractions of antigens.

  12. Immunoassay for determination of trilobolide

    Czech Academy of Sciences Publication Activity Database

    Huml, L.; Jurášek, M.; Mikšátková, P.; Zimmermann, T.; Tomanová, P.; Buděšínský, Miloš; Rottnerová, Z.; Šimková, M.; Harmatha, Juraj; Kmoníčková, Eva; Lapčík, O.; Drašar, P. B.

    2017-01-01

    Roč. 117, Jan (2017), s. 105-111 ISSN 0039-128X. [Conference on Isoprenoids /23./. Minsk, 04.09.2016-07.09.2016] Institutional support: RVO:61388963 ; RVO:68378041 Keywords : trilobolide * avidin-biotin * ELISA * Laser trilobum * synthesis * immunoassay Subject RIV: CE - Biochemistry; FR - Pharmacology ; Medidal Chemistry (UEM-P) OBOR OECD: Biochemical research methods; Pharmacology and pharmacy (UEM-P) Impact factor: 2.282, year: 2016

  13. Lectin-enzyme binding assays : development of the technique and applications in biochemistry and medicine

    NARCIS (Netherlands)

    J.M. Pekelharing

    1989-01-01

    textabstractThe aim of this work is to determine if lectins can be used in "sandwich" ELISA techniques so that the glycosylation of specific proteins in mixtures could be characterised in a fast and sensitive way without prior purification of the protein. Furthermore, the feasability of

  14. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with /sup 125/I-labelled Clq binding and Raji-cell RIA tests

    Energy Technology Data Exchange (ETDEWEB)

    Casali, P; Bossus, A; Carpentier, N A; Lambert, P H [Hopital Cantonal Geneve (Switzerland)

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample was incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes were detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test did not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG was 3 ..mu..g/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 ..mu..g/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the /sup 125/I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, /sup 125/I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application.

  15. Enzyme-Linked Immunosorbent Assay (ELISA).

    Science.gov (United States)

    Konstantinou, George N

    2017-01-01

    Food allergy is a public health concern especially after recognizing its constantly increased prevalence and severity. Despite careful reading of food ingredient statements, food allergic individuals may experience reactions caused by "hidden", "masked", or "contaminated" proteins that are known major allergens. Many techniques have been developed to detect even small traces of food allergens, for clinical or laboratory purposes. Enzyme-linked immunosorbent assay (ELISA) is one of the best validated and most routinely used immunoassay in allergy research, in allergy diagnosis in allergy-related quality control in various industries. Although as a technique it has been implemented for the last 45 years, the evolution in biochemistry allowed the development of ultrasensitive ELISA variations that are capable of measuring quantities in the scale of picograms, rendering ELISA attractive, robust, and very famous.

  16. Development of immunoassays for detecting clothianidin residue in agricultural products.

    Science.gov (United States)

    Li, Ming; Sheng, Enze; Cong, Lujing; Wang, Minghua

    2013-04-17

    Two enzyme-linked immunosorbent assays (ELISAs) based on polyclonal antibodies (PcAbs) for clothianidin are described: colorimetric detection format (ELISA) and pattern of chemiluminescent assay (CLEIA). Clothianidin hapten was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to produce immunogen and coating antigen. Anticlothianidin PcAbs were obtained from immunized New Zealand white rabbits. Under optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (LOD, IC₂₀) of clothianidin were 0.046 and 0.0028 mg/L for the ELISA and 0.015 and 0.0014 mg/L for the CLEIA, respectively. There were no obvious cross-reactivities of the antibodies with its analogues except for dinotefuran. Recoveries of 76.4-116.4% for the immunoassays were achieved from spiked samples. The results of immunoassays for the spiked and authentic samples were largely consistent with gas chromatography. Therefore, the proposed immunoassays would be convenient and satisfactory analytical methods for the monitoring of clothianidin in agricultural products.

  17. Immunoassay: Principles, development and potential applications in the applied plant sciences

    Energy Technology Data Exchange (ETDEWEB)

    Hofman, P J

    1986-02-01

    The article briefly discusses the general principles of, and the methods involved in, immunoassay, and their development. Emplasis is placed on radioimmunoassay (RIA) and to a lesser extent, enzyme-linked immunosorbent assay (ELISA). The practical applications, with special reference to the citrus and subtropical fruit industries are discussed.

  18. Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: hskim76@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)

    2017-01-15

    Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.

  19. Comparison of an enzyme-immunoassay with a radio-immunoassay ...

    African Journals Online (AJOL)

    1990-07-21

    Jul 21, 1990 ... patient's serum is added to beads coated with HBsAg and, after a period of incubation, HBsAg conjugated with iodine-. 125 which binds to any anti-HBs on the bead, is added. Department of Virology, University of the Witwatersrand and National Institute for Virology, Johannesburg. N. K. BLACKBURN, F.LM ...

  20. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  1. Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

    Science.gov (United States)

    Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

    2017-01-01

    Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

  2. Evaluation of four immunoassays for diagnosis of brucellosis in Cuba

    International Nuclear Information System (INIS)

    Peraza, C.; Valdes, O.; Fonseca, N.; Garcia, M.; Alvarez, M.; Izquierdo, D.L.

    1998-01-01

    Four immunoassays (two indirect and two competitive ones) were evaluated by samples from areas free of disease, free by vaccination and affected areas using as reference techniques the Bengal Rose Tests, the Antigen in Buffered Plate Tests and the Complement Fixation Reaction Test. The evaluated samples demonstrated that the competitive assays (ELISAC-1 and ELISAC-2) detected less false positives than the indirect ones (ELISAI-1 and ELISAI-2). Of the competitive ELISAS, version 2 presented better sensitivity and specificity results in affected areas for 95% confidence: 80.9 - 96.9% and 97.5 - 99.4% respectively with positive predictive value in the range of 76 to 94% and negative predictive one between 98.1 and 99.7%. It was concluded that this assay can be used for brucellosis control because it gives higher assurance than the other evaluated immunoassays and it can discriminate infected from vaccinated animals. (author)

  3. Immunoassay of β-endorphin

    International Nuclear Information System (INIS)

    Hoellt, V.; Gramsch, C.; Herz, A.

    1979-01-01

    The present paper describes the characteristics of a series of antisera against β-endorphin (β-E) developed in our laboratory which all recognize β-lipotropin and their use in (1) the determination of β-E in tissue and in body fluids and in (2) the immunocytochemical localization of β-E containing neurons in the rat brain and in (3) the study of the conversion of the β-E/β-LPH precursor into β-LPH and β-E in the pars intermedia/nervosa of the rat pituitary. It is the purpose of the present report to critically analyze the pitfalls and drawbacks, as well as the advantage, of the use of radioimmunoassay and other immunoassays in the determination of β-E. (Auth.)

  4. Materials for Microfluidic Immunoassays: A Review.

    Science.gov (United States)

    Mou, Lei; Jiang, Xingyu

    2017-08-01

    Conventional immunoassays suffer from at least one of these following limitations: long processing time, high costs, poor user-friendliness, technical complexity, poor sensitivity and specificity. Microfluidics, a technology characterized by the engineered manipulation of fluids in channels with characteristic lengthscale of tens of micrometers, has shown considerable promise for improving immunoassays that could overcome these limitations in medical diagnostics and biology research. The combination of microfluidics and immunoassay can detect biomarkers with faster assay time, reduced volumes of reagents, lower power requirements, and higher levels of integration and automation compared to traditional approaches. This review focuses on the materials-related aspects of the recent advances in microfluidics-based immunoassays for point-of-care (POC) diagnostics of biomarkers. We compare the materials for microfluidic chips fabrication in five aspects: fabrication, integration, function, modification and cost, and describe their advantages and drawbacks. In addition, we review materials for modifying antibodies to improve the performance of the reaction of immunoassay. We also review the state of the art in microfluidic immunoassays POC platforms, from the laboratory to routine clinical practice, and also commercial products in the market. Finally, we discuss the current challenges and future developments in microfluidic immunoassays. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Oligopeptide-based enzyme immunoassay for ovine lentivirus antibody detection.

    OpenAIRE

    Kwang, J; Torres, J V

    1994-01-01

    Ovine progressive pneumonia virus (OPPV) is a lentivirus which causes a progressive disease in sheep. Immunodominant epitopes have been identified in the envelope gp40 glycoprotein. Synthetic peptides representing these regions are able to detect the presence of OPPV antibodies in 96% of infected sheep.

  6. Effective gasoline site assessment using the D TECH trademark BTEX immunoassay

    International Nuclear Information System (INIS)

    Hudak, R.T.; Melby, J.M.; Stave, J.W.

    1994-01-01

    The application of immunoassay to environmental testing can greatly aid in assessing sites for various contaminants including PCB, TNT, RDX, PAH and BTEX. Immunoassay offers several benefits to site assessment: it is accurate, reproducible and relatively inexpensive compared to instrumental analysis. In addition, because of its ease-of-use, this technique is ideal for the field and requires minimal user training. To demonstrate not only the effectiveness of the BTEX immunoassay, but also the reliability of the field results, a gasoline contaminated site was assessed comparing the BTEX immunoassay to gas chromatography. All sampling and site related activities were executed in accordance to the USEPA SW-846 guidelines. Three (3) analyses were performed on each sample. One immunoassay analysis was performed in the field by an individual who received two (2) hours of training prior to the start of the study. A technician familiar with the immunoassay ran the second analysis in a laboratory. Finally, an independent GC laboratory certified for BTEX method 8020 and 602 performed the GC analyses. One hundred one (101) samples were analyzed: thirty-nine (39) samples were water, the other sixty-two (62) were soils ranging from clay to silt. The results and costs of the methods are compared

  7. History of inductively coupled plasma mass spectrometry-based immunoassays

    International Nuclear Information System (INIS)

    Giesen, Charlotte; Waentig, Larissa; Panne, Ulrich; Jakubowski, Norbert

    2012-01-01

    The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA‐ICP‐MS. - Highlights: ► We discuss the fundamentals of elemental tagging for ICP‐MS applications. ► We propose a definition for the expressions “label” and “tag”. ► We highlight LA‐ICP‐MS‐based heteroelement detection. ► We give an historic overview on ICP-MS and LA‐ICP‐MS-based immunoassays. ► In a personal outlook, we discuss future improvements realistically attainable.

  8. A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide

    DEFF Research Database (Denmark)

    Barington, T; Sparholt, S; Juul, L

    1992-01-01

    A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary...... antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality...... and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection...

  9. A comparative study of two advanced spraying techniques for the deposition of biologically active enzyme coatings onto bone-substituting implants

    International Nuclear Information System (INIS)

    Jonge, Lise T. de; Ju, J.; Leeuwenburgh, S.C.G.; Yamagata, Y.; Higuchi, T.; Wolke, J.G.C.; Inoue, K.; Jansen, J.A.

    2010-01-01

    Surface modification of implant materials with biomolecule coatings is of high importance to improve implant fixation in bone tissue. In the current study, we present two techniques for the deposition of biologically active enzyme coatings onto implant materials. The well-established thin film ElectroSpray Deposition (ESD) technique was compared with the SAW-ED technique that combines high-frequency Surface Acoustic Wave atomization with Electrostatic Deposition. By immobilizing the enzyme alkaline phosphatase (ALP) onto implant surfaces, the influence of both SAW-ED and ESD deposition parameters on ALP deposition efficiency and ALP biological activity was investigated. ALP coatings with preserved enzyme activity were deposited by means of both the SAW-ED and ESD technique. The advantages of SAW-ED over ESD include the possibility to spray highly conductive protein solutions, and the 60-times faster deposition rate. Furthermore, significantly higher deposition efficiencies were observed for the SAW-ED technique compared to ESD. Generally, it was shown that protein inactivation is highly dependent on both droplet dehydration and the applied electrical field strength. The current study shows that SAW-ED is a versatile and flexible technique for the fabrication of functionally active biomolecule coatings.

  10. Variance function estimation for immunoassays

    International Nuclear Information System (INIS)

    Raab, G.M.; Thompson, R.; McKenzie, I.

    1980-01-01

    A computer program is described which implements a recently described, modified likelihood method of determining an appropriate weighting function to use when fitting immunoassay dose-response curves. The relationship between the variance of the response and its mean value is assumed to have an exponential form, and the best fit to this model is determined from the within-set variability of many small sets of repeated measurements. The program estimates the parameter of the exponential function with its estimated standard error, and tests the fit of the experimental data to the proposed model. Output options include a list of the actual and fitted standard deviation of the set of responses, a plot of actual and fitted standard deviation against the mean response, and an ordered list of the 10 sets of data with the largest ratios of actual to fitted standard deviation. The program has been designed for a laboratory user without computing or statistical expertise. The test-of-fit has proved valuable for identifying outlying responses, which may be excluded from further analysis by being set to negative values in the input file. (Auth.)

  11. Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

    Science.gov (United States)

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

    2015-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Directory of Open Access Journals (Sweden)

    Ratthaphol Charlermroj

    Full Text Available Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac, chilli vein-banding mottle virus (CVbMV, potyvirus, watermelon silver mottle virus (WSMoV, tospovirus serogroup IV and melon yellow spot virus (MYSV, tospovirus. An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour was much shorter than that of ELISA (4 hours. This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  13. Multiplex detection of plant pathogens using a microsphere immunoassay technology.

    Science.gov (United States)

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R; Karoonuthaisiri, Nitsara; Elliott, Christopher T

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.

  14. Development of an Ultrasensitive Immunoassay for Detecting Tartrazine

    Directory of Open Access Journals (Sweden)

    Chuanlai Xu

    2013-06-01

    Full Text Available We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages.

  15. A redox-mediated chromogenic reaction and application in immunoassay.

    Science.gov (United States)

    Yu, Ru-Jia; Ma, Wei; Peng, Mao-Pan; Bai, Zhi-Shan; Long, Yi-Tao

    2016-08-31

    A novel redox-mediated chromogenic reaction was demonstrated based on the reaction between HAuCl4 and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which generate various color responses from red to green in the resulting solutions. Various redox substance could be used to mediate the reaction and trigger a distinct color response. We established a sensitive hydrogen peroxide colorimetric sensor based on the redox-mediated chromogenic reaction and depicted the application both in detection of enzyme and in an immunoassay. Combining the traditional chromogenic reagent with gold nanoparticles, our assay has the advantage in short response time (within three minutes), high sensitivity (10(-12) g mL(-1) for HBsAg) and stability. Copyright © 2016. Published by Elsevier B.V.

  16. Development of an ultrasensitive immunoassay for detecting tartrazine.

    Science.gov (United States)

    Li, Zhuokun; Song, Shanshan; Xu, Liguang; Kuang, Hua; Guo, Shidong; Xu, Chuanlai

    2013-06-25

    We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages.

  17. Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

    Science.gov (United States)

    Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

    2002-12-01

    To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

  18. Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

    Science.gov (United States)

    Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

    2018-05-23

    Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

  19. Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation

    NARCIS (Netherlands)

    Dittmer, W.U.; Kievit, de P.; Prins, M.W.J.; Vissers, J.L.M.; Mersch, M.E.C.; Martens, M.F.W.C.

    2008-01-01

    A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions

  20. Estimation of Tissue Distribution of mRNA Transcripts for Desaturase and Elongase Enzymes in Channa striata (Bloch, 1793 Fingerlings using PCR Technique

    Directory of Open Access Journals (Sweden)

    M. Aliyu-Paiko

    2013-06-01

    Full Text Available Fish species are varied in their capacity to biosynthesize n-3 highlyunsaturated fatty acids (HUFA such as eicosapentaenoic and docosahexaenoic acids (EPA & DHA that are crucial to the health and well-being of all higher vertebrates. Experts report that HUFA metabolism involves enzyme-mediated fatty acyl desaturation (FAD and elongation (FAE processes. In previous studies, different workers cloned, characterized, identified and reported several genes for FAD and FAE enzymes in different fish species such as Atlantic salmon, gilthead seabream, rainbow trout and zebrafish, and also demonstrated the up- and down-regulation in the activity of these enzymes in response to fluctuations in dietary HUFA. In this paper, we report on the expression of genes (mRNA transcripts for FAD and FAE enzymes in different tissues of Channa striata (Bloch, 1793 fingerling, to evaluate the tissues of the fish in which activity of both enzymes are high. To achieve this objective, we used conventional polymerase chain reaction (PCR technique to isolate and quantify the absolute copy number for each gene transcripts from 8 different tissues of the fish (reared with a commercial feed. Our estimate show that the distribution of the 2 enzyme transcripts were significantly (P < 0.05 higher in the liver and brain of C. striata than detected in the 6 other tissues evaluated (muscle, ovary, testis, intestine, kidney and skin. Subsequently, we discuss here extensively, the implication of this observation with respect to the use of vegetable oils (VO as substitute to fish oil (FO in diets for freshwater fish species.

  1. The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Xiaoling Fu

    2018-03-01

    Full Text Available The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA was presented for the clinical determination and analysis of human epididymis protein 4 (HE4 in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0–1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA, with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum. Keywords: Chemiluminescence immunoassay, Magnetic particles, Human epididymis protein 4

  2. Trends in interfacial design for surface plasmon resonance based immunoassays

    International Nuclear Information System (INIS)

    Shankaran, Dhesingh Ravi; Miura, Norio

    2007-01-01

    Immunosensors based on surface plasmon resonance (SPR) have become a promising tool in sensor technology for biomedical, food, environmental, industrial and homeland security applications. SPR is a surface sensitive optical technique, suitable for real-time and label-free analysis of biorecognition events at functional transducer surfaces. Fabrication of highly active and robust sensing surfaces is an important part in immunoassays because the quality, quantity, chemistry and topography of the interfacial biomembranes play a major role in immunosensor performance. Eventually, a variety of immobilization methods such as physical adsorption, covalent coupling, Langmuir-Blodgett film, polymer thin film, self-assembly, sol-gel, etc, have been introduced over the years for the immobilization of biomolecules (antibody or antigen) on the transducer surfaces. The selection of an immobilization method for an immunoassay is governed by several factors such as nature and stability of the biomolecules, target analyte, application, detection principle, mode of signal transduction, matrix complexity, etc. This paper provides an overview of the various surface modification methods for SPR based immunosensor fabrication. The preparation, structure and application of different functional interfacial surfaces have been discussed along with a brief introduction to the SPR technology, biomolecules and detection principles. (review article)

  3. Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

    Science.gov (United States)

    Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

    2004-06-01

    The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

  4. Trends in interfacial design for surface plasmon resonance based immunoassays

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Dhesingh Ravi [Art, Science and Technology Center for Cooperative Research, Kyushu University, Kasuga-shi, Fukuoka, 816-8580 (Japan); Miura, Norio [Art, Science and Technology Center for Cooperative Research, Kyushu University, Kasuga-shi, Fukuoka, 816-8580 (Japan)

    2007-12-07

    Immunosensors based on surface plasmon resonance (SPR) have become a promising tool in sensor technology for biomedical, food, environmental, industrial and homeland security applications. SPR is a surface sensitive optical technique, suitable for real-time and label-free analysis of biorecognition events at functional transducer surfaces. Fabrication of highly active and robust sensing surfaces is an important part in immunoassays because the quality, quantity, chemistry and topography of the interfacial biomembranes play a major role in immunosensor performance. Eventually, a variety of immobilization methods such as physical adsorption, covalent coupling, Langmuir-Blodgett film, polymer thin film, self-assembly, sol-gel, etc, have been introduced over the years for the immobilization of biomolecules (antibody or antigen) on the transducer surfaces. The selection of an immobilization method for an immunoassay is governed by several factors such as nature and stability of the biomolecules, target analyte, application, detection principle, mode of signal transduction, matrix complexity, etc. This paper provides an overview of the various surface modification methods for SPR based immunosensor fabrication. The preparation, structure and application of different functional interfacial surfaces have been discussed along with a brief introduction to the SPR technology, biomolecules and detection principles. (review article)

  5. Diagnóstico de leptospirosis: evaluación de un enzimoinmunoensayo en fase sólida en diferentes etapas de la enfermedad Diagnosis of leptospirosis: evaluation of a solid-phase enzyme immunoassay in different stages of the disease

    Directory of Open Access Journals (Sweden)

    Norma B. Vanasco

    2007-06-01

    solid-phase enzyme immunoassay (ELISA for genus-specific immunoglobulin G (IgG determination with leptospirosis and to evaluate the ELISA in different stages of the disease. METHODS: A total of 1 077 serum samples from 812 patients with suspected leptospirosis were analyzed. The samples had come from diagnoses done in the laboratory of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias, in the city of Santa Fe, Argentina, between 1999 and 2005. Included in the study were 182 confirmed cases (267 samples, 167 negative cases (293 samples, and 40 probable cases (60 samples (based on case definitions based on the results from the microscopic agglutination test (MAT, leukocyte counts, and neutrophilia values. Each sample was classified, according to the days of the natural history of disease, into one of three stages: first ( 25 days. The antigen used in the ELISA was an extract of a mixture of pyrogenes and tarassovi serovars cultivated in a liquid medium, treated with ultrasound, and immobilized by adsorption on polystyrene plates. As a secondary antibody, a peroxidase-conjugated goat anti-human IgG monoclonal antibody was used. The cutoff value, sensitivity, and specificity of the ELISA were determined using the definitions of confirmed cases and of negatives cases as the standard. In order to determine the optimal cutoff value, the area under the receiver operating characteristic curve was calculated. RESULTS: The sensitivity of the evaluated test was much higher in the second stage (93.2% than in either the first stage (68.1% or the third stage (78.8%. The specificity increased gradually from 96.3% in the first stage to 100% in the third stage. CONCLUSIONS: Our results indicate that this ELISA test can be a very useful complement to the MAT for the diagnosis of leptospirosis in all the stages and, in particular, in order to diagnose acute disease sooner.

  6. Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemaglutination techniques for detecting cytomegalovirus IgG antibody

    Energy Technology Data Exchange (ETDEWEB)

    Booth, J C; Hannington, G; Bakir, T M.F.; Stern, H; Kangro, H; Griffiths, P D; Heath, R B [Saint George' s Hospital Medical School, London (UK); Saint Bartholomew' s Hospital, London (UK))

    1982-12-01

    The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems.

  7. The future development of immunoassay

    International Nuclear Information System (INIS)

    Ekins, R.P.

    1977-01-01

    To summarise these brief observations on the relative merits of isotopic and non-isotopic labels, the logistic advantages of non-isotopic labels are likely to be combined with sensitivities comparable with, or even greatly superior to, the corresponding radioisotopic techniques. However, in assessing the general directions that assay methodology is likely to take in the next few years, the nature of the label that is likely to be preferred in techniques of this kind is a matter of domestic rather than fundamental importance. Of greater interest is the challenge presented by the need to develop techniques which are of far higher structural specificity than those currently available - this requirement being imposed not only by the need to elucidate the physiological importance of structural heterogeneity in the molecules of substances of biological interest, but also by the need to develop 'rugged' standardisable assays for routine clinical use. Because radioimmunoassays and other saturation assays rely, in practice, on the interaction between a single binding site on the analyte molecule with the specific reagent, such techniques lack the overall higher structural specificity of 'sandwich', excess-reagent, assays. Moreover the ease of preparation and the long-term chemical stability of the labelled reagents used in the 'excess-reagent' assay system, and their intrinsically higher sensitivity, suggests that these will ultimately displace 'saturation assay' methods such as radioimmunoassay in those cases in which the analyte is capable of simultaneous reaction with two structurally specific recognition sites. On the other hand, for analytes of small molecular size, capable of binding to, or reacting with, only a single molecule of a specific reagent, the intrinsically higher specificity of limited reagent systems is likely to ensure that these will remain the methods of choice. (orig.) [de

  8. Determination of triiodothyronine in serum by enzyme- and radioimmunoassay

    International Nuclear Information System (INIS)

    Oellerich, M.; Haindl, H.; Medizinische Hochschule Hannover

    1981-01-01

    An evaluation of a heterogeneous enzyme immunoassay for determination of triiodothyronine in serum (Enzymun-Test T 3 , Boehringer Mannheim) is presented. The enzyme immunoassay was compared with the laboratory routine radioimmunoassay. The precision of both assays was satisfactory at triiodothyronine concentrations between 1.0 and 8.0 nmol/l (coefficients of variation from day to day 3 from 96-104% and with the radioimmunoassay from 88-111%. A comparison of the results obtained by Enzymun-Test T 3 and the radioimmunoassay in a series of 103 patients showed a good correlation between both methods. L-thyroxine did not cause a relevant cross-reaction in the enzyme immunoassay. About 20 unknown samples can be analyzed in triplicate by Enzymun-Test T 3 within 260 minutes. (orig.) [de

  9. Development of national immunoassay reagent programmes

    International Nuclear Information System (INIS)

    Sufi, S.B.; Micallef, J.V.; Ahsan, R.; Goncharov, N.P.

    1992-01-01

    Despite the existence of networks of fully equipped laboratories with well-trained staff, the availability of immunodiagnostic services in developing countries is often limited by the high cost of imported kits. There are a number of ways of tackling this problem, ranging from bulk purchase of kits or reagents to local development and production of assay systems. Argentina/Chile, China, Cuba/Mexico, and Thailand are amongst the countries which have established local immunoassay reagent programmes to manufacture low cost, high quality immunoassay reagents. Kits from these projects are now beginning to become available, and it is hoped that they will promote national diagnostic services and research, as well as stimulating the development of reagent programmes for other analytes. (author). 4 refs, 1 tab

  10. An artificial-intelligence technique for qualitatively deriving enzyme kinetic mechanisms from initial-velocity measurements and its application to hexokinase.

    Science.gov (United States)

    Garfinkel, L; Cohen, D M; Soo, V W; Garfinkel, D; Kulikowski, C A

    1989-01-01

    We have developed a computer method based on artificial-intelligence techniques for qualitatively analysing steady-state initial-velocity enzyme kinetic data. We have applied our system to experiments on hexokinase from a variety of sources: yeast, ascites and muscle. Our system accepts qualitative stylized descriptions of experimental data, infers constraints from the observed data behaviour and then compares the experimentally inferred constraints with corresponding theoretical model-based constraints. It is desirable to have large data sets which include the results of a variety of experiments. Human intervention is needed to interpret non-kinetic information, differences in conditions, etc. Different strategies were used by the several experimenters whose data was studied to formulate mechanisms for their enzyme preparations, including different methods (product inhibitors or alternate substrates), different experimental protocols (monitoring enzyme activity differently), or different experimental conditions (temperature, pH or ionic strength). The different ordered and rapid-equilibrium mechanisms proposed by these experimenters were generally consistent with their data. On comparing the constraints derived from the several experimental data sets, they are found to be in much less disagreement than the mechanisms published, and some of the disagreement can be ascribed to different experimental conditions (especially ionic strength). PMID:2690819

  11. Device for use in immunoassays

    International Nuclear Information System (INIS)

    Tu, J.I.

    1981-01-01

    A novel double antibody-coated test tube is described for use in radioimmunoassays. The solid phase separation technique of this invention is based on a test tube which has been coated on its internal surface with two antibody layers: a first layer of nonspecific antibodies which is bound to the internal surface of the test tube and a second layer of more specific antibodies which are bound to the nonspecific antibodies. The invention is illustrated in the preparation of double antibody-coated test tubes for use in the radioimmunoassays of plasma digoxin levels and serum triiodothyronine levels. (U.K.)

  12. Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays.

    Science.gov (United States)

    Chen, Hui; Hagström, Anna E V; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C; Atmar, Robert L; Willson, Richard C

    2016-04-14

    In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 10(6) virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings.

  13. Integrated immunoassay using tuneable surface acoustic waves and lensfree detection.

    Science.gov (United States)

    Bourquin, Yannyk; Reboud, Julien; Wilson, Rab; Zhang, Yi; Cooper, Jonathan M

    2011-08-21

    The diagnosis of infectious diseases in the Developing World is technologically challenging requiring complex biological assays with a high analytical performance, at minimal cost. By using an opto-acoustic immunoassay technology, integrating components commonly used in mobile phone technologies, including surface acoustic wave (SAW) transducers to provide pressure driven flow and a CMOS camera to enable lensfree detection technique, we demonstrate the potential to produce such an assay. To achieve this, antibody functionalised microparticles were manipulated on a low-cost disposable cartridge using the surface acoustic waves and were then detected optically. Our results show that the biomarker, interferon-γ, used for the diagnosis of diseases such as latent tuberculosis, can be detected at pM concentrations, within a few minutes (giving high sensitivity at a minimal cost). This journal is © The Royal Society of Chemistry 2011

  14. Silver nanoparticles deposited on graphene oxide for ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.

    Science.gov (United States)

    Yang, Lin; Zhen, Shu Jun; Li, Yuan Fang; Huang, Cheng Zhi

    2018-06-14

    Graphene oxide (GO) exhibits distinctive Raman scattering features for its high frequency D (disordered) and tangential modes (G-band), which are characteristically sharp at 1580 cm-1 and 1350 cm-1, respectively, but are too weak for sensitive quantitation purposes. By depositing silver nanoparticles on the surface of GO in this contribution, both D and G bands of GO become enhanced. The enzyme label of this method controls the dissolution of silver nanoparticles on the surface of GO through hydrogen peroxide which is produced by the oxidation of the enzyme substrate. With the dissolution of the silver nanoparticles a greatly decreased SERS signal of GO was obtained. This strategy involves dual signal amplification of the enzyme and nanocomposites to improve the detection sensitivity. As a proof of concept, prostate specific antigen (PSA), a biomarker for prostate cancer, is successfully detected as a target by forming a sandwich structure in immunoassay. The SERS immunoassay possesses excellent analytical performance in the range 0.5 pg mL-1 to 500 pg mL-1 with a limit of detection of 0.23 pg mL-1, making the detection of PSA serum samples from prostate cancer patients satisfactory, demonstrating that the sensitive enzyme-assisted dissolved AgNPs SERS immunoassay of PSA has potential applications in clinical diagnosis.

  15. An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads.

    Science.gov (United States)

    Tao, Xiaoqi; Jiang, Haiyang; Yu, Xuezhi; Zhu, Jinghui; Wang, Xia; Wang, Zhanhui; Niu, Lanlan; Wu, Xiaoping; Shen, Jianzhong

    2013-05-01

    A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Trypanosomosis surveillance on Zanzibar island, using the trypanosomal antigen detection ELISA (enzyme-linked immunosorbent assay) technique

    Energy Technology Data Exchange (ETDEWEB)

    Mbwambo, H A [Animal Disease Research Inst. (ADRI), Dar-es-Salaam (Tanzania, United Republic of)

    1997-02-01

    The effectiveness of trypanosomosis control programs depends greatly on prior knowledge of basic data of the epidemiological situation of the disease, which in turns depends, among others, on the use of techniques that give a fairly quick and accurate diagnosis. An antigen-detection (Ag) ELISA was first introduced into Tanzania and validated at the Animal Disease Research Institute (ADRI) through the FAO/IAEA Research Contract (RC) No. 5030/NL. Incorporation of the Ag-ELISA technique into a FAO animal disease control project (1986-1993) on Unguja island, in 1992, revealed useful information of high trypanosomosis prevalence in an area previously declared free of the disease using just stained blood smears and buffy coat examinations. This triggered further efforts into more intensive surveys of the tsetse and trypanosomosis situation on Unguja island. The present study is a continuation of previous work in an effort to confirm the practical application of Ag-ELISA in trypanosomosis control operations. Results obtained from a known tsetse and trypanosomosis-free area, on Pemba island, showed a high specificity of the test for Trypanosoma congolense, T. vivax and T. brucei. A preliminary cut-off value of 10% (Percent Positivity = PP) was used. When the PP of 10 was used on sera of trypanosomosis-endemic areas (Mangapwani, Ndijani, Dunga and Kikungwi) on Unguja island, the results reflected the `real` trypanosomis situation in the affected area. This was most strongly felt in the Mangapwani area, where tsetse and trypanosomosis were considered under control by 1994 (no tsetse flies were caught and no samples were encountered positive by the buffy coat technique). However, it should be stressed that the buffy coat technique and the Ag-ELISA complement each other and should be used in conjunction. (author). 8 refs, 1 fig., 5 tabs.

  17. Trypanosomosis surveillance on Zanzibar island, using the trypanosomal antigen detection ELISA (enzyme-linked immunosorbent assay) technique

    International Nuclear Information System (INIS)

    Mbwambo, H.A.

    1997-01-01

    The effectiveness of trypanosomosis control programs depends greatly on prior knowledge of basic data of the epidemiological situation of the disease, which in turns depends, among others, on the use of techniques that give a fairly quick and accurate diagnosis. An antigen-detection (Ag) ELISA was first introduced into Tanzania and validated at the Animal Disease Research Institute (ADRI) through the FAO/IAEA Research Contract (RC) No. 5030/NL. Incorporation of the Ag-ELISA technique into a FAO animal disease control project (1986-1993) on Unguja island, in 1992, revealed useful information of high trypanosomosis prevalence in an area previously declared free of the disease using just stained blood smears and buffy coat examinations. This triggered further efforts into more intensive surveys of the tsetse and trypanosomosis situation on Unguja island. The present study is a continuation of previous work in an effort to confirm the practical application of Ag-ELISA in trypanosomosis control operations. Results obtained from a known tsetse and trypanosomosis-free area, on Pemba island, showed a high specificity of the test for Trypanosoma congolense, T. vivax and T. brucei. A preliminary cut-off value of 10% (Percent Positivity = PP) was used. When the PP of 10 was used on sera of trypanosomosis-endemic areas (Mangapwani, Ndijani, Dunga and Kikungwi) on Unguja island, the results reflected the 'real' trypanosomis situation in the affected area. This was most strongly felt in the Mangapwani area, where tsetse and trypanosomosis were considered under control by 1994 (no tsetse flies were caught and no samples were encountered positive by the buffy coat technique). However, it should be stressed that the buffy coat technique and the Ag-ELISA complement each other and should be used in conjunction. (author). 8 refs, 1 fig., 5 tabs

  18. Detection of alpha-fetoprotein in magnetic immunoassay of thin channels using biofunctional nanoparticles

    Science.gov (United States)

    Tsai, H. Y.; Gao, B. Z.; Yang, S. F.; Li, C. S.; Fuh, C. Bor

    2014-01-01

    This paper presents the use of fluorescent biofunctional nanoparticles (10-30 nm) to detect alpha-fetoprotein (AFP) in a thin-channel magnetic immunoassay. We used an AFP model biomarker and s-shaped deposition zones to test the proposed detection method. The results show that the detection using fluorescent biofunctional nanoparticle has a higher throughput than that of functional microparticle used in previous experiments on affinity reactions. The proposed method takes about 3 min (versus 150 min of previous method) to detect 100 samples. The proposed method is useful for screening biomarkers in clinical applications, and can reduce the run time for sandwich immunoassays to less than 20 min. The detection limits (0.06 pg/ml) and linear ranges (0.068 pg/ml-0.68 ng/ml) of AFP using fluorescent biofunctional nanoparticles are the same as those of using functional microparticles within experimental errors. This detection limit is substantially lower and the linear range is considerably wider than those of enzyme-linked immunosorbent assay (ELISA) and other methods in sandwich immunoassay methods. The differences between this method and an ELISA in AFP measurements of serum samples were less than 12 %. The proposed method provides simple, fast, and sensitive detection with a high throughput for biomarkers.

  19. Which amphetamine-type stimulants can be detected by oral fluid immunoassays?

    Science.gov (United States)

    Souza, Daniele Z; Boehl, Paula O; Comiran, Eloisa; Prusch, Débora S; Zancanaro, Ivomar; Fuentefria, Alexandre M; Pechansky, Flavio; Duarte, Paulina C A V; De Boni, Raquel B; Fröehlich, Pedro E; Limberger, Renata P

    2012-02-01

    The use of oral fluid for monitoring drug consumption on roads has many advantages over conventional biological fluids; therefore, several immunoassays have been developed for this purpose. In this work, the ability of 3 commercial immunoassays to detect amphetamine-type stimulants (ATSs) in oral fluid was assessed. In addition, it was reviewed the main controlled ATSs available worldwide, as well as the oral fluid immunological screening tests that have been used for identifying ATSs in drivers. The analytical specificity of amphetamine direct enzyme-linked immunosorbent assay (ELISA), methamphetamine direct ELISA (Immunalysis Corporation), and Oral-View saliva multidrug of abuse test (Alfa Scientific Designs) was evaluated using ATS-spiked oral fluid. Legislation and published articles that report the use of immunological screening tests to detect ATS consumption in conductors were reviewed, including the kit's technical information, project reports, police and drug databases. Even at high concentrations, the tested assays were not able to detect methylphenidate, fenproporex, or diethylpropion, controlled ATSs legally marketed in many countries. This evidences the need to develop new kits that enable one to control the misuse of prescription ATSs on roads through oral fluid immunoassays.

  20. Massively multi-parametric immunoassays using ICPMS

    International Nuclear Information System (INIS)

    Tanner, S.D.; Ornatsky, O.; Bandura, D.R.; Baranov, V.I.

    2009-01-01

    The use of stable isotopes as tags in immunoassays, and their determination by ICPMS, is poised to have a huge impact on multi-parametric bioanalysis. A new technology, which we term 'mass cytometry', enables high throughput, highly multiplexed individual cell analysis. Preliminary results for T-cell immunophenotyping in peripheral blood mononuclear cells (PBMC), agonist influence on concomitant phosphorylation pathways, and sub-classification of acute myeloid leukemia patients' samples will be presented. The significance of individual cell analysis is demonstrated by the identification of populations of rogue cells in PBMC samples through the use of multidimensional neural network cluster analysis. (author)

  1. Gliadin Detection in Food by Immunoassay

    Science.gov (United States)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  2. Pancreatic Enzymes

    Science.gov (United States)

    ... Contact Us DONATE NOW GENERAL DONATION PURPLESTRIDE Pancreatic enzymes Home Facing Pancreatic Cancer Living with Pancreatic Cancer ... and see a registered dietitian. What are pancreatic enzymes? Pancreatic enzymes help break down fats, proteins and ...

  3. COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY METHODS FOR MEASURING 3,5,6-TRICHLORO-2PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    Science.gov (United States)

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by a commercial RaPID immunoassay testing kit. ...

  4. Nanoparticle-based immunosensors and immunoassays for aflatoxins

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xu; Niessner, Reinhard [Institute of Hydrochemistry and Chair of Analytical Chemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München (Germany); Tang, Dianping [Key Laboratory of Analysis and Detection for Food Safety, MOE & Fujian Province, Department of Chemistry, Fuzhou University, Fuzhou 350108 (China); Knopp, Dietmar, E-mail: dietmar.knopp@ch.tum.de [Institute of Hydrochemistry and Chair of Analytical Chemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München (Germany)

    2016-03-17

    Aflatoxins are naturally existing mycotoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, present in a wide range of food and feed products. Because of their extremely high toxicity and carcinogenicity, strict control of maximum residue levels of aflatoxins in foodstuff is set by many countries. In daily routine, different chromatographic methods are used almost exclusively. As supplement, in several companies enzyme immunoassay-based sample testing as primary screening is performed. Recently, nanomaterials such as noble metal nanoparticles, magnetic particles, carbon nanomaterials, quantum dots, and silica nanomaterials are increasingly utilized for aflatoxin determination to improve the sensitivity and simplify the detection. They are employed either as supports for the immobilization of biomolecules or as electroactive or optical labels for signal transduction and amplification. Several nanoparticle-based electrochemical, piezoelectric, optical, and immunodipstick assays for aflatoxins have been developed. In this review, we summarize these recent advances and illustrate novel concepts and promising applications in the field of food safety. - Highlights: • Novel concepts and promising applications of nanoparticle-based immunological methods for the determination of aflatoxins. • Inclusion of most important nanomaterials and hybrid nanostructures. • Inclusion of electrochemical, optical and mass-sensitive biosensors as well as optical and immunochromatographic assays.

  5. Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

    Directory of Open Access Journals (Sweden)

    Na Kong

    2015-04-01

    Full Text Available An indirect competitive enzyme-linked immunosorbent assay (icELISA and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed.

  6. Fluorescence immunoassay for detecting periodontal bacterial pathogens in plaque.

    OpenAIRE

    Wolff, L F; Anderson, L; Sandberg, G P; Aeppli, D M; Shelburne, C E

    1991-01-01

    A particle concentration fluorescence immunoassay has been modified into a bacterial concentration fluorescence immunoassay (BCFIA) to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilizes fluorescently tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected gram-negative plaque bacteria. Microorganisms closely associated with periodontal disease that can be identified in plaque with the BCFIA include Porphyromonas gingivalis, Bac...

  7. Fake news? Biotin interference in thyroid immunoassays.

    Science.gov (United States)

    Koehler, Viktoria F; Mann, Ulrike; Nassour, Ayham; Alexander Mann, W

    2018-05-29

    We report on a 47 year old male patient with multiple sclerosis (MS) presenting in our outpatient neurology clinic in Frankfurt/Main for therapy evaluation. Before change of treatment laboratory investigations were performed. Thyroid function tests (TFTs) with a streptavidin/biotin based immunoassay revealed severe hyperthyroidism with positive thyroid autoantibodies suggestive for Graves' disease. Clinical presentation and thyroid sonography were unremarkable. Due to the discordance between clinical presentation and TFTs, we repeated medical history, in which the patient reported taking high-doses of biotin (300 mg/day) for MS. Recent studies with patients suffering from primary and secondary progressive MS, indicated promising effects of high-dose biotin on MS-related disability. In immunoassays relaying on streptavidin-biotin interaction, biotin intake can cause falsely high or low results. Two weeks after withdrawing biotin, biotin/streptavidin dependant assays showed no longer the biochemical picture of severe hyperthyroidism. Biotin intake should be paused for at least two to five days prior to the use of biotin/streptavidin dependant assays. Alternatively, non-biotin/streptavidin dependant assays (radioimmunoassay, gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry) may be used. Copyright © 2017. Published by Elsevier B.V.

  8. Chemiluminescence immunoassay for prostate-specific antigen

    International Nuclear Information System (INIS)

    Zhang Xuefeng; Liu Yibing; Jia Juanjuan; Xu Wenge; Li Ziying; Chen Yongli; Han Shiquan

    2008-01-01

    The chemiluminescence immunoassay (CLIA) for serum total prostate-specific antigen (T-PSA) was developed. The reaction of luminol with hydrogen peroxide was introduced into this chemiluminescence system. The detection limit is established as 0.12 μg/L (n=10, mean of zero standard + 2SD) and the analytical recovery of PSA is 83.8%-118.7%. The intra-assay and inter-assay CVs vary from 4.4%-5.0% and 6.2%-11.7%, respectively. The experimental correlation coefficient of dilution is found to be 0.999. Compared with immunoradiometric assay (IRMA) kits, the correlative equation is y=1.07x+0.68, and correlation coefficient r=0.97. The standard range for the method is 1.5-80 μg/L, and it presents good linearity. (authors)

  9. The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer.

    Science.gov (United States)

    Fu, Xiaoling; Liu, Yangyang; Qiu, Ruiyun; Foda, Mohamed F; Zhang, Yong; Wang, Tao; Li, Jinshan

    2018-03-01

    The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0-1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.

  10. Rapid Salmonella detection in experimentally inoculated equine faecal and veterinary hospital environmental samples using commercially available lateral flow immunoassays.

    Science.gov (United States)

    Burgess, B A; Noyes, N R; Bolte, D S; Hyatt, D R; van Metre, D C; Morley, P S

    2015-01-01

    Salmonella enterica is the most commonly reported cause of outbreaks of nosocomial infections in large animal veterinary teaching hospitals and the closure of equine hospitals. Rapid detection may facilitate effective control practices in equine populations. Shipping and laboratory testing typically require ≥48 h to obtain results. Lateral flow immunoassays developed for use in food-safety microbiology provide an alternative that has not been evaluated for use with faeces or environmental samples. We aimed to identify enrichment methods that would allow commercially available rapid Salmonella detection systems (lateral flow immunoassays) to be used in clinical practice with equine faecal and environmental samples, providing test results in 18-24 h. In vitro experiment. Equine faecal and environmental samples were inoculated with known quantities of S. enterica serotype Typhimurium and cultured using 2 different enrichment techniques for faeces and 4 enrichment techniques for environmental samples. Samples were tested blindly using 2 different lateral flow immunoassays and plated on agar media for confirmatory testing. In general, commercial lateral flow immunoassays resulted in fewer false-negative test results with enrichment of 1 g faecal samples in tetrathionate for 18 h, while all environmental sample enrichment techniques resulted in similar detection rates. The limit of detection from spiked samples, ∼4 colony-forming units/g, was similar for all methods evaluated. The lateral flow immunoassays evaluated could reliably detect S. enterica within 18 h, indicating that they may be useful for rapid point-of-care testing in equine practice applications. Additional evaluation is needed using samples from naturally infected cases and the environment to gain an accurate estimate of test sensitivity and specificity and to substantiate further the true value of these tests in clinical practice. © 2014 EVJ Ltd.

  11. Detection of narcotics with an immunoassay film badge

    International Nuclear Information System (INIS)

    Lukens, H.R.

    1993-01-01

    Efficient personnel performance, a major requirement for a safe nuclear industry, is jeopardized where personnel use narcotics. However, detection of narcotics at nuclear plants is a challenge. The unique specificity and sensitivity of an immunoassay has been implemented in the form of a small, dry immunoassay film badge (IFB) for the detection of vapors emitted by narcotics. The device is suitable as an area monitor, and its characteristics are suitable for use as a breath monitor for the detection of drug use

  12. Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

    Directory of Open Access Journals (Sweden)

    John Cooper

    2010-03-01

    Full Text Available The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs specifically on electrodes using complementary DNA sequences conjugated to the Abs.An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different.Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

  13. Design of novel hybrid organic-inorganic nanostructured biomaterials for immunoassay applications

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, G [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Barbosa-Stancioli, E F [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Piscitelli Mansur, A A [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Vasconcelos, W L [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Mansur, H S [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil)

    2006-12-01

    The purpose of this study was to develop novel hybrid organic-inorganic materials based on poly(vinyl alcohol) (PVA) polymer chemically crosslinked network to be tested as solid support on bovine herpesvirus immunoassay. Hybrids were synthesized by reacting PVA with three different alkoxysilanes modifying chemical groups: tetraethoxysilane (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS). PVA-derived hybrids were also modified by chemically crosslinking with glutaraldehyde (GA) during the synthesis reaction. In order to investigate the structure in the nanometer-scale, PVA-derived hybrids were characterized by using small-angle x-ray scattering synchrotron radiation (SAXS) and x-ray diffraction (XRD). PVA hybrids' chemical functionalities and their interaction with herpesviruses were also characterized by Fourier transform infrared spectroscopy (FTIR). The bioactivity assays were tested through enzyme linked immunosorbent assay (ELISA). SAXS results have indicated nano-ordered disperse domains for PVA hybrids with different x-ray scattering patterns for PVA polymer and PVA-derived hybrids. FTIR spectra have shown major vibration bands associated with organic-inorganic chemical groups present in the PVA, PVA-derived by silane modifier and PVA chemically crosslinked by GA. The immunoassay results have shown that PVA hybrids with chemically functionalized structures regulated to some extent the specific bioimmobilization of herpesvirus onto solid phase. We think that it is due to the overall balance of forces associated with van der Waals interaction, hydrophilic and hydrophobic forces and steric hindrance acting at the surface. PVA and PVA-derived hybrid materials were successfully produced with GA crosslinking in a nanometer-scale network. Also, such a PVA-based material could be advantageously used in immunoassays with enhanced specificity for diagnosis.

  14. Design of novel hybrid organic-inorganic nanostructured biomaterials for immunoassay applications

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, G [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Barbosa-Stancioli, E F [Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, PO Box 486, 31270.901, Belo Horizonte, MG (Brazil); Piscitelli Mansur, A A [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Vasconcelos, W L [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Mansur, H S [Department of Metallurgical and Materials Engineering, Biomaterials and Tissue Engineering Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil)

    2006-12-01

    The purpose of this study was to develop novel hybrid organic-inorganic materials based on poly(vinyl alcohol) (PVA) polymer chemically crosslinked network to be tested as solid support on bovine herpesvirus immunoassay. Hybrids were synthesized by reacting PVA with three different alkoxysilanes modifying chemical groups: tetraethoxysilane (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS). PVA-derived hybrids were also modified by chemically crosslinking with glutaraldehyde (GA) during the synthesis reaction. In order to investigate the structure in the nanometer-scale, PVA-derived hybrids were characterized by using small-angle x-ray scattering synchrotron radiation (SAXS) and x-ray diffraction (XRD). PVA hybrids' chemical functionalities and their interaction with herpesviruses were also characterized by Fourier transform infrared spectroscopy (FTIR). The bioactivity assays were tested through enzyme linked immunosorbent assay (ELISA). SAXS results have indicated nano-ordered disperse domains for PVA hybrids with different x-ray scattering patterns for PVA polymer and PVA-derived hybrids. FTIR spectra have shown major vibration bands associated with organic-inorganic chemical groups present in the PVA, PVA-derived by silane modifier and PVA chemically crosslinked by GA. The immunoassay results have shown that PVA hybrids with chemically functionalized structures regulated to some extent the specific bioimmobilization of herpesvirus onto solid phase. We think that it is due to the overall balance of forces associated with van der Waals interaction, hydrophilic and hydrophobic forces and steric hindrance acting at the surface. PVA and PVA-derived hybrid materials were successfully produced with GA crosslinking in a nanometer-scale network. Also, such a PVA-based material could be advantageously used in immunoassays with enhanced specificity for diagnosis.

  15. Comparison of Immunoassay methods for T3, T4 and TSH

    International Nuclear Information System (INIS)

    Alonso Rodríguez, Celia A.

    2016-01-01

    Measurements of T3, T4 and TSH have been considered very important in the diagnosis and monitoring of thyroid diseases both overt and subclinical. These subclinical diseases are actively seeking for years, both in healthy patients and hospitalized for other illnesses; and in the population over 35 years, especially women, in health checkups. The active search for these diseases requires the use of rapid and reliable techniques; that can be developed massively, with good level of detectability and comparable. The overall objective is to present the evaluation of different immunoassay techniques with respect to the RIA and IRMA: ELISA, chemiluminescence, Amplified Chemiluminescence, electrochemiluminescence Immunofluorescence. Compare including automatic methods and analyze the cost and feasibility of them for laboratory immunoassay. ELISA colorimetric technique for dosing was comparable to RIA T4, not for T3. Chemiluminescence (AMERLITE) compared to dosing RIA and IRMA T4 to TSH proved to be valid for both. Amplified Chemiluminescence (Immulite) compared to IRMA for TSH was no significant difference. Electrochemiluminescence (Elecsys 2010) compared to T3 and T4 RIA and IRMA for TSH, no significant differences for T4 and TSH; but no variation to T3. Immunofluorescence (AIA-600) used to compare with RIA for T3 and T4, and TSH IRMA, no significant differences for the measured analytes. Benchmarking of automatic methods suggests that the most thrifty of trials is Immunofluorescence the AIA-600, regarding calibration and control, programming time, randomization and the ability to save the value of the fluorescence deferred calculations for tests without valid at the time of realizing calibration. Analyzing the cost and feasibility of these methods for laboratory immunoassay, we must consider that their characteristics electrochemiluminescence is the fastest, but its price is prohibitive for our health systems. The AIA-600 appears to be the method of choice for its

  16. A summary report on the 23rd quality control survey for immunoassays in Japan, 2001

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-10-01

    The purpose of the survey is to improve the quality of in vitro tests and this report is its summary of immunoassays (the old name: RI in vitro tests) conducted in 2001. The survey was performed in 143 facilities out of 1,655 in Japan, which involved 20 national and public university hospitals, 16 private university hospitals, 21 national and public hospitals, 26 private hospitals, 41 hygiene test institutes and 19 reagent manufacturers. Tests examined were on 6 substances related to functions of pituitary, 5 of thyroid, 1 of parathyroid, 4 of gastro-intestine and pancreas, 5 of gonad and placenta, 4 of adrenal, 1 of renal-blood pressure regulation, on IgE, on digoxin and on 12 tumor-related substances. Tests were done on 2-3 samples supplied from the Committee and the mean, standard deviation and coefficient of variation were calculated for one way analysis of variance of within- and between-kit. Methods included those (65.4% vs 62.7% in 2000) with non-radioisotope like enzyme immunoassay (non-RI) and with radioisotopes like radioimmunoassay (RI). Dissociation between manufacturers of non-RI values without a common standard substance and between non-RI and RI values even with the same antibody was noted: the Committee considered these problems as their future task. (K.H.)

  17. Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

    Science.gov (United States)

    Barroso, Javier; Saa, Laura; Grinyte, Ruta; Pavlov, Valeri

    2016-03-15

    We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP). Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

    Science.gov (United States)

    Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

    2018-04-01

    In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

  19. Comparison of immunoassays for differentiation of herpes simplex virus type 1 and 2 antibodies

    International Nuclear Information System (INIS)

    Klapper, Paul E.; Valley, Pam J.; Cleator, Gerham M.; Mandall, D.; Qutub, Mohammed O.

    2006-01-01

    To asses the commercial available enzyme-linked immunosorbent assays (ELISA) for differentiation of herpes simplex virus type 1 (Hs-1) and type 2 (HSV-2) antibodies. The study was performed between January 1997 to November 2002 in the Division ofVirology,Department of Pathological Sciences, Central Manchester Healthcare Trust and University of Manchester, Manchester, United Kingdom. Assays based upon type-specific glycoprotein G-1 (gG-1) for HSV-1, and glycoprotein G-2 (gG-2) from HSV-2 were evaluated to differentiate between HSV-1 and HSV-2 antibodies. Using 5 different ELISA tests, 2 panels of serum samples were tested. Panel one consisted of 88 sera, selected from the serum bank of the Clinical Virology Laboratory, Manchester Royal Infirmary; panel 2 comprised of 90 sera selected from samples collected from Bangladeshi female commercial workers.The data of this study showed that a high rate of gG-1 based immunoassays ranged from 87.9-100% for sensitivity and 51.5-100% specificity. Although there are several immunoassays were claimed to differentiate between HSV-1 and HSV-2 antibodies, selection of these assays should be carefully interpreted with the overall clinical framework provided by detailed sexual history and genital examination. (author)

  20. Increasing of sensitivity of fluorescent immunoassay analysis of alpha-fetoprotein by means of plasmonical silver nanoparticles

    International Nuclear Information System (INIS)

    Vashchenko, S.V.; Min'ko, A.A.; Romanenko, A.A.; Gaponenko, S.V.; Kulakovich, O.S.

    2014-01-01

    A test system is proposed based on metal enhanced fluorescence to analyze low concentrations of alpha-fetoprotein (AFP), a tumor marker. Antigen-antibody reaction was performed on polystyrene plates coated with silver nanoparticles to increase sensitivity of fluorescent immunoassay and signal-to-noise ratio as compared to silver-free system. As compared to widely used ELISA technique and other immunoassay techniques the proposed approach is characterized by smaller probe volume, fast analysis and simplicity. The proposed test system uses layer-by-layer assembly approach, LED excitation and nanowatt photodetection set-up. The proposed test system offers AFP detection at concentrations used in clinical practice. Fluorescence enhancement for labeled AFP antibodies on a silver substrate was found to depend on antibodies concentration and was up to 6 times. (authors)

  1. Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

    Science.gov (United States)

    Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

    2016-10-21

    This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

  2. False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer.

    Science.gov (United States)

    Arndt, Torsten; Grüner, Joachim; Schröfel, Stefanie; Stemmerich, Karsten

    2012-11-30

    Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer. EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use. False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented

  3. Potential applications of immunoassays in studies of flatfish recruitment

    Science.gov (United States)

    Feller, Robert J.

    The fisheries recruitment-stock problem, a lack of correlation between measures of reproductive output of the parent stock and recruitment to the fishery, has several potential biotic and abiotic causes. Immunoassays may be useful in examining several aspects of this and several other problems in flatfish ecology: stock identification, parasitism and disease, and trophic interactions. Given stage-specific antisera capable of recognozing antigenic moieties of, for instance, eggs, larvae, or newly-settled juveniles, it is possible to screen stomach contents of many putative predators ( e.g., shrimp or crabs) rapidly for the presence and amounts of platfish prey. This trophic application of immunological methods has great promise for measuring loss of potential recruits to predation. All immunoassays are limited by the quality of antisera used and the researcher's ability to interpret quantitative data in an ecologically meaningful way. Key references for applications of immunoassays in fish-related questions are provided with recommendations for their utilization.

  4. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

    Science.gov (United States)

    Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

    2018-01-01

    Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

  5. Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

    Science.gov (United States)

    Wang, Guochun; Das, Champak; Ledden, Bradley; Sun, Qian; Nguyen, Chien

    2017-10-01

    Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

  6. Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens.

    Science.gov (United States)

    Wang, Zhanhui; Zhang, Huiyan; Ni, Hengjia; Zhang, Suxia; Shen, Jianzhong

    2014-04-11

    In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 μg L(-1) to as low as 0.07 μg L(-1). The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Vertical microreactor stack consist of poly-(tetrafluoroethylene) microfluidics for immunoassay

    International Nuclear Information System (INIS)

    Ukita, Yoshiaki; Kondo, Saki; Utsumi, Yuichi; Takeo, Masahiro; Negoro, Seiji; Kataoka, Chiwa

    2010-01-01

    This paper reports the first application of high-aspect ratio PTFE microstruscute, which fabricated by synchrotron radiation induced photo-evaporation process, to enzyme-linked immunosorvent assay. The advantages of PTFE microstructure for the development of lab-on-a-chip due to the extremely high-aspect ratio microstructure and chemical stability of PTFE is discussed. The results of immunoassay shows the successful detection of analyte (mouse IgG) with detection range with 0-100ng/ml. This result suggests the successful immobilization of antibody (anti-mouse IgG goat antibody) onto the x-ray exposed surface of PTFE microstructure and successful demonstration of antigen-antibody reaction in the PTFE high-aspect ratio microstructure. We also demonstrated the detection of polychlorinated biphenyl (PCB). As the result of demonstration, we successfully detected PCB with ranging analyte concentration of 0.1-10 ng/ml. (author)

  8. Synthesis-based approach toward direct sandwich immunoassay for ciguatoxin CTX3C.

    Science.gov (United States)

    Oguri, Hiroki; Hirama, Masahiro; Tsumuraya, Takeshi; Fujii, Ikuo; Maruyama, Megumi; Uehara, Hisatoshi; Nagumo, Yoko

    2003-06-25

    Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of ciguatoxin CTX3C were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens, 3 and 4, in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C itself. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C at the ppb level with no cross-reactivity against other related marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.

  9. Measurement of enzyme activity.

    Science.gov (United States)

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  10. Evaluation of an enzyme immunoassay for clinical diagnosis of neurocysticercosis in symptomatic patients Avaliação de um teste imunoenzimático para o diagnóstico clínico de neurocisticercose em pacientes sintomáticos

    Directory of Open Access Journals (Sweden)

    Reynaldo Mendes de Carvalho Junior

    2010-12-01

    Full Text Available INTRODUCTION: Neurocysticercosis is an infection of the human central nervous system caused by the metacestode larvae of Taenia solium. Neurocysticercosis is the most common parasitic disease in developing countries. Epilepsy is the most common clinical manifestation. Difficulties in confirming the diagnosis motivated the evaluation of the enzyme-linked immunosorbent assay on cerebral spinal fluid (CSF. METHODS: Twenty-two patients with NCC and 44 control patients were studied. CSF was analyzed using a commercial ELISA kit developed for NCC. Sensitivity and specificity were measured and a multivariate logistic regression was performed. RESULTS: Sensitivity and specificity of ELISA were 31.8% and 100%, respectively, with accuracy of 77.3%. Only the size of the lesions proved to be important for performance of the test. CONCLUSIONS: The results showed that ELISA contributes to the diagnosis of neurocysticercosis if the result is negative or if the patient has a lesion of 2 cm or more.INTRODUÇÃO: Neurocisticercose é a infecção do sistema nervoso central causada pela larva metacestódea da Taenia solium. Neurocisticercose é a parasitose mais comum nos países em desenvolvimento. Epilepsia é a sua manifestação clínica mais comum. A dificuldade para confirmar o diagnóstico motivou a avaliação do ensaio imunoenzimático ligado à enzima no líquido cérebro-espinhal. MÉTODOS: Vinte e dois pacientes com NCC e 44 pacientes controles foram estudados. Líquido cérebro-espinhal foi analisado por um kit ELISA comercial desenvolvido para NCC. A sensibilidade e especificidade foram medidas e uma análise multivariada de regressão logística foi realizada. RESULTADOS: A sensibilidade e a especificidade de ELISA foram, respectivamente, 31,8% e 100%, com acurácia de 77,3%. Apenas o tamanho das lesões mostrou-se importante para o desempenho do teste. CONCLUSÕES: Este estudo concluiu que ELISA contribui para o diagnóstico de NCC, caso o teste

  11. Nuclear techniques in animal production and health and food irradiation

    International Nuclear Information System (INIS)

    Cetinkaya, N.

    2002-01-01

    Nuclear techniques applied to animal production and health are concentrated in three main fields: Animal nutrition, reproduction and animal health. Isotopic markers, both radioactive (''1''4C, ''5 1 Cr, 32 P and 35 S) and stable ( 15 N), have been used in the development of feeding strategies by understanding the rumen fermentation process, and how protein and other nutrients are utilized to determine a balanced diet for meeting animal requirements for growth, pregnancy and lactation. The simple and easily applicable technology was developed for the preparation of a urea mineral multi nutrient block as a supplement and animal cake for the replacement of concentrate feed used by dairy cattle holders. The model was developed in Yerli Kara Cattle and its cross-breeds to estimate protein requirements of animals. Progesterone immunoassays (RIA/EIA) make it possible to control the reproductive performance of cattle, sheep and goats. A milk progesterone enzyme immunoassay kit known as Reprokon was developed at our Center. The kit has licensed by the Ministry of Agriculture and Rural Affairs. As for animal diseases, especially parasitic infections, nuclear techniques have proved to be of great value, namely in the production of irradiated vaccines against helminitic diseases. The Enzyme Linked Immunosorbent assay (ELISA) diagnostic techniques were used on the diagnosis of babesiosis, a disease which cause great economic loss in livestock in Turkey. Food irradiation is the treatment of raw, semi-processed or processed food or food ingredients with ionizing radiation to achieve a reduction of losses due to insect infestation, germination of root crops, spoilage and deterioration of perishable produce, and/or the control of microorganisms and other organisms that cause food borne diseases

  12. NiCoBP-doped carbon nanotube hybrid: A novel oxidase mimetic system for highly efficient electrochemical immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bing; He, Yu; Liu, Bingqian; Tang, Dianping, E-mail: dianping.tang@fzu.edu.cn

    2014-12-03

    Highlights: • We report a new oxidase mimetic system for highly efficient electrochemical immunoassay. • NiCoBP-doped carbon nanotube hybrids were used as the nanocatalysts. • NiCoBP-doped carbon nanotube hybrids were used as the mimic oxidase. - Abstract: NiCoBP-doped multi-walled carbon nanotube (NiCoBP–MWCNT) was first synthesized by using induced electroless-plating method and functionalized with the biomolecules for highly efficient electrochemical immunoassay of prostate-specific antigen (PSA, used as a model analyte). We discovered that the as-synthesized NiCoBP–MWCNT had the ability to catalyze the glucose oxidization with a stable and well-defined redox peak. The catalytic current increased with the increment of the immobilized NiCoBP–MWCNT on the electrode. Transmission electron microscope (TEM) and energy dispersive X-ray spectrometry (EDX) were employed to characterize the as-prepared NiCoBP–MWCNT. Using the NiCoBP–MWCNT-conjugated anti-PSA antibody as the signal-transduction tag, a new enzyme-free electrochemical immunoassay protocol could be designed for the detection of target PSA on the capture antibody-functionalized immunosensing interface. Experimental results revealed that the designed immunoassay system could exhibit good electrochemical responses toward target PSA, and allowed the detection of PSA at a concentration as low as 0.035 ng mL{sup −1}. More importantly, the NiCoBP-MWCNT-based oxidase mimetic system could be further extended for the monitoring of other low-abundance proteins or disease-related biomarkers by tuning the target antibody.

  13. Polyethylene imine/graphene oxide layer-by-layer surface functionalization for significantly improved limit of detection and binding kinetics of immunoassays on acrylate surfaces.

    Science.gov (United States)

    Miyazaki, Celina M; Mishra, Rohit; Kinahan, David J; Ferreira, Marystela; Ducrée, Jens

    2017-10-01

    Antibody immobilization on polymeric substrates is a key manufacturing step for microfluidic devices that implement sample-to-answer automation of immunoassays. In this work, a simple and versatile method to bio-functionalize poly(methylmethacrylate) (PMMA), a common material of such "Lab-on-a-Chip" systems, is proposed; using the Layer-by-Layer (LbL) technique, we assemble nanostructured thin films of poly(ethylene imine) (PEI) and graphene oxide (GO). The wettability of PMMA surfaces was significantly augmented by the surface treatment with (PEI/GO) 5 film, with an 81% reduction of the contact angle, while the surface roughness increased by 600%, thus clearly enhancing wettability and antibody binding capacity. When applied to enzyme-linked immunosorbent assays (ELISAs), the limit of detection of PMMA surface was notably improved from 340pgmL -1 on commercial grade polystyrene (PS) and 230pgmL -1 on plain PMMA surfaces to 130pgmL -1 on (PEI/GO) 5 treated PMMA. Furthermore, the accelerated antibody adsorption kinetics on the LbL films of GO allowed to substantially shorten incubation times, e.g. for anti-rat IgG adsorption from 2h down to 15min on conventional and treated surfaces, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Distribution of Phytophthora spp. in Field Soils Determined by Immunoassay.

    Science.gov (United States)

    Miller, S A; Madden, L V; Schmitthenner, A F

    1997-01-01

    ABSTRACT Populations of Phytophthora spp. were determined by enzyme-linked immunosorbent assay (ELISA) in field soils used for pepper and soybean production in Ohio. Soybean fields were sampled extensively (64 fields, n = 6 samples per field over 2 years) and intensively (4 fields, n = 64 samples per field in 1 year) to assess heterogeneity of P. sojae populations. Four pepper fields (n = 64), three of which had a history of Phytophthora blight (caused by P. capsici), also were sampled intensively during a 6-month period. Mean (m), variance (v), and measures of aggregation (e.g., variance-to-mean ratio [v/m]) of immunoassay values, translated to Phytophthora antigen units (PAU), were related to the disease history in each of the pepper and soybean fields. Mean PAU values for fields in which Phytophthora root rot (soybean) or blight (pepper) had been moderate to severe were higher than in fields in which disease incidence had been low or not observed. A detection threshold value of 11.3 PAU was calculated with values for 64 samples from one pepper field, all of which tested negative for Phytophthora by bioassay and ELISA. Seven of the eight intensively sampled fields contained at least some detectable Phytophthora propagules, with the percentage of positive samples ranging from 1.6 to 73.4. Mean PAU values ranged from 1 to 84 (extensive soybean field sampling), 6 to 24 (intensive soybean field sampling), and 4 to 30 (intensive pepper field sampling); however, variances ranged from 0 to 7,774 (extensive sampling), 30 to 848 (intensive soybean field sampling), and 5 to 2,401 (intensive pepper field sampling). Heterogeneity of PAU was high in most individual soybean and pepper fields, with values of v/m greater than 1, and log(v) increasing with log(m), with a slope of about 2.0. Spatial autocorrelation coefficients were not significant, indicating there was no relationship of PAU values in neighboring sampling units (i.e., field locations) of the intensively sampled

  15. Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

    Science.gov (United States)

    Stephen, Laurie; Guest, Paul C

    2018-01-01

    Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

  16. Optimization of an immunoassay of 2,6-dichlorobenzamide (BAM) and development of regenerative surfaces by immunosorbent modification with newly synthesised BAM hapten library

    DEFF Research Database (Denmark)

    Uthuppu, Basil; Aamand, Jens; Jørgensen, Claus

    2012-01-01

    Dichlobenil is an extensively used herbicide worldwide which is transformed to the mobile 2,6-dichlorobenzamide (BAM) in soil. BAM has been found in many European groundwater resources that are exploited for drinking water. Currently, immunoassay based monitoring technique (plate based ELISA) is ...

  17. Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

    International Nuclear Information System (INIS)

    Tahara, Yusuke; Huang, Zhe; Kiritoshi, Tetsuro; Onodera, Takeshi; Toko, Kiyoshi

    2014-01-01

    The monitoring of salivary cortisol as a key biomarker of an individual’s stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt–100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

  18. Development of Indirect Competitive Immuno-Assay Method Using SPR Detection for Rapid and Highly Sensitive Measurement of Salivary Cortisol Levels

    Energy Technology Data Exchange (ETDEWEB)

    Tahara, Yusuke; Huang, Zhe; Kiritoshi, Tetsuro [Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka (Japan); Onodera, Takeshi [Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka (Japan); Toko, Kiyoshi, E-mail: toko@ed.kyushu-u.ac.jp [Graduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka (Japan); Research and Development Center for Taste and Odor Sensing, Kyushu University, Fukuoka (Japan)

    2014-05-30

    The monitoring of salivary cortisol as a key biomarker of an individual’s stress response has been increasingly focused on. This paper describes the development of a novel cortisol immuno-assay method based on an indirect competitive method using a commercially available surface plasmon resonance instrument. The surface of an Au chip was modified with PEG6-COOH aromatic dialkanethiol self-assembled monolayers and hydrocortisone 3-(O-carboxymethyl) oxime (hydrocortisone 3-CMO) as a cortisol analog. A detection limit of 38 ppt range with a measurement range of 10 ppt–100 ppb was accomplished without the incubation of a mixing solution consisting of standard cortisol and an anti-cortisol antibody, and the time for quantification of cortisol concentration was 8 min from the sample injection. We experimentally compared our immuno-assay with a commercialized salivary cortisol enzyme-linked immunosorbent assay (ELISA) kit using human saliva samples. It was found that the results obtained by the cortisol immuno-assay had a good correlation with those obtained by ELISA assay (R = 0.96). Our findings indicate the potential utility of the cortisol immuno-assay for measurements of human salivary cortisol levels.

  19. Biosensor immunoassay for flumequine in broiler serum and muscle

    NARCIS (Netherlands)

    Haasnoot, W.; Gercek, H.; Cazemier, G.; Nielen, M.W.F.

    2007-01-01

    Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5 min per sample) and specific (no cross-reactivity

  20. Feasibility of a simple microsieve-based immunoassay platform

    NARCIS (Netherlands)

    Zweitzig, D.R.; Tibbe, Arjan G.J.; Nguyen, A.T.; van Rijn, C.J.M.; Kopnitsky, M.J.; Cichonski, K.; Terstappen, Leonardus Wendelinus Mathias Marie

    2016-01-01

    The intrinsic properties of silicon microsieves, such as an optically flat surface, high overall porosity, and low flow resistance have led to an increasing number of biotechnology applications. In this report, the feasibility of creating a microsieve-based immunoassay platform was explored.

  1. Feasibility of a simple microsieve-based immunoassay platform

    NARCIS (Netherlands)

    Zweitzig, Daniel R.; Tibbe, Arjan G.; Nguyen, Ai T.; Rijn, van Cees J.M.; Kopnitsky, Mark J.; Cichonski, Kathleen; Terstappen, Leon W.M.M.

    2016-01-01

    The intrinsic properties of silicon microsieves, such as an optically flat surface, high overall porosity, and low flow resistance have led to an increasing number of biotechnology applications. In this report, the feasibility of creating a microsieve-based immunoassay platform was explored.

  2. IMMUNOASSAYS FOR THE DETECTION OF UROKINASE RECEPTOR FORMS

    DEFF Research Database (Denmark)

    2004-01-01

    amounts of uPAR forms in a sample with data indicative of the presence of the cancer disease. The method can be used for staging, prognosis or diagnosis of prostate cancer. Two novel monoclonal antibodies and a kit and immunoassays for detecting at least one uPAR form(s) are also described in the present...

  3. Rapid micromotor-based naked-eye immunoassay.

    Science.gov (United States)

    de Ávila, Berta Esteban-Fernández; Zhao, Mingjiao; Campuzano, Susana; Ricci, Francesco; Pingarrón, José M; Mascini, Marcello; Wang, Joseph

    2017-05-15

    A dynamic micromotor-based immunoassay, exemplified by cortisol detection, based on the use of tubular micromotors functionalized with a specific antibody is described. The use of antibody-functionalized micromotors offers huge acceleration of both direct and competitive cortisol immunoassays, along with greatly enhanced sensitivity of direct and competitive immunoassays. The dramatically improved speed and sensitivity reflect the greatly increased likelihood of antibody-cortisol contacts and fluid mixing associated with the dynamic movement of these microtube motors and corresponding bubble generation that lead to a highly efficient and rapid recognition process. Rapid naked-eye detection of cortisol in the sample is achieved in connection to use of horseradish peroxidase (HRP) tag and TMB/H 2 O 2 system. Key parameters of the competitive immunoassay (e.g., incubation time and reaction volume) were optimized. This fast visual micromotor-based sensing approach enables "on the move" specific detection of the target cortisol down to 0.1μgmL -1 in just 2min, using ultrasmall (50µL) sample volumes. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Direct salivary cortisol radio-immunoassay determination. Clinical applications

    International Nuclear Information System (INIS)

    Simon, C.; Cherfan, J.; Kurtz, F.; Vignon, F.; Schlienger, J.L.; Chabrier, G.

    1987-01-01

    Salivary cortisol levels reflect the biologically active free fraction of blood cortisol. The authors describe the results obtained with the aim of a radio-immunoassay commercial serum cortisol kit, without prealable extraction in different physiological and pathological situations. Salivary cortisol determination appears performant both in nycthemeral studies and in stimulation or freination tests [fr

  5. Evaluation of Six Different Immunoassays for Serum Thyrotropin

    International Nuclear Information System (INIS)

    Ma Donghong; Lu Hankui; Gao Yunchao; Ge Wenli; Xiong Jiang; Liu Qiaoping; Gu Qing

    2010-01-01

    To analyzes the discrepancy and association among six different thyrotropin (TSH) immunoassay methods and to study their impact on the clinical diagnoses of thyroid diseases, the 150 serum samples from three groups consisting of hyperthyroidism, hypothyroidism and healthy subjects, 50 samples in each group were included in this study. The serum TSH levels were measured simultaneously by radioimmunoassay (RIA), immunoradiometric assay (IRMA), three-type chemilumiminescence immunoassay (CLIA) and electrochemiluminescence immunoassay (ECLIA). The results showed that individual serum TSH level varied significantly from one assay to another. There was no correlation between TSH RIA and other five assays in groups of hyperthyroidism and healthy subjects(P>0.05). The correlations between TSH IRMA and four automatic assays in hyperthyroidism group were relatively low (r= 0.38∼0.41). However, among the four automatic assays, TSH levels were well correlated (r= 0.92∼0.99). For clinical diagnoses, TSH RIA alone was not useful in the differentiation of hyperthyroidism and normal subjects, and TSH IRMA was misleading in some hyperthyroidism. There were no significant differences for four TSH automatic immunoassays in differential diagnoses of thyroid diseases. (authors)

  6. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    Science.gov (United States)

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  7. Comparison of capillary electrophoresis-based immunoassay with fluorescence polarization immunoassay for the immunodetermination of methamphetamine using various methamphetamine antibodies.

    Science.gov (United States)

    Choi, J; Kim, C; Choi, M J

    1998-11-01

    An accurate and simple immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) was performed for the detection of methamphetamine (MA) in urine. The CE-LIF was conducted with an untreated fused-silica column using antiserum and a tracer of fluorescein isothiocyanate (FITC)-labeled MA. This CE-LIF system was compared with fluorescence polarization immunoassay (FPIA) in a TDx analyzer in the photo-check mode using the same FITC-labeled tracer and the same antiserum. Various antibodies, not only those prepared by our own immunogens but also those from commercial sources, were screened and characterized in both assay systems with regard to sensitivity, precision, and cross-reactivity. Both systems satisfied analytical precision and gave similar cross-reactivity patterns. However, the CE-LIF-based immunoassay was approximately one order superior to FPIA in sensitivity, requiring less volume of sample, antiserum, and tracer for the assay. Considering that the FPIA system is well known to be a useful tool for screening antibodies and detecting drugs, the CE-LIF-based immunoassay system, which is seemingly more advantageous than the FPIA system, appears to have great power for the characterization of antibodies and for the detection of MA in urine.

  8. Zymography methods for visualizing hydrolytic enzymes

    OpenAIRE

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E.; Opdenakker, Ghislain

    2013-01-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful., but often misinterpreted, tool. yielding information on potential. hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tis...

  9. Rapid screening of aflatoxin B1 in beer by fluorescence polarization immunoassay.

    Science.gov (United States)

    Beloglazova, N V; Eremin, S A

    2015-09-01

    This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for the mycotoxin aflatoxin B1 (AFB1) in various beer samples, both lager and dark. The highest sensitivity was determined for six poly- and monoclonal antibodies selective towards aflatoxins. The sample pretreatment design was emphasized since beer samples are characterized by extremely diverse matrices. Herein, the choice of sorbent for effective removal of matrix interferences prior to analysis was crucial. The samples were diluted with a borate buffer solution containing 1% PEG 6000 and passed through the clean-up column packed with NH2-derivated silica. This sample pretreatment technique was perfectly suitable for the FPIA of lager beer samples, but for dark beer and ale it did not suffice. An artificial matrix was constructed to plot a calibration curve and quantify the results of the latter samples. The developed immunoassay was characterized by a limit of detection of 1 ng mL(-1). Apparent recovery values of 89-114% for lager and 80-125% for dark beer were established. The FPIA data for AFB1 was characterized by elevated linear regression coefficients, 0.9953 for spiked lager and 0.9895 for dark beer samples respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Quantification of trypsin with a radioimmunoassay in herring larvae (Clupea harengus L.) compared with a highly sensitive fluorescence technique to determine tryptic enzyme activity

    International Nuclear Information System (INIS)

    Ueberschaer, B.; Pedersen, B.H.; Hjelmeland, K.

    1993-01-01

    Enzymatic activity and quantity of the protease trypsin were measured in individual herring larvae (Clupea harengus L.). The enzymatic activity assay was done by a fluorescence technique, and a radioimmunoassay was used for quantification of trypsin. The results are compared and the differences between the techniques discussed. Both methods have similar results, as high or low values in trypsin quantity were reflected in high or low values of tryptic activity. Quantity and activity were linearly and positively correlated, but small differences between methods were found at the lowest detection limits. Both techniques reflect the high variability between individual larvae. (orig.)

  11. Development of a Nanobody-Based Lateral Flow Immunoassay for Detection of Human Norovirus.

    Science.gov (United States)

    Doerflinger, Sylvie Y; Tabatabai, Julia; Schnitzler, Paul; Farah, Carlo; Rameil, Steffen; Sander, Peter; Koromyslova, Anna; Hansman, Grant S

    2016-01-01

    Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. These viruses are usually detected by molecular methods, including reverse transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Human noroviruses are genetically and antigenically diverse, with two main genogroups that are further subdivided into over 40 different genotypes. During the past decade, genogroup 2 genotype 4 (GII.4) has dominated in most countries, but recently, viruses belonging to GII.17 have increased in prevalence in a number of countries. A number of commercially available ELISAs and lateral flow immunoassays were found to have lower sensitivities to the GII.17 viruses, indicating that the antibodies used in these methods may not have a high level of cross-reactivity. In this study, we developed a rapid Nanobody-based lateral flow immunoassay (Nano-immunochromatography [Nano-IC]) for the detection of human norovirus in clinical specimens. The Nano-IC assay detected virions from two GII.4 norovirus clusters, which included the current dominant strain and a novel variant strain. The Nano-IC method had a sensitivity of 80% and specificity of 86% for outbreak specimens. Norovirus virus-like particles (VLPs) representing four genotypes (GII.4, GII.10, GII.12, and GII.17) could be detected by this method, demonstrating the potential in clinical screening. However, further modifications to the Nano-IC method are needed in order to improve this sensitivity, which may be achieved by the addition of other broadly reactive Nanobodies to the system. IMPORTANCE We previously identified a Nanobody (termed Nano-85) that bound to a highly conserved region on the norovirus capsid. In this study, the Nanobody was biotinylated and gold conjugated for a lateral flow immunoassay (termed Nano-IC). We showed that the Nano-IC assay was capable of detecting at least four antigenically distinct GII genotypes, including the newly emerging GII.17. In the clinical setting, the

  12. Aspergillus Galactomannan Enzyme Immunoassay and Quantitative PCR for Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid

    OpenAIRE

    Musher, Benjamin; Fredricks, David; Leisenring, Wendy; Balajee, S. Arunmozhi; Smith, Caitlin; Marr, Kieren A.

    2004-01-01

    Invasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillus species from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillus antigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for addit...

  13. Performance evaluation of enzyme immunoassay for voriconazole therapeutic drug monitoring with automated clinical chemistry analyzers

    Directory of Open Access Journals (Sweden)

    Yongbum Jeon

    2017-08-01

    Full Text Available Objective: Voriconazole is a triazole antifungal developed for the treatment of fungal infectious disease, and the clinical utility of its therapeutic drug monitoring has been evaluated. Recently, a new assay for analyzing the serum voriconazole concentration with an automated clinical chemistry analyzer was developed. We evaluated the performance of the new assay based on standardized protocols. Methods: The analytical performance of the assay was evaluated according to its precision, trueness by recovery, limit of quantitation, linearity, and correlation with results from liquid chromatography-tandem mass spectrometry (LC-MS/MS. The evaluation was performed with the same protocol on two different routine chemistry analyzers. All evaluations were performed according to CLSI Guidelines EP15, EP17, EP6, and EP9 [1–4]. Results: Coefficients of variation for within-run and between-day imprecision were 3.2–5.1% and 1.5–3.0%, respectively, on the two different analyzers for pooled serum samples. The recovery rates were in the range of 95.4–102.2%. The limit of blank was 0.0049 μg/mL, and the limit of detection of the samples was 0.0266–0.0376 μg/mL. The percent recovery at three LoQ levels were 67.9–74.6% for 0.50 μg/mL, 75.5–80.2% for 0.60 μg/mL, and 89.9–96.6% for 0.70 μg/mL. A linear relationship was demonstrated between 0.5 μg/mL and 16.0 μg/mL (R2=0.9995–0.9998. The assay correlated well with LC-MS/MS results (R2=0.9739–0.9828. Conclusions: The assay showed acceptable precision, trueness, linearity, and limit of quantification, and correlated well with LC-MS/MS. Therefore, its analytical performance is satisfactory for monitoring the drug concentration of voriconazole. Keywords: Voriconazole, Antifungal agents, Therapeutic drug monitoring

  14. Development and evaluation of modern enzyme immunoassays for comprehensive syphilis serology

    NARCIS (Netherlands)

    O.E. IJsselmuiden

    1989-01-01

    textabstractIn the previous Chapters of this dissertation the major shortcomings of the current serological tests for syphilis were investigated. These include an insufficient sensitivity and specificity, reactivity of a diagnostic test long after the syphilitic infection had been

  15. Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein.

    Science.gov (United States)

    Smith, D S; Kitts, D D

    1994-03-01

    A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

  16. Performance evaluation of enzyme immunoassay for voriconazole therapeutic drug monitoring with automated clinical chemistry analyzers.

    Science.gov (United States)

    Jeon, Yongbum; Han, Minje; Han, Eun Young; Lee, Kyunghoon; Song, Junghan; Song, Sang Hoon

    2017-08-01

    Voriconazole is a triazole antifungal developed for the treatment of fungal infectious disease, and the clinical utility of its therapeutic drug monitoring has been evaluated. Recently, a new assay for analyzing the serum voriconazole concentration with an automated clinical chemistry analyzer was developed. We evaluated the performance of the new assay based on standardized protocols. The analytical performance of the assay was evaluated according to its precision, trueness by recovery, limit of quantitation, linearity, and correlation with results from liquid chromatography-tandem mass spectrometry (LC-MS/MS). The evaluation was performed with the same protocol on two different routine chemistry analyzers. All evaluations were performed according to CLSI Guidelines EP15, EP17, EP6, and EP9 [1-4]. Coefficients of variation for within-run and between-day imprecision were 3.2-5.1% and 1.5-3.0%, respectively, on the two different analyzers for pooled serum samples. The recovery rates were in the range of 95.4-102.2%. The limit of blank was 0.0049 μg/mL, and the limit of detection of the samples was 0.0266-0.0376 μg/mL. The percent recovery at three LoQ levels were 67.9-74.6% for 0.50 μg/mL, 75.5-80.2% for 0.60 μg/mL, and 89.9-96.6% for 0.70 μg/mL. A linear relationship was demonstrated between 0.5 μg/mL and 16.0 μg/mL ( R 2 =0.9995-0.9998). The assay correlated well with LC-MS/MS results ( R 2 =0.9739-0.9828). The assay showed acceptable precision, trueness, linearity, and limit of quantification, and correlated well with LC-MS/MS. Therefore, its analytical performance is satisfactory for monitoring the drug concentration of voriconazole.

  17. The effect of storage conditions on salivary cortisol concentrations using an enzyme immunoassay

    DEFF Research Database (Denmark)

    Nalla, Anjana A; Thomsen, Gerda; Knudsen, Karen Birgitte Moos

    2015-01-01

    it was stored at - 80°C. The last tube was stored directly at - 80°C and served as the 'gold standard'. The saliva samples were assayed using Salivary Cortisol Diagnostic EIA. Differences in cortisol measurements between each of the five conditions and the 'gold standard' (- 80°C) were evaluated by one-sample t...

  18. Effects of saliva collection using cotton swabs on melatonin enzyme immunoassay.

    Science.gov (United States)

    Kozaki, Tomoaki; Lee, Soomin; Nishimura, Takayuki; Katsuura, Tetsuo; Yasukouchi, Akira

    2011-01-10

    Although various acceptable and easy-to-use devices have been used for saliva collection, cotton swabs are among the most common ones. Previous studies reported that cotton swabs yield a lower level of melatonin detection. However, this statistical method is not adequate for detecting an agreement between cotton saliva collection and passive saliva collection, and a test for bias is needed. Furthermore, the effects of cotton swabs have not been examined at lower melatonin level, a level at which melatonin is used for assessment of circadian rhythms, namely dim light melatonin onset (DLMO). In the present study, we estimated the effect of cotton swabs on the results of salivary melatonin assay using the Bland-Altman plot at lower level. Nine healthy males were recruited and each provided four saliva samples on a single day to yield a total of 36 samples. Saliva samples were directly collected in plastic tubes using plastic straws, and subsequently pipetted onto cotton swabs (cotton saliva collection) and into clear sterile tubes (passive saliva collection). The melatonin levels were analyzed in duplicate using commercially available ELISA kits. The mean melatonin concentration in cotton saliva collection samples was significantly lower than that in passive saliva collection samples at higher melatonin level (>6 pg/mL). The Bland-Altman plot indicated that cotton swabs causes relative and proportional biases in the assay results. For lower melatonin level (<6 pg/mL), although the BA plots didn't show proportional and relative biases, there was no significant correlation between passive and cotton saliva collection samples. Our findings indicate an interference effect of cotton swabs on the assay result of salivary melatonin at lower melatonin level. Cotton-based collection devices might, thus, not be suitable for assessment of DLMO.

  19. Effects of saliva collection using cotton swabs on melatonin enzyme immunoassay

    Directory of Open Access Journals (Sweden)

    Katsuura Tetsuo

    2011-01-01

    Full Text Available Abstract Background Although various acceptable and easy-to-use devices have been used for saliva collection, cotton swabs are among the most common ones. Previous studies reported that cotton swabs yield a lower level of melatonin detection. However, this statistical method is not adequate for detecting an agreement between cotton saliva collection and passive saliva collection, and a test for bias is needed. Furthermore, the effects of cotton swabs have not been examined at lower melatonin level, a level at which melatonin is used for assessment of circadian rhythms, namely dim light melatonin onset (DLMO. In the present study, we estimated the effect of cotton swabs on the results of salivary melatonin assay using the Bland-Altman plot at lower level. Methods Nine healthy males were recruited and each provided four saliva samples on a single day to yield a total of 36 samples. Saliva samples were directly collected in plastic tubes using plastic straws, and subsequently pipetted onto cotton swabs (cotton saliva collection and into clear sterile tubes (passive saliva collection. The melatonin levels were analyzed in duplicate using commercially available ELISA kits. Results The mean melatonin concentration in cotton saliva collection samples was significantly lower than that in passive saliva collection samples at higher melatonin level (>6 pg/mL. The Bland-Altman plot indicated that cotton swabs causes relative and proportional biases in the assay results. For lower melatonin level ( Conclusion Our findings indicate an interference effect of cotton swabs on the assay result of salivary melatonin at lower melatonin level. Cotton-based collection devices might, thus, not be suitable for assessment of DLMO.

  20. Urinary growth hormone levels in children with leukemia determined by a sensitive sandwich enzyme immunoassay

    International Nuclear Information System (INIS)

    Umezawa, Sachiko; Yano, Hisako; Tanaka, Toshiaki; Hibi, Itsuo; Tsukimoto, Ichiro; Ishikawa, Eiji; Mouri, Yoshikazu; Murakami, Yoshiaki.

    1989-01-01

    Elective cranial irradiation in central nervous leukemia leads to a long term survival; however, the influence upon the hypothalamus and hypophysis remains uncertain. Therefore, changes in SD scores of height and urinary growth hormone (U-GH) levels were determined in 23 leukemic patients previously treated with cranial elective irradition of 18 or 24 Gy, whose ages ranged from 4 to 16 years. Decreased SD scores of height was observed in 9 (90%) of 10 patients in whom treatment has been continuing (within 5 years after the onset) and in 6 (50%) of 12 patients in whom treatment had been completed (5 years or more after the onset). The level of U-GH was significantly decreased in leukemic patients during complete remission than in healthy children. U-HG levels were independent of radiation doses. In evaluable 14 patients, the blood concentration of somatomedin C (SM-C) was within the normal range. Although there was a discrepancy between U-CH and SM-C levels, further follow-up of these leukemic patients is required. (N.K.)

  1. Microcapillary blood sampling for serological examinations by radioimmunoassay (RIA) and enzyme immunoassay (ELISA)

    Energy Technology Data Exchange (ETDEWEB)

    Rodak, L; Smid, B; Valicek, L; Jurak, E [Vyzkumny Ustav Veterinarniho Lekarstvi, Brno-Medlanky (Czechoslovakia)

    1984-01-01

    Methods were tested of sampling blood and blood serum for serological examinations on filtration paper and into heparinized glass capillaries with transfer into the dilution solution of the given composition. Samples were also examined for ACH virus antibodies. The suitability of the sampling was verified by an examination of samples using ELISA and RIA methods. The results showed the suitability of sampling using microcapillaries. The titres of virus antibodies found using the ELISA and RIA methods were identical and the sensitivity of antibody detection was not reduced even after the sample had been stored for 60 days at a temperature of 20 degC.

  2. Determination of serum DNA concentration by enzyme immunoassay (ELISA) in gamma irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Gabor, J; Misurova, E

    1987-01-01

    A sensitive and specific ELISA method was used to determine changes in the serum DNA concentration in rats at hours 6 and 9 and on the days 1, 3, 7, 10, 15 and 30 after acute whole-body gamma irradiation with a dose of 8 Gy. Changes in the DNA serum concentration were determined also on day 10 after irradiation with doses of 4, 6, 8, 10 and 12 Gy. The present results indicate that the pattern of changes in the serum DNA concentration is characterized by an initial decrease, typical also of the leukocyte count, followed by a statistically significant increase in the DNA concentration on day 10 and in later periods of time. These data confirmed, in principle, the authors' previous findings on changes in the DNA concentration in the rat blood plasma after acute X-ray irradiation assessed by the fluorimetric method with ethidium bromide. (author). 4 figs., 14 refs.

  3. Microcapillary blood sampling for serological examinations by radioimmunoassay (RIA) and enzyme immunoassay (ELISA)

    International Nuclear Information System (INIS)

    Rodak, L.; Smid, B.; Valicek, L.; Jurak, E.

    1984-01-01

    Methods were tested of sampling blood and blood serum for serological examinations on filtration paper and into heparinized glass capillaries with transfer into the dilution solution of the given composition. Samples were also examined for ACH virus antibodies. The suitability of the sampling was verified by an examination of samples usiOg ELISA and RIA methods. The results showed the suitability of sampling using microcapillaries. The titres of virus antibodies found using the ELISA and RIA methods were identical and the sensitivity of antibody detection was not reduced even after the sample had been stored for 60 days at a temperature of 20 degC. (B.S.)

  4. System-on-fluidics immunoassay device integrating wireless radio-frequency-identification sensor chips.

    Science.gov (United States)

    Yazawa, Yoshiaki; Oonishi, Tadashi; Watanabe, Kazuki; Shiratori, Akiko; Funaoka, Sohei; Fukushima, Masao

    2014-09-01

    A simple and sensitive point-of-care-test (POCT) device for chemiluminescence (CL) immunoassay was devised and tested. The device consists of a plastic flow-channel reactor and two wireless-communication sensor chips, namely, a photo-sensor chip and a temperature-sensor chip. In the flow-channel reactor, a target antigen is captured by an antibody immobilized on the inner wall of the flow-channel and detected with enzyme labeled antibody by using CL substrate. The CL signal corresponding to the amount of antigen is measured by a newly developed radio-frequency-identification (RFID) sensor, which enables batteryless operation and wireless data communication with an external reader. As for the POCT device, its usage environment, especially temperature, varies for each measurement. Hence, temperature compensation is a key issue in regard to eliminating dark-signal fluctuation, which is a major factor in deterioration of the precision of the POCT device. A two-stage temperature-compensation scheme was adopted. As for the first stage, the signals of two photodiodes, one with an open window and one with a sealed window, integrated on the photo-sensor chip are differentiated to delete the dark signal. As for the second stage, the differentiated signal fluctuation caused by a temperature variation is compensated by using the other sensor chip (equipped with a temperature sensor). The dark-level fluctuation caused by temperature was reduced from 0.24 to 0.02 pA/°C. The POCT device was evaluated as a CL immunoassay of thyroid-stimulating hormone (TSH). The flow rate of the CL reagent in the flow channel was optimized. As a result, the detection limit of the POCT device was 0.08 ng/ml (i.e., 0.4 μIU/ml). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Mobile Phone Ratiometric Imaging Enables Highly Sensitive Fluorescence Lateral Flow Immunoassays without External Optical Filters.

    Science.gov (United States)

    Shah, Kamal G; Singh, Vidhi; Kauffman, Peter C; Abe, Koji; Yager, Paul

    2018-05-14

    Paper-based diagnostic tests based on the lateral flow immunoassay concept promise low-cost, point-of-care detection of infectious diseases, but such assays suffer from poor limits of detection. One factor that contributes to poor analytical performance is a reliance on low-contrast chromophoric optical labels such as gold nanoparticles. Previous attempts to improve the sensitivity of paper-based diagnostics include replacing chromophoric labels with enzymes, fluorophores, or phosphors at the expense of increased fluidic complexity or the need for device readers with costly optoelectronics. Several groups, including our own, have proposed mobile phones as suitable point-of-care readers due to their low cost, ease of use, and ubiquity. However, extant mobile phone fluorescence readers require costly optical filters and were typically validated with only one camera sensor module, which is inappropriate for potential point-of-care use. In response, we propose to couple low-cost ultraviolet light-emitting diodes with long Stokes-shift quantum dots to enable ratiometric mobile phone fluorescence measurements without optical filters. Ratiometric imaging with unmodified smartphone cameras improves the contrast and attenuates the impact of excitation intensity variability by 15×. Practical application was shown with a lateral flow immunoassay for influenza A with nucleoproteins spiked into simulated nasal matrix. Limits of detection of 1.5 and 2.6 fmol were attained on two mobile phones, which are comparable to a gel imager (1.9 fmol), 10× better than imaging gold nanoparticles on a scanner (18 fmol), and >2 orders of magnitude better than gold nanoparticle-labeled assays imaged with mobile phones. Use of the proposed filter-free mobile phone imaging scheme is a first step toward enabling a new generation of highly sensitive, point-of-care fluorescence assays.

  6. Nanogold–polyaniline–nanogold microspheres-functionalized molecular tags for sensitive electrochemical immunoassay of thyroid-stimulating hormone

    International Nuclear Information System (INIS)

    Cui Yuling; Chen Huafeng; Hou Li; Zhang Bing; Liu Bingqian; Chen Guonan; Tang Dianping

    2012-01-01

    Highlights: ► A novel immunosensing strategy was designed for detection of thyroid-stimulating hormone. ► Using nanogold–polyaniline–nanogold microspheres as molecular tags. ► Improvement of electrochemical activity of nanolabels. ► Combination enzyme labels with nanolabels for signal amplification. - Abstract: Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold–polyaniline–nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab 2 ) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L −1 H 2 O 2 by using GPG-labeled HRP-Ab 2 as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab 2 . Several labeling protocols comprising HRP-Ab 2 , nanogold particle-labeled HRP-Ab 2 , and polyaniline nanospheres-labeled HRP-Ab 2 , were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab 2 . With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab 2 to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 μIU mL −1 with a detection limit (LOD) of 0.005 μIU mL −1 TSH at the 3s B criterion

  7. Enzyme Molecules in Solitary Confinement

    Directory of Open Access Journals (Sweden)

    Raphaela B. Liebherr

    2014-09-01

    Full Text Available Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

  8. Design and Fabrication of a PDMS Microchip Based Immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Guocheng; Wang, Wanjun; Wang, Jun; Lin, Yuehe

    2010-07-01

    In this paper, we describe the design and fabrication process of a polydimethylsiloxane (PDMS) microchip for on-chip multiplex immunoassay application. The microchip consists of a PDMS microfluidic channel layer and a micro pneumatic valve control layer. By selectively pressurizing the pneumatic microvalves, immuno reagents were controlled to flow and react in certain fluidic channel sites. Cross contamination was prevented by tightly closed valves. Our design was proposed to utilize PDMS micro channel surface as the solid phase immunoassay substrate and simultaneously detect four targets antigens on chip. Experiment result shows that 20psi valve pressure is sufficient to tightly close a 200µm wide micro channel with flow rate up to 20µl/min.

  9. Label-Free Electrochemical Immunoassay for C-Reactive Protein

    Directory of Open Access Journals (Sweden)

    Madasamy Thangamuthu

    2018-03-01

    Full Text Available C-reactive protein (CRP is one of the most expressed proteins in blood during acute phase inflammation, and its minute level increase has also been recognized for the clinical diagnosis of cardio vascular diseases. Unfortunately, the available commercial immunoassays are labour intensive, require large sample volumes, and have practical limitations, such as low stability and high production costs. Hence, we have developed a simple, cost effective, and label-free electrochemical immunoassay for the measurement of CRP in a drop of serum sample using an immunosensor strip made up of a screen printed carbon electrode (SPE modified with anti-CRP functionalized gold nanoparticles (AuNPs. The measurement relies on the decrease of the oxidation current of the redox indicator Fe3+/Fe2+, resulting from the immunoreaction between CRP and anti-CRP. Under optimal conditions, the present immunoassay measures CRP in a linear range from 0.4–200 nM (0.047–23.6 µg mL−1, with a detection limit of 0.15 nM (17 ng mL−1, S/N = 3 and sensitivity of 90.7 nA nM−1, in addition to a good reproducibility and storage stability. The analytical applicability of the presented immunoassay is verified by CRP measurements in human blood serum samples. This work provides the basis for a low-priced, safe, and easy-to-use point-of-care immunosensor assay to measure CRP at clinically relevant concentrations.

  10. Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

    Science.gov (United States)

    Li, Dongyang; Cui, Yongliang; Morisseau, Christophe; Gee, Shirley J; Bever, Candace S; Liu, Xiangjiang; Wu, Jian; Hammock, Bruce D; Ying, Yibin

    2017-06-06

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

  11. Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer.

    Science.gov (United States)

    Khalil, O S; Zurek, T F; Tryba, J; Hanna, C F; Hollar, R; Pepe, C; Genger, K; Brentz, C; Murphy, B; Abunimeh, N

    1991-09-01

    We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.

  12. Current status and future developments in radiolabelled immunoassays

    International Nuclear Information System (INIS)

    Edwards, R.

    1998-01-01

    Radioisotopes are used extensively in medical practice and their use in RIA or IRMA usually represent a small proportion of the total. Radiolabelled immunoassays based on 125 I constitute a simple didactic, cost effective and robust technology which is still regarded as the reference method in many clinical applications. The IAEA has implemented many successful programmes using the ''bulk reagent'' approach, involving 68 countries in all the different regions. The main achievements have been in technology transfer with self sufficiency in production for some countries; training of large numbers of staff; quality control and quality assurance schemes; devolution of screening programmes for neonatal congenital hypothryoidism. Alternatives to the use of radioisotopic tracers are constrained by many factors and are often only available in restricted commercial packages. They are often not suitable for technology transfer programmes and often lack any didactic component in addition to a relative high cost. The production of radiolabels using 125 I is both simple and adaptable. In addition expertise in their preparation and purification is widespread even in developing countries. Together with the ease of producing antibodies, the facts have made 125 I-radiolabelled immunoassays ideal for investigative procedures for many research activities (30,31) particularly in the medical context where radioisotopes are commonly used. In conclusion, even a superficial examination of public health statistics for various countries throughout the continents indicates a need for a simple, inexpensive and robust analytical tool. In this light, there is a predicted continuing role for radiolabelled immunoassays. (author)

  13. Simple patterned nanofiber scaffolds and its enhanced performance in immunoassay.

    Directory of Open Access Journals (Sweden)

    Jing Wang

    Full Text Available Cancer has become the leading cause of death worldwide; early diagnosis and treatment of cancers is critical for the survival of the patients. The concentration of cancer markers in easy-to-access biological fluids can provide great assistance in screening for occult primary cancers, distinguishing malignant from benign findings, determining prognosis and prediction for cancer patients. The multiplex detection technology of a panel of cancer markers can greatly increase the accuracy of disease diagnosis. Herein, we briefly fabricate a high-throughput micro-immunoassay based on the electrospun polystyrene (PS substrates to improve detection sensitivity. The immunoassay was evaluated by analyzing three different cancer biomarkers (AFP, CEA, VEGF. For AFP, CEA, VEGF immunofluorescence assay, the LOD of assay conducted on electrospun PS substrates before or after plasma and the conventional PS substrates were 0.42, 0.10, 1.12 ng/mL, 0.57, 0.09, 1.24 ng/mL, and 159.75, 26.19, 385.59 pg/mL, respectively (P < 0.05. Due to the high porosity and large surface area-to-volume ratio which is the foremost merit of nanostructures, and the plasma treatment which make the hydrophobic PS nanofibers hydropholic, the nanofibers substrates showed sufficient retention of immunoassay functionality and high potential for capture molecules immobilization. Consequently, the immunofluorescence assay conducted on electrospun PS substrates could significantly enhance the sensitivity and limits of detection.

  14. Functionalization of single-walled carbon nanotubes with protein by click chemistry as sensing platform for sensitized electrochemical immunoassay

    International Nuclear Information System (INIS)

    Qi Honglan; Ling Chen; Huang Ru; Qiu Xiaoying; Shangguan Li; Gao Qiang; Zhang Chengxiao

    2012-01-01

    Highlights: ► Single-walled carbon nanotubes were functionalized with protein by click chemistry. ► The SWNTs conjugated with protein showed excellent dispersion in water and kept good bioacitvity. ► A competitive electrochemical immunoassay for the determination of anti-IgG was developed with high sensitivity and good stability. - Abstract: The application of the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition to the functionalization of single-walled carbon nanotubes (SWNTs) with the protein and the use of the artificial SWNTs as a sensing platform for sensitive immunoassay were reported. Covalent functionalization of azide decorated SWNTs with alkyne modified protein was firstly accomplished by the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition. FT-IR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron micrograph were used to characterize the protein-functionalized SWNTs. It was found that the SWNTs conjugated with the proteins showed excellent dispersion in water and kept good bioacitivity when immunoglobulin (IgG) and horseradish peroxidase (HRP) were chosen as model proteins. As a proof-of-concept, IgG-functionalized SWNTs were immobilized onto the surface of a glassy carbon electrode by simple casting method as immunosensing platform and a sensitive competitive electrochemical immunoassay was developed for the determination of anti-immunoglobulin (anti-IgG) using HRP as enzyme label. The fabrication of the immunosensor were characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the redox probe [Fe(CN) 6 ] 3−/4− . The SWNTs as immobilization platform showed better sensitizing effect, a detection limit of 30 pg mL −1 (S/N = 3) was obtained for anti-IgG. The proposed strategy provided a stable immobilization method and sensitized recognition platform for analytes. This work demonstrated that the click coupling of SWNTs with protein was an effective

  15. Implementation of a rapid HIT immunoassay at a university hospital - Retrospective analysis of HIT laboratory orders in patients with thrombocytopenia.

    Science.gov (United States)

    Black, Anne; Heimerl, Susanne; Oertli, Linnéa; Wilczek, Wolf; Greinacher, Andreas; Spannagl, Michael; Herr, Wolfgang; Hart, Christina

    2017-10-01

    Heparin-induced thrombocytopenia (HIT) is a rare cause of thrombocytopenia and a potentially life-threatening adverse drug reaction. Clinical overdiagnosis of HIT results in costly laboratory tests and anticoagulation. Criteria and algorithms for diagnosis are established, but their translation into clinical practice is still challenging. In a retrospective approach we studied all HIT related laboratory test requests within four years and evaluated data before (1st period, 24month) and after (2nd period, 24month) replacing particle gel immunoassay (PaGIA) and enzyme-linked immunosorbent assay (ELISA) by a chemiluminescent immunoassay (CLIA). HIT was confirmed by heparin-induced platelet activation (HIPA) test. Clinical pretest probability for HIT using an implemented simplified 4Ts score and platelet count were evaluated. Costs for laboratory tests and alternative anticoagulation were calculated. In 1850 patients with suspected HIT, 2327 laboratory orders were performed. In 87.2% of these orders an intermediate/high simplified 4Ts score was found. Thrombocytopenia was present in 87.1%. After replacing PaGIA and ELISA by CLIA the number of immunological and functional laboratory tests was reduced by 38.2%. The number of positive HIT immunoassays declined from 22.6% to 6.0%, while the number of positive HIPA tests among positive immunological tests increased by 19%. Altogether, acute HIT was confirmed in 59 patients. A decline in the use of alternative anticoagulants was observed in the 2nd period. Our study shows that in a university hospital setting HIT is well-known, but diagnosis requires a precise laboratory confirmation. Replacing PaGIA and ELISA by CLIA did not influence laboratory order behavior but results in reduced overall costs for laboratory diagnostics and alternative anticoagulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W.

    Science.gov (United States)

    Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

    2018-01-01

    Using compound W (a 3,3'-diiodothyronine sulfate [T 2 S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Photosensitive particles (donor beads) coated with compound W or T 2 S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

  17. Evaluation of three gentamicin serum assay techniques

    International Nuclear Information System (INIS)

    Matzke, G.R.; Gwizdala, C.; Wery, J.; Ferry, D.; Starnes, R.

    1982-01-01

    This investigation was designed to compare the enzyme-modified immunoassay (Syva--EMIT) with a radioimmunoassay (New England Nuclear--RIA) and the radiometric assay (Johnston--BACTEC) to determine the optimal assay for use in our aminoglycoside dosing service. The serum concentration determinations obtained via the three assay methods were analyzed by linear regression analysis. Significant positive correlations were noted between the three assay techniques (p less than 0.005) during both sample collection phases. The coefficients of determination for EMIT vs BACTEC and RIA vs BACTEC were 0.73 and 0.83 during phase 1, respectively, and 0.65 and 0.68 during phase 2, respectively. The slope of the regression lines also varied markedly during the two phases; 0.49 and 0.42 for EMIT and for RIA vs BACTEC, respectively, during phase 1 compound with 1.12 and 0.77, respectively, during phase 2. The differences noted in these relationships during phase 1 and 2 may be related to the alteration of the pH of the control sera utilized in the BACTEC assay. In contrast, RIA vs EMIT regression analysis indicated that existence of a highly significant relationship (p less than 0.0005 and r2 . 0.90). The EMIT technique was the easiest and most accurate for determination of serum gentamicin concentrations, whereas the BACTEC method was judged unacceptable for clinical use

  18. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  19. An immunoassay for dibutyl phthalate based on direct hapten linkage to the polystyrene surface of microtiter plates.

    Directory of Open Access Journals (Sweden)

    Chenxi Wei

    Full Text Available BACKGROUND: Dibutyl phthalate (DBP is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. METHODOLOGY/PRINCIPAL FINDINGS: A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC. Compared with conjugate coated format (IC(50=106 ng/mL, the direct hapten coated format (IC(50=14.6 ng/mL improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. CONCLUSIONS/SIGNIFICANCE: The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed

  20. Advances in enzyme bioelectrochemistry

    Directory of Open Access Journals (Sweden)

    ANDRESSA R. PEREIRA

    Full Text Available ABSTRACT Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

  1. Rational design of Raman-labeled nanoparticles for a dual-modality, light scattering immunoassay on a polystyrene substrate.

    Science.gov (United States)

    Israelsen, Nathan D; Wooley, Donald; Hanson, Cynthia; Vargis, Elizabeth

    2016-01-01

    Surface-enhanced Raman scattering (SERS) is a powerful light scattering technique that can be used for sensitive immunoassay development and cell labeling. A major obstacle to using SERS is the complexity of fabricating SERS probes since they require nanoscale characterization and optical uniformity. The light scattering response of SERS probes may also be modulated by the substrate used for SERS analysis. A typical SERS substrate such as quartz can be expensive. Polystyrene is a cheaper substrate option but can decrease the SERS response due to interfering Raman emission peaks and high background fluorescence. The goal of this research is to develop an optimized process for fabricating Raman-labeled nanoparticles for a SERS-based immunoassay on a polystyrene substrate. We have developed a method for fabricating SERS nanoparticle probes for use in a light scattering immunoassay on a polystyrene substrate. The light scattering profile of both spherical gold nanoparticle and gold nanorod SERS probes were characterized using Raman spectroscopy and optical absorbance spectroscopy. The effects of substrate interference and autofluorescence were reduced by selecting a Raman reporter with a strong light scattering response in a spectral region where interfering substrate emission peaks are minimized. Both spherical gold nanoparticles and gold nanorods SERS probes used in the immunoassay were detected at labeling concentrations in the low pM range. This analytical sensitivity falls within the typical dynamic range for direct labeling of cell-surface biomarkers using SERS probes. SERS nanoparticle probes were fabricated to produce a strong light scattering signal despite substrate interference. The optical extinction and inelastic light scattering of these probes was detected by optical absorbance spectroscopy and Raman spectroscopy, respectively. This immunoassay demonstrates the feasibility of analyzing strongly enhanced Raman signals on polystyrene, which is an

  2. Hapten design and indirect competitive immunoassay for parathion determination: Correlation with molecular modeling and principal component analysis

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yihua [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Jin Maojun [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Gui Wenjun [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Cheng Jingli [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Guo Yirong [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China); Zhu Guonian [Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029 (China)]. E-mail: zhugn@zju.edu.cn

    2007-05-22

    A novel procedure for parathion hapten design is described. The optimal antigen for parathion was selected after molecular modeling studies of six types of potentially immunizing haptens with the aim to identify the best mimicking target analyte. Heterologous competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed after screening a battery of competitors as coating antigens. The relationship between the heterology degree of the competitor and the resulting immunoassay detectability was investigated according to the electronic similarities of the competitor haptens and the target analyte. Molecular modeling and principal component analysis were performed to understand the electronic distribution and steric parameters of the haptens at their minimum energetic levels. The results suggested that the competitors should have a high heterology to produce assays with good detectability values. An indirect competitive ELISA was finally selected for further investigation. The immunoassay had an IC{sub 50} value of 4.79 ng mL{sup -1} and a limit of detection of 0.31 ng mL{sup -1}. There was little or no cross-reactivity to similar compounds tested except for the insecticide parathion-methyl, which showed a cross-reactivity of 7.8%.

  3. Effect of forage type, harvesting time and exogenous enzyme application on degradation characteristics measured using in vitro technique

    DEFF Research Database (Denmark)

    Moharrery, Ali; Hvelplund, Torben; Weisbjerg, Martin Riis

    2009-01-01

    Five forage species cut at different harvest times were studied for their degradation characteristics using in vitro digestibility technique. The forage species were two grasses and three legumes growing in two seasons (spring growth and second re-growth). Grass and legume forages were harvested...... at three harvesting times being early (E), middle (M) and late (L), both during the spring growth and the second re-growth. The grasses included perennial ryegrass (Lolium perenne), and festulolium (XFestulolium), and the legumes included white clover (Trifolium repens), red clover (Trifolium pratense......) and neutral detergent fibre (aNDFom) degradation profiles were fitted to an exponential equation. The fractional rate of degradation (c) of DM or aNDFom did vary among the forage species and was highest for the legumes. The potential degradability ranged from 580 to 870 g/kg for DM and from 380 to 900 g...

  4. Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin

    Directory of Open Access Journals (Sweden)

    Sultana Akter

    2017-09-01

    Full Text Available Nodularin (NOD is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E,6(E-dienoic acid monoclonal antibody (Mab. One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR. It was expressed in Escherichia coli as a single chain antibody fragment (scFv fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.

  5. Micromotor-based lab-on-chip immunoassays

    Science.gov (United States)

    García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

    2013-01-01

    Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic

  6. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

    Science.gov (United States)

    Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

    2016-12-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Enzymes: principles and biotechnological applications

    Science.gov (United States)

    Robinson, Peter K.

    2015-01-01

    Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. PMID:26504249

  8. Novel redox species polyaniline derivative-Au/Pt as sensing platform for label-free electrochemical immunoassay of carbohydrate antigen 199

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Liyuan; Shan, Jiao; Feng, Feng; Ma, Zhanfang, E-mail: mazhanfang@cnu.edu.cn

    2016-03-10

    A novel electrochemical redox-active nanocomposite was synthesized by a one-pot method using N,N′-diphenyl-p-phenylediamine as monomer, and HAuCl{sub 4} and K{sub 2}PtCl{sub 4} as co-oxidizing agents. The as-prepared poly(N,N′-diphenyl-p-phenylediamine)-Au/Pt exhibited admirable electrochemical redox activity at 0.15 V, excellent H{sub 2}O{sub 2} electrocatalytic ability and favorable electron transfer ability. Based on these, the evaluation of the composite as sensing substrate for label-free electrochemical immunosensing to the sensitive detection of carbohydrate antigen 199 was described. This technique proved to be a prospective detection tool with a wide liner range from 0.001 U mL{sup −1} to 40 U mL{sup −1}, and a low detection limit of 2.3 × 10{sup −4} U mL{sup −1} (S/N = 3). In addition, this method was used for the analysis of human serum sample, and good agreement was obtained between the values and those of enzyme-linked immunosorbent assay, implying the potential application in clinical research. Importantly, the strategy of the present substrate could be extended to other polymer-based nanocomposites such as polypyrrole derivatives or polythiophene derivatives, and this could be of great significance for the electrochemical immunoassay. - Highlights: • A novel electrochemical redox composite PPPD-Au/Pt was synthesized by one-pot method. • PPPD-Au/Pt was used as sensing substrate for label-free electrochemical immunosensor. • The immunosensor showed wide detection range and ultralow detection limit for the detection of CA199.

  9. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to immunoassay for the detection of cTnI and decreased matrix related interferences, thus resulting in a lower number of falsely elevated cTnI-values. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  10. Tumor specific lung cancer diagnostics with multiplexed FRET immunoassays

    Science.gov (United States)

    Geißler, D.; Hill, D.; Löhmannsröben, H.-G.; Thomas, E.; Lavigne, A.; Darbouret, B.; Bois, E.; Charbonnière, L. J.; Ziessel, R. F.; Hildebrandt, N.

    2010-02-01

    An optical multiplexed homogeneous (liquid phase) immunoassay based on FRET from a terbium complex to eight different fluorescent dyes is presented. We achieved highly sensitive parallel detection of four different lung cancer specific tumor markers (CEA, NSE, SCC and CYFRA21-1) within a single assay and show a proof-of-principle for 5- fold multiplexing. The method is well suited for fast and low-cost miniaturized point-of-care testing as well as for highthroughput screening in a broad range of in-vitro diagnostic applications.

  11. Linkage of biomolecules to solid phases for immunoassay

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    Topics covered by this lecture include a brief review of the principal methods of linkage of biomolecules to solid phase matrices. Copies of the key self explanatory slides are presented as figures together with reprints of two publications by the author dealing with a preferred chemistry for the covalent linkage of antibodies to hydroxyl and amino functional groups and the effects of changes in solid phase matrix and antibody coupling chemistry on the performance of a typical excess reagent immunoassay for thyroid stimulating hormone

  12. On-Chip Immunoassay for Determination of Urinary Albumin

    OpenAIRE

    Laiwattanapaisal, Wanida; Songjaroen, Temsiri; Maturos, Thitima; Lomas, Tanom; Sappat, Assawapong; Tuantranont, Adisorn

    2009-01-01

    An immunoassay performed on a portable microfluidic device was evaluated for the determination of urinary albumin. An increase in absorbance at 500 nm resulting from immunoagglutination was monitored directly on the poly(dimethylsiloxane) (PDMS) microchip using a portable miniature fibre-optic spectrometer. A calibration curve was linear up to 10 mg L–1 (r2 = 0.993), with a detection limit of 0.81 mg L–1 (S/N = 3). The proposed system showed good precision, with relative standard deviations (...

  13. Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay

    OpenAIRE

    Ruzzenente, Orazio; Salvagno, Gian Luca; Gelati, Matteo; Lippi, Giuseppe

    2016-01-01

    Objectives: This study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT) immunoassay. Design and methods: This analytical evaluation encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. Results: The LOB, LOD and functional sensitivity were 0.0010 ng/mL, 0.0016 ng/mL and ...

  14. Enzyme-MOF (metal-organic framework) composites.

    Science.gov (United States)

    Lian, Xizhen; Fang, Yu; Joseph, Elizabeth; Wang, Qi; Li, Jialuo; Banerjee, Sayan; Lollar, Christina; Wang, Xuan; Zhou, Hong-Cai

    2017-06-06

    The ex vivo application of enzymes in various processes, especially via enzyme immobilization techniques, has been extensively studied in recent years in order to enhance the recyclability of enzymes, to minimize enzyme contamination in the product, and to explore novel horizons for enzymes in biomedical applications. Possessing remarkable amenability in structural design of the frameworks as well as almost unparalelled surface tunability, Metal-Organic Frameworks (MOFs) have been gaining popularity as candidates for enzyme immobilization platforms. Many MOF-enzyme composites have achieved unprecedented results, far outperforming free enzymes in many aspects. This review summarizes recent developments of MOF-enzyme composites with special emphasis on preparative techniques and the synergistic effects of enzymes and MOFs. The applications of MOF-enzyme composites, primarily in transferation, catalysis and sensing, are presented as well. The enhancement of enzymatic activity of the composites over free enzymes in biologically incompatible conditions is emphasized in many cases.

  15. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    Science.gov (United States)

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  16. Design of a surface plasmon resonance immunoassay for therapeutic drug monitoring of amikacin.

    Science.gov (United States)

    Losoya-Leal, Adrian; Estevez, M-Carmen; Martínez-Chapa, Sergio O; Lechuga, Laura M

    2015-08-15

    The therapeutic drug monitoring (TDM) of pharmaceutical drugs with narrow therapeutic ranges is of great importance in the clinical setting. It provides useful information towards the enhancement of drug therapies, aiding in dosage control and toxicity risk management. Amikacin is an aminoglycoside antibiotic commonly used in neonatal therapies that is indicated for TDM due to the toxicity risks inherent in its use. Current techniques for TDM such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are costly, time consuming, and cannot be performed at the site of action. Over the last decades, surface plasmon resonance (SPR) biosensors have become increasingly popular in clinical diagnostics due to their ability to detect biomolecular interactions in real-time. We present an SPR-based competitive immunoassay for the detection of the antibiotic amikacin, suitable for TDM in both adults and neonates. We have obtained high specificity and sensitivity levels with an IC50 value of 1.4ng/mL and a limit of detection of 0.13ng/mL, which comfortably comply with the drug's therapeutic range. Simple dilution of serum can therefore be sufficient to analyze low-volume real samples from neonates, increasing the potential of the methodology for TDM. Compared to current TDM conventional methods, this SPR-based immunoassay can provide advantages such as simplicity, potential portability, and label-free measurements with the possibility of high throughput. This work is the foundation towards the development of an integrated, simple use, highly sensitive, fast, and point-of-care sensing platform for the opportune TDM of antibiotics and other drugs in a clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. A Portable Immunoassay Platform for Multiplexed Detection of Biotoxins in Clinical and Environmental Samples

    Energy Technology Data Exchange (ETDEWEB)

    Koh, Chung-Yan [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Piccini, Matthew Ernest [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Cepheid, Sunnyvale, CA (United States); Schaff, Ulrich Y. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandstone Diagnostics, Livermore, CA (United States); Stanker, Larry H. [US Dept. of Agriculture, Albany, CA (United States). Western Regional Research Center, Foodborne Contaminants Research Unit; Cheng, Luisa W. [US Dept. of Agriculture, Albany, CA (United States). Western Regional Research Center, Foodborne Contaminants Research Unit; Ravichandran, Easwaran [Univ. of Massachusetts, Dartmouth, MA (United States); Singh, Bal-Ram [Univ. of Massachusetts, Dartmouth, MA (United States); Sommer, Greg J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandstone Diagnostics, Livermore, CA (United States); Singh, Anup K. [Sandia National Lab. (SNL-CA), Livermore, CA (United States)

    2015-01-01

    Multiple cases of attempted bioterrorism events using biotoxins have highlighted the urgent need for tools capable of rapid screening of suspect samples in the field (e.g., mailroom and public events). We present a portable microfluidic device capable of analyzing environmental (e.g., white powder), food (e.g., milk) and clinical (e.g., blood) samples for multiplexed detection of biotoxins. The device is rapid (<15-30 min sample-to-answer), sensitive (< 0.08 pg/mL detection limit for botulinum toxin), multiplexed (up to 64 parallel assays) and capable of analyzing small volume samples (< 20 μL total sample input). The immunoassay approach (SpinDx) is based on binding of toxins in a sample to antibody-laden capture particles followed by sedimentation of particles through a density-media in a microfluidic disk and quantification using a laser-induced fluorescence detector. A direct, blinded comparison with a gold standard ELISA revealed a 5-fold more sensitive detection limit for botulinum toxin while requiring 250-fold less sample volume and a 30 minute assay time with a near unity correlation. A key advantage of the technique is its compatibility with a variety of sample matrices with no additional sample preparation required. Ultrasensitive quantification has been demonstrated from direct analysis of multiple clinical, environmental and food samples, including white powder, whole blood, saliva, salad dressing, whole milk, peanut butter, half and half, honey, and canned meat. We believe that this device can met an urgent need in screening both potentially exposed people as well as suspicious samples in mail-rooms, airports, public sporting venues and emergency rooms. The general-purpose immunodiagnostics device can also find applications in screening of infectious and systemic diseases or serve as a lab device for conducting rapid immunoassays.

  18. Production of monoclonal antibodies for sandwich immunoassay detection of Pacific ciguatoxins.

    Science.gov (United States)

    Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

    2010-10-01

    Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin. Copyright 2009 Elsevier Ltd. All rights reserved.

  19. Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

    Science.gov (United States)

    Xie, Zhen-ming; Yu, Lin; Fang, Li-sha

    2015-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 μg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

  20. Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues.

    Science.gov (United States)

    Zhang, Hongyan; Zhang, Yan; Wang, Shuo

    2008-08-20

    Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip-stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2-horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1-5 microg L(-1) or 1-10 microg L(-1) in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high-performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle (flow-through or dip-stick) or milk (only dip-stick).

  1. Direct analysis of airborne mite allergen (Der f1) in the residential atmosphere by chemifluorescent immunoassay using bioaerosol sampler.

    Science.gov (United States)

    Miyajima, Kumiko; Suzuki, Yurika; Miki, Daisuke; Arai, Moeka; Arakawa, Takahiro; Shimomura, Hiroji; Shiba, Kiyoko; Mitsubayashi, Kohji

    2014-06-01

    Dermatophagoides farinae allergen (Der f1) is one of the most important indoor allergens associated with allergic diseases in humans. Mite allergen Der f1 is usually associated with particles of high molecular weight; thus, Der f1 is generally present in settled dust. However, a small quantity of Der f1 can be aerosolized and become an airborne component. Until now, a reliable method of detecting airborne Der f1 has not been developed. The aim of this study was to develop a fiber-optic chemifluorescent immunoassay for the detection of airborne Der f1. In this method, the Der f1 concentration measured on the basis of the intensity of fluorescence amplified by an enzymatic reaction between the labeled enzyme by a detection antibody and a fluorescent substrate. The measured Der f1 concentration was in the range from 0.49 to 250 ng/ml and a similar range was found by enzyme-linked immunosorbent assay (ELISA). This method was proved to be highly sensitive to Der f1 compared with other airborne allergens. For the implementation of airborne allergen measurement in a residential environment, a bioaerosol sampler was constructed. The airborne allergen generated by a nebulizer was conveyed to a newly sampler we developed for collecting airborne Der f1. The sampler was composed of polymethyl methacrylate (PMMA) cells for gas/liquid phases and some porous membranes which were sandwiched in between the two phases. Der f1 in air was collected by the sampler and measured using the fiber-optic immunoassay system. The concentration of Der f1 in aerosolized standards was in the range from 0.125 to 2.0 mg/m(3) and the collection rate of the device was approximately 0.2%. © 2013 Elsevier B.V. All rights reserved.

  2. Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay Diagnóstico da hepatite por vírus C em doadores de sangue brasileiros, usando a reação de transcrição reversa e a reação em cadeia da polimerase "nested": comparação com os ensaios imunoenzimáticos e imunoblot recombinante

    Directory of Open Access Journals (Sweden)

    Neiva S. L. GONÇALES

    2000-10-01

    Full Text Available Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT serum levels generally prevents the transmission of hepatitis C virus (HCV by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT-nested polymerase chain reaction (PCR to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2 results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value. ALT results were expressed as the ALT quotient (qALT, calculated dividing the ALT value of the samples by the maximum normal value (53UI/l for the method. Donors (n=178 were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT or = 3, ALT>1, group C (11 and group E (EIA or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%, especially when the c22.3 band was detected.Na prevenção da transmissão de Hepatite por Vírus C (HCV em transfusões de hemocomponentes, utiliza-se rotineiramente, como testes de triagem de doadores de sangue, ensaios que detectam anticorpos anti-HCV e dosagens da enzima alanina-aminotransferase (ALT. O presente estudo tem como objetivo principal avaliar a eficiência do ensaio imunoenzimático de segunda geração (EIA-2 como teste de triagem, na identificação de doadores de sangue potencialmente infectados, e portanto, capazes de transmitir hepatite C pelos hemocomponentes. Nós utilizamos o ensaio de transcri

  3. Redox-Magnetohydrodynamic Microfluidics Without Channels and Compatible with Electrochemical Detection Under Immunoassay Conditions

    Science.gov (United States)

    Weston, Melissa C.; Nash, Christena K.; Fritsch, Ingrid

    2010-01-01

    A unique capability of redox-magnetohydrodynamics (redox-MHD) for handling liquids on a small scale was demonstrated. A 1.2-μL solution plug was pumped from an injection site to a detector without the need for a channel to direct the flow. The redox pumping species did not interfere with enzymatic activity in a solution compatible with enzyme-linked immunoassays. Alkaline phosphatase (AP), a common enzyme label, converted p-aminophenyl phosphate (PAPP) to p-aminophenol (PAPR) in the presence of 2.5 mM Ru(NH3)6Cl2 and 2.5 mM Ru(NH3)6 Cl3, in 0.1 M Tris buffer (pH=9). A solution plug containing PAPP (no AP) was pumped through the surrounding solution containing AP (no PAPP), and the enzymatically-generated PAPR was easily detected and distinguishable electrochemically from the pumping species with square wave voltammetry down to 0.1 mM concentrations. The test device consisted of a silicon chip containing individually-addressable microband electrodes, placed on a 0.5-T NdFeB permanent magnet with the field oriented perpendicular to the chip. A 8.0-mm wide × 15.5-mm long × 1.5-mm high volume of solution was contained by a poly(dimethylsiloxane) gasket and capped with a glass slide. A steady-state fluid velocity of ~30 μm/s was generated in a reinforcing flow configuration between oppositely polarized sets of pumping electrodes with ~2.1 μA. PMID:20681513

  4. Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees

    Directory of Open Access Journals (Sweden)

    Khaled DJELOUAH

    2015-01-01

    Full Text Available A direct tissue blot immunoassay (DTBIA technique has been compared with ELISA and PCR for detection of Xylella fastidiosa in olive trees from Apulia (southern Italy. Fresh cross-sections of young twigs and leaf petioles were printed onto nitrocellulose membranes and analyzed in the laboratory. Analyses of a first group of 61 samples gave similar efficiency for the three diagnostic techniques for detection the bacterium (24 positive and 36 negative samples, except for a single sample which was positive only with DTBIA and PCR. Similar results were obtained by separately analyzing suckers and twigs collected from different sectors of tree canopies of a second group of 20 olive trees (ten symptomatic and ten symptomless. In this second test the three diagnostic techniques confirmed the irregular distribution of the bacterium in the tree canopies and erratic detectability of the pathogen in the young suckers. It is therefore necessary to analyse composite samples per tree which should be prepared with twigs collected from different sides of the canopy. The efficiency comparable to ELISA and PCR, combined with the advantages of easier handling, speed and cost, make DTBIA a valid alternative to ELISA in large-scale surveys for occurrence of X. fastidiosa. Moreover, the printing of membranes directly in the field prevents infections spreading to Xylella-free areas, through movement of plant material with pathogen vectors for laboratory testing.

  5. AN ENVIRONMENTAL TECHNOLOGY VERIFICATION (ETV) TESTING OF THREE IMMUNOASSAY TEST KITS FOR ANTHRAX, BOTULINUM TOXIN AND RICIN

    Science.gov (United States)

    Immunoassay test kits are based on immunoassay methods, where specific antibodies are used to detect and measure the contaminants of interest. Immunoassay test kits rely on the reaction of a contaminant or antigen with a selective antibody to give a product that can be measures....

  6. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Mehrangiz Rajaii

    2015-01-01

    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

  7. Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food

    International Nuclear Information System (INIS)

    Meimaridou, Anastasia; Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios; Pulkrabova, Jana; Hajslova, Jana; Nielen, Michel W.F.

    2010-01-01

    Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC 50 of 0.3 μg L -1 using a monoclonal antibody (Mab22F12) against BaP, similar to the IC 50 of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are

  8. de novo computational enzyme design.

    Science.gov (United States)

    Zanghellini, Alexandre

    2014-10-01

    Recent advances in systems and synthetic biology as well as metabolic engineering are poised to transform industrial biotechnology by allowing us to design cell factories for the sustainable production of valuable fuels and chemicals. To deliver on their promises, such cell factories, as much as their brick-and-mortar counterparts, will require appropriate catalysts, especially for classes of reactions that are not known to be catalyzed by enzymes in natural organisms. A recently developed methodology, de novo computational enzyme design can be used to create enzymes catalyzing novel reactions. Here we review the different classes of chemical reactions for which active protein catalysts have been designed as well as the results of detailed biochemical and structural characterization studies. We also discuss how combining de novo computational enzyme design with more traditional protein engineering techniques can alleviate the shortcomings of state-of-the-art computational design techniques and create novel enzymes with catalytic proficiencies on par with natural enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Enzyme structure and interaction with inhibitors

    International Nuclear Information System (INIS)

    London, R.E.

    1983-01-01

    This article reviews some of the results of studies on the 13 C-labeled enzyme dihydrofolate reductase (DHFR). Nuclear magnetic resonance (NMR) techniques are used in combination with isotopic labeling to learn about the structure and dynamics of this enzyme. 13 C-labeling is used for the purpose of studying enzyme/substrate and enzyme/inhibitor interactions. A second set of studies with DHFR was designed to investigate the basis for the high affinity between the inhibitor methotrexate and DHFR. The label was placed on the inhibitor, rather than the enzyme

  10. Evaluation of envelope glycoprotein E(rns) of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus.

    Science.gov (United States)

    Vijayaraghavan, Balaje; Xia, Hongyan; Harimoorthy, Rajiv; Liu, Lihong; Belák, Sándor

    2012-11-01

    Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)

    2013-01-01

    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  12. Zymography methods for visualizing hydrolytic enzymes.

    Science.gov (United States)

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E; Opdenakker, Ghislain

    2013-03-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tissue sections with in situ zymography. In vivo zymography can pinpoint proteolytic activity to sites in an intact organism. Future development of novel substrate probes and improvement in detection and imaging methods will increase the applicability of zymography for (reverse) degradomics studies.

  13. Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.; Fillmore, Thomas L.; Sokoll, Lori J.; Kronewitter, Scott R.; Izmirlian, Grant; Shi, Tujin; Qian, Weijun; Leach, Robin; Thompson, Ian M.; Chan, Daniel W.; Smith, Richard D.; Kagan, Jacob; Srinivastava, Sudhir; Rodland, Karin D.; Camp, David G.

    2012-08-03

    Sandwich immunoassay is the standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention. Albeit highly sensitive, the development of a specific immunoassay is rather time-consuming and associated with extremely high cost due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissue and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and great multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed very good correlation (R2 values ranging from 0.90 to 0.99) in several independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, simultaneous measurement of free and total PSA and many other biomarkers can be performed in a single analysis using high-resolution liquid chromatographic separation coupled with SRM-MS.

  14. PIXE analysis of Zn enzymes

    International Nuclear Information System (INIS)

    Solis, C.; Oliver, A.; Andrade, E.; Ruvalcaba-Sil, J.L.; Romero, I.; Celis, H.

    1999-01-01

    Zinc is a necessary component in the action and structural stability of many enzymes. Some of them are well characterized, but in others, Zn stoichiometry and its association is not known. PIXE has been proven to be a suitable technique for analyzing metallic proteins embedded in electrophoresis gels. In this study, PIXE has been used to investigate the Zn content of enzymes that are known to carry Zn atoms. These include the carbonic anhydrase, an enzyme well characterized by other methods and the cytoplasmic pyrophosphatase of Rhodospirillum rubrum that is known to require Zn to be stable but not how many metal ions are involved or how they are bound to the enzyme. Native proteins have been purified by polyacrylamide gel electrophoresis and direct identification and quantification of Zn in the gel bands was performed with an external proton beam of 3.7 MeV energy

  15. Lateral Flow Immunoassays for Ebola Virus Disease Detection in Liberia.

    Science.gov (United States)

    Phan, Jill C; Pettitt, James; George, Josiah S; Fakoli, Lawrence S; Taweh, Fahn M; Bateman, Stacey L; Bennett, Richard S; Norris, Sarah L; Spinnler, David A; Pimentel, Guillermo; Sahr, Phillip K; Bolay, Fatorma K; Schoepp, Randal J

    2016-10-15

    Lateral flow immunoassays (LFIs) are point-of-care diagnostic assays that are designed for single use outside a formal laboratory, with in-home pregnancy tests the best-known example of these tests. Although the LFI has some limitations over more-complex immunoassay procedures, such as reduced sensitivity and the potential for false-positive results when using complex sample matrices, the assay has the benefits of a rapid time to result and ease of use. These benefits make it an attractive option for obtaining rapid results in an austere environment. In an outbreak of any magnitude, a field-based rapid diagnostic assay would allow proper patient transport and for safe burials to be conducted without the delay caused by transport of samples between remote villages and testing facilities. Use of such point-of-care instruments in the ongoing Ebola virus disease (EVD) outbreak in West Africa would have distinct advantages in control and prevention of local outbreaks, but proper understanding of the technology and interpretation of results are important. In this study, a LFI, originally developed by the Naval Medical Research Center for Ebola virus environmental testing, was evaluated for its ability to detect the virus in clinical samples in Liberia. Clinical blood and plasma samples and post mortem oral swabs submitted to the Liberian Institute for Biomedical Research, the National Public Health Reference Laboratory for EVD testing, were tested and compared to results of real-time reverse transcription-polymerase chain reaction (rRT-PCR), using assays targeting Ebola virus glycoprotein and nucleoprotein. The LFI findings correlated well with those of the real-time RT-PCR assays used as benchmarks. Rapid antigen-detection tests such as LFIs are attractive alternatives to traditional immunoassays but have reduced sensitivity and specificity, resulting in increases in false-positive and false-negative results. An understanding of the strengths, weaknesses, and

  16. Elevated Liver Enzymes

    Science.gov (United States)

    Symptoms Elevated liver enzymes By Mayo Clinic Staff Elevated liver enzymes may indicate inflammation or damage to cells in the liver. Inflamed or ... than normal amounts of certain chemicals, including liver enzymes, into the bloodstream, which can result in elevated ...

  17. Clinical development of placental malaria vaccines and immunoassays harmonization

    DEFF Research Database (Denmark)

    Chêne, Arnaud; Houard, Sophie; Nielsen, Morten A

    2016-01-01

    Placental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies...... that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently...... in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays...

  18. On-Chip Immunoassay for Determination of Urinary Albumin

    Directory of Open Access Journals (Sweden)

    Adisorn Tuantranont

    2009-12-01

    Full Text Available An immunoassay performed on a portable microfluidic device was evaluated for the determination of urinary albumin. An increase in absorbance at 500 nm resulting from immunoagglutination was monitored directly on the poly(dimethylsiloxane (PDMS microchip using a portable miniature fibre-optic spectrometer. A calibration curve was linear up to 10 mg L–1 (r2 = 0.993, with a detection limit of 0.81 mg L–1 (S/N = 3. The proposed system showed good precision, with relative standard deviations (RSDs of 5.1%, when evaluated with 10 mg L–1 albumin (n = 10. Determination of urinary albumin with the proposed system gave results highly similar to those determined by the conventional spectrophotometric method using immunoturbidimetric detection (r2 = 0.995; n = 15.

  19. Performance of immunoassay kits for site characterization and remediation

    International Nuclear Information System (INIS)

    Waters, L.C.; Palausky, A.; Counts, R.W.; Jenkins, R.A.

    1995-01-01

    The US Department of Energy (DOE) is supporting efforts to identify, validate and implement the use of effective, low-cost alternatives to currently used analytical methods for environmental management. As part of that program, we have evaluated the performances of a number of immunoassay (IA) kits with specificities for environmental contaminants of concern to the DOE. The studies were done in the laboratory using both spiked and field test samples. The analyte specificity and manufacturers of the kits evaluated were the following: mercury, BioNebraska; polychlorinated biphenyls (PCBs), EnSys and Millipore; petroleum fuel hydrocarbons, Millipore and Ohmicron; and polyaromatic hydrocarbons (PAHs), Ohmicron and Millipore. The kits were used in either a semiquantitative or quantitative format according to the preference of the manufacturers

  20. Streptavidin-functionalized capillary immune microreactor for highly efficient chemiluminescent immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Yang Zhanjun [State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); College of Chemistry and Engineering, Yangzhou University, 88 South University Avenue, Yangzhou 225002 (China); Zong Chen [State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); Ju Huangxian, E-mail: hxju@nju.edu.cn [State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093 (China); Yan Feng, E-mail: yanfeng2007@sohu.com [Jiangsu Institute of Cancer Prevention and Cure, Nanjing 210009 (China)

    2011-11-07

    Highlights: {yields} A novel capillary immune microreactor was proposed for highly efficient flow-through chemiluminescent immunoassay. {yields} The microreactor was prepared by functionalizing capillary inner wall with streptavidin for capture of biotinylated antibody. {yields} The proposed immunoassay method showed wide dynamic range, good reproducibility, stability and practicality. {yields} The microreactor was low-cost and disposable, and possessed several advantages over the conventional immunoreactors. - Abstract: A streptavidin functionalized capillary immune microreactor was designed for highly efficient flow-through chemiluminescent (CL) immunoassay. The functionalized capillary could be used as both a support for highly efficient immobilization of antibody and a flow cell for flow-through immunoassay. The functionalized inner wall and the capture process were characterized using scanning electron microscopy. Compared to conventional packed tube or thin-layer cell immunoreactor, the proposed microreactor showed remarkable properties such as lower cost, simpler fabrication, better practicality and wider dynamic range for fast CL immunoassay with good reproducibility and stability. Using {alpha}-fetoprotein as model analyte, the highly efficient CL flow-through immunoassay system showed a linear range of 3 orders of magnitude from 0.5 to 200 ng mL{sup -1} and a low detection limit of 0.1 ng mL{sup -1}. The capillary immune microreactor could make up the shortcoming of conventional CL immunoreactors and provided a promising alternative for highly efficient flow-injection immunoassay.

  1. Application of an immunoassay method to improve the diagnosis and control of African trypanosomosis. Proceedings of the workshop

    International Nuclear Information System (INIS)

    1997-02-01

    The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture supports the introduction and use of nuclear and related techniques in developing countries through co-ordinated research programmes. A co-ordinated research programme (CRP) entitled ''Improving the diagnosis and control of trypanosomosis and other research programme (CRP) entitled ''Improving the diagnosis and control of trypanosomosis and other vector-borne diseases of African livestock using immunoassay methods'' was executed from 1987 to 1993 with funding from the Directorate General of Development Co-operation (DGIS) of the Netherlands. A total of 12 reports by the various Research Contract holders were compiled and published as a Technical Document (IAEA-TECDOC-707) together with conclusions, recommendations and reports by experts in the field. Refs, figs tabs

  2. Application of an immunoassay method to improve the diagnosis and control of African trypanosomosis. Proceedings of the workshop

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-02-01

    The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture supports the introduction and use of nuclear and related techniques in developing countries through co-ordinated research programmes. A co-ordinated research programme (CRP) entitled ``Improving the diagnosis and control of trypanosomosis and other research programme (CRP) entitled ``Improving the diagnosis and control of trypanosomosis and other vector-borne diseases of African livestock using immunoassay methods`` was executed from 1987 to 1993 with funding from the Directorate General of Development Co-operation (DGIS) of the Netherlands. A total of 12 reports by the various Research Contract holders were compiled and published as a Technical Document (IAEA-TECDOC-707) together with conclusions, recommendations and reports by experts in the field. Refs, figs tabs.

  3. Determination of telmisartan in human blood plasma: Part I: Immunoassay development

    NARCIS (Netherlands)

    Hempen, C.M.; Gläsle-Schwarz, Liane; Kunz, Ulrich; Karst, U.

    2006-01-01

    Telmisartan is an angiotensin II receptor antagonist and a known drug against high blood pressure. In this report, the development of a new and rapid analytical technique, an enzyme-linked immunosorbent assay (ELISA) for the determination of telmisartan in human blood plasma is described. The

  4. Aquaculture investigations with nuclear energy techniques

    International Nuclear Information System (INIS)

    Heredia Salazar, Brunilda

    1997-01-01

    The culture of aquatic organisms, especially that of fishes under controlled conditions, up to their harvesting, processing, commercialization and consumption, has been pointed out as an activity that produces a lot of benefits, among them: the obtention high proteic valued food, the incorporation to the economy of lands not usefull for agriculture activities, the increment of fishing resources, the recycling of organic matter produced in the units, the regional development, the generation of employment, technologies and foreign currencies. Several research areas are identified that can be developed, using the nuclear technologies, for example in the reproduction, nutrition, diagnose and control of illnesses, environmental monitoring and quality certification of products. In the concerning to the Venezuelan aquaculture, investigations are required that need to use those techniques. For example: 1) Production of autochthonous inductive agents, by means of radioinmunoenssay (RIA), to determine the gonadotropines coming from the hypophysis of fish cultivated with the purpose of gathering the glands in its best moment, to generate the final maturation and spawn in autochthonous species. 2) Genetic improvement of cultivated species through the knowledge of the genetic load of different lines and breeds found in the natural means, and to achieve its maintenance to solve inbreeding problems, in autochthonous species aswell in as in exotic ones, by the use of marking techniques (ADE, RFLA and microsatellite techniques). 3) Nutritional and feeding studies of species under commertial culture, especially on the effect of the aflatoxins in the inputs or the portions, substances that influence in a negative way the aquatic nutrition. In this case, competitive immunoassays of enzymes bounded (ELISA) and radioinmunoessays. 4) Illness diagnose, by means of the ELISA kit, specifically of the more common illness in fishes cultivated in the country [es

  5. Optical waveguide lightmode spectroscopy technique-based immunosensor development for aflatoxin B1 determination in spice paprika samples.

    Science.gov (United States)

    Majer-Baranyi, Krisztina; Zalán, Zsolt; Mörtl, Mária; Juracsek, Judit; Szendrő, István; Székács, András; Adányi, Nóra

    2016-11-15

    Optical waveguide lightmode spectroscopy (OWLS) technique has been applied to label-free detection of aflatoxin B1 in a competitive immunoassay format, with the aim to compare the analytical goodness of the developed OWLS immunosenor with HPLC and enzyme-linked immunosorbent assay (ELISA) methods for the detection of aflatoxin in spice paprika matrix. We have also assessed applicability of the QuEChERS method prior to ELISA measurements, and the results were compared to those obtained by traditional solvent extraction followed by immunoaffinity clean-up. The AFB1 content of sixty commercial spice paprika samples from different countries were measured with the developed and optimized OWLS immunosensor. Comparing the results from the indirect immunosensor to that obtained by HPLC or ELISA provided excellent correlation (with regression coefficients above 0.94) indicating that the competitive OWLS immunosensor has a potential for quick determination of aflatoxin B1 in paprika samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Ultrasensitive fluorescence immunoassay for detection of ochratoxin A using catalase-mediated fluorescence quenching of CdTe QDs

    Science.gov (United States)

    Huang, Xiaolin; Zhan, Shengnan; Xu, Hengyi; Meng, Xianwei; Xiong, Yonghua; Chen, Xiaoyuan

    2016-04-01

    Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to perform a dynamic linear detection of OTA ranging from 0.05 pg mL-1 to 10 pg mL-1. The half maximal inhibitory concentration was 0.53 pg mL-1 and the limit of detection was 0.05 pg mL-1. These values were approximately 283- and 300-folds lower than those of horseradish peroxidase (HRP)-based conventional ELISA, respectively. The reported method is accurate, highly reproducible, and specific against other mycotoxins in agricultural products as well. In summary, the developed fluorescence immunoassay based on H2O2-induced fluorescence quenching of CdTe QDs can be used for the rapid and highly sensitive detection of mycotoxins or haptens in food safety monitoring.Herein, for the first time we report an improved competitive fluorescent enzyme linked immunosorbent assay (ELISA) for the ultrasensitive detection of ochratoxin A (OTA) by using hydrogen peroxide (H2O2)-induced fluorescence quenching of mercaptopropionic acid-modified CdTe quantum dots (QDs). In this immunoassay, catalase (CAT) was labeled with OTA as a competitive antigen to connect the fluorescence signals of the QDs with the concentration of the target. Through the combinatorial use of H2O2-induced fluorescence quenching of CdTe QDs as a fluorescence signal output and the ultrahigh catalytic activity of CAT to H2O2, our proposed method could be used to

  7. Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays

    Science.gov (United States)

    Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

    2012-01-01

    Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

  8. Comparison of two enzyme-linked immunosorbent assays and one rapid immunoblot assay for detection of herpes simplex virus type 2-specific antibodies in serum

    NARCIS (Netherlands)

    Groen, J; Van Dijk, G; Niesters, H G; Van Der Meijden, W I; Osterhaus, A D

    The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull

  9. Evaluation of an enzyme-linked immunosorbent assay for serological surveillance of infection with Actinobacillus pleuropneumoniae serotype 5 in pig herds

    DEFF Research Database (Denmark)

    Klausen, Joan; Andresen, Lars Ole; Barfod, Kristen

    2002-01-01

    An indirect enzyme-linked immunoassay for serological surveillance of infection of pigs with Actinobacillus pleuropneumoniae (Ap) serotype 5 was developed. The antigen used was prepared from Ap serotype 5b strain L20. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis...

  10. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Rui [College of Life Science, Nanchang University, Nanchang, 330031 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua [State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China); Xiong, Yonghua, E-mail: yhxiongchen@163.com [College of Life Science, Nanchang University, Nanchang, 330031 (China); State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047 (China)

    2016-12-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H{sub 2}O{sub 2}) and H{sub 2}O{sub 2}-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H{sub 2}O{sub 2} and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H{sub 2}O{sub 2}, as well as the incubation time between H{sub 2}O{sub 2} and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10{sup 2} CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10{sup 3} CFU/mL to 1.18 × 10{sup 6} CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. - Highlights: • A novel fluorescence immunoassay was developed for the ultrasensitive detection of E. coli O157:H7. • This detection was achieved through the combination of the high bioactivity of CAT and H{sub 2}O{sub 2}-sensitive QDs. • The activity of CAT to H{sub 2}O{sub 2} is 1000 folds higher than that of the HRP

  11. A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots

    International Nuclear Information System (INIS)

    Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

    2016-01-01

    Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H_2O_2) and H_2O_2-sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H_2O_2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H_2O_2, as well as the incubation time between H_2O_2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10"2 CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10"3 CFU/mL to 1.18 × 10"6 CFU/mL were in the range of 65.88%–105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. - Highlights: • A novel fluorescence immunoassay was developed for the ultrasensitive detection of E. coli O157:H7. • This detection was achieved through the combination of the high bioactivity of CAT and H_2O_2-sensitive QDs. • The activity of CAT to H_2O_2 is 1000 folds higher than that of the HRP to tetramethylbenzidine. • The limit of detection of the proposed method could

  12. Immunoassay of paralytic shellfish toxins by moving magnetic particles in a stationary liquid-phase lab-on-a-chip.

    Science.gov (United States)

    Kim, Myoung-Ho; Choi, Suk-Jung

    2015-04-15

    In this study, we devised a stationary liquid-phase lab-on-a-chip (SLP LOC), which was operated by moving solid-phase magnetic particles in the stationary liquid phase. The SLP LOC consisted of a sample chamber to which a sample and reactants were added, a detection chamber containing enzyme substrate solution, and a narrow channel connecting the two chambers and filled with buffer. As a model system, competitive immunoassays of saxitoxin (STX), a paralytic shellfish toxin, were conducted in the SLP LOC using protein G-coupled magnetic particles (G-MPs) as the solid phase. Anti-STX antibodies, STX-horseradish peroxidase conjugate, G-MPs, and a STX sample were added to the sample chamber and reacted by shaking. While liquids were in the stationary state, G-MPs were transported from the sample chamber to the detection chamber by moving a magnet below the LOC. After incubation to allow the enzymatic reaction to occur, the absorbance of the detection chamber solution was found to be reciprocally related to the STX concentration of the sample. Thus, the SLP LOC may represent a novel, simple format for point-of-care testing applications of enzyme-linked immunosorbent assays by eliminating complicated liquid handling steps. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Comparative performance of electrochemiluminescence immunoassay and EIA for HIV screening in a multiethnic region of China.

    Directory of Open Access Journals (Sweden)

    Xiaohui Bi

    Full Text Available The recent approval of 4th generation HIV tests has forced many laboratories to decide whether to shift from 3rd to these tests. There are limited published studies on the comparative evaluation of these two different assays. We compare the performance of fourth-generation electrochemiluminescence immunoassay (ChIA and third-generation enzyme linked immunosorbent assay (EIA for human immunodeficiency virus (HIV screening and gauge whether the shift from EIA to ChIA could be better in a multiethnic region of China.We identified a large number of routine specimens (345,492 using two different assays from Jan 2008 to Aug 2011 in a teaching hospital with high sample throughput. Of the 344,596 specimens with interpretable HIV test results, 526(0.23% of 228,761 using EIA and 303(0.26% of 115,835 using ChIA were HIV-1 positive. The false-positive rate of EIA was lower than that of ChIA [0.03% vs. 0.08%, odds ratio 0.33 (95% confidence interval 0.24, 0.45]. The positive predictive value (PPV of EIA (89.6% was significantly higher than that of ChIA (76.1% (<0.001, reflecting the difference between the two assays. The clinical sensitivities of two assays in this study were 99.64% for EIA and 99.88% for ChIA.Caution is needed before shifting from 3rd to 4th generation HIV tests. Since none of these tests are perfect, different geographic and ethnic area probably require different considerations with regard to HIV testing methods, taking into account the local conditions.

  14. Comparative performance of electrochemiluminescence immunoassay and EIA for HIV screening in a multiethnic region of China.

    Science.gov (United States)

    Bi, Xiaohui; Ning, Hongxia; Wang, Tingting; Li, Dongdong; Liu, Yongming; Yang, Tingfu; Yu, Jiansheng; Tao, Chuanmin

    2012-01-01

    The recent approval of 4th generation HIV tests has forced many laboratories to decide whether to shift from 3rd to these tests. There are limited published studies on the comparative evaluation of these two different assays. We compare the performance of fourth-generation electrochemiluminescence immunoassay (ChIA) and third-generation enzyme linked immunosorbent assay (EIA) for human immunodeficiency virus (HIV) screening and gauge whether the shift from EIA to ChIA could be better in a multiethnic region of China. We identified a large number of routine specimens (345,492) using two different assays from Jan 2008 to Aug 2011 in a teaching hospital with high sample throughput. Of the 344,596 specimens with interpretable HIV test results, 526(0.23%) of 228,761 using EIA and 303(0.26%) of 115,835 using ChIA were HIV-1 positive. The false-positive rate of EIA was lower than that of ChIA [0.03% vs. 0.08%, odds ratio 0.33 (95% confidence interval 0.24, 0.45)]. The positive predictive value (PPV) of EIA (89.6%) was significantly higher than that of ChIA (76.1%) (<0.001), reflecting the difference between the two assays. The clinical sensitivities of two assays in this study were 99.64% for EIA and 99.88% for ChIA. Caution is needed before shifting from 3rd to 4th generation HIV tests. Since none of these tests are perfect, different geographic and ethnic area probably require different considerations with regard to HIV testing methods, taking into account the local conditions.

  15. Multiantigen Print Immunoassay for Comparison of Diagnostic Antigens for Taenia solium Cysticercosis and Taeniasis▿

    Science.gov (United States)

    Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N.; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E.; Garcia, Hector H.; Gilman, Robert H.; Tsang, Victor C. W.; Wilkins, Patricia P.

    2010-01-01

    One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases. PMID:19906893

  16. Multiantigen print immunoassay for comparison of diagnostic antigens for Taenia solium cysticercosis and taeniasis.

    Science.gov (United States)

    Handali, Sukwan; Klarman, Molly; Gaspard, Amanda N; Noh, John; Lee, Yeuk-Mui; Rodriguez, Silvia; Gonzalez, Armando E; Garcia, Hector H; Gilman, Robert H; Tsang, Victor C W; Wilkins, Patricia P

    2010-01-01

    One of the best-characterized tests for the diagnosis of neurocysticercosis is the enzyme-linked immunoelectrotransfer blot assay, developed at the CDC, which uses lentil lectin-purified glycoproteins (LLGPs) extracted from Taenia solium cysticerci. The purification of the LLGP antigens has been difficult to standardize, and the polyacrylamide gel system used for the immunoblot assay is not easily transferable to other laboratories. In this study, we developed a multiantigen printing immunoassay (MAPIA) to compare the performance of multiple recombinant Taenia solium proteins with the potential for the detection of cysticercosis and taeniasis. We prepared MAPIA strips using six cysticercosis and two taeniasis diagnostic proteins and compared the performance of the proteins with sera collected from defined cysticercosis and taeniasis cases. Of the six cysticercosis antigens, rT24H performed well in detecting cases with two or more viable cysts in the brain (sensitivity and specificity, 97% and 99.4%, respectively); the use of a combination of cysticercosis antigens did not improve the sensitivity of the test and decreased the specificity. None of the antigens could differentiate the different clinical presentations of cysticercosis. Both of the taeniasis antigens (rES33 and rES38) had the same sensitivity of 99.4% and specificities of 93.9% and 94.5%, respectively. Some cross-reactivity against rES33 and rES38 was found, especially with sera from cases infected with Schistosoma mansoni. We conclude that MAPIA is a simple and effective tool that may be used to compare antibody responses to different cysticercosis and taeniasis antigens and, in this case, may be useful for the rapid detection of T. solium cases.

  17. Control of (pre-analytical aspects in immunoassay measurements of metabolic hormones in rodents

    Directory of Open Access Journals (Sweden)

    Maximilian Bielohuby

    2018-04-01

    Full Text Available The measurement of circulating hormones by immunoassay remains a cornerstone in preclinical endocrine research. For scientists conducting and interpreting immunoassay measurements of rodent samples, the paramount aim usually is to obtain reliable and meaningful measurement data in order to draw conclusions on biological processes. However, the biological variability between samples is not the only variable affecting the readout of an immunoassay measurement and a considerable amount of unwanted or unintended variability can be quickly introduced during the pre-analytical and analytical phase. This review aims to increase the awareness for the factors ‘pre-analytical’ and ‘analytical’ variability particularly in the context of immunoassay measurement of circulating metabolic hormones in rodent samples. In addition, guidance is provided how to gain control over these variables and how to avoid common pitfalls associated with sample collection, processing, storage and measurement. Furthermore, recommendations are given on how to perform a basic validation of novel single and multiplex immunoassays for the measurement of metabolic hormones in rodents. Finally, practical examples from immunoassay measurements of plasma insulin in mice address the factors ‘sampling site and inhalation anesthesia’ as frequent sources of introducing an unwanted variability during the pre-analytical phase. The knowledge about the influence of both types of variability on the immunoassay measurement of circulating hormones as well as strategies to control these variables are crucial, on the one hand, for planning and realization of metabolic rodent studies and, on the other hand, for the generation and interpretation of meaningful immunoassay data from rodent samples.

  18. A study using an isotope probe comparing immunoassay with serology in detection of Brucella Abortus antibody

    International Nuclear Information System (INIS)

    Devlin, J.G.; Redington, F.; Stephenson, M.

    1986-01-01

    We report a comparison of radio-immunoassay with conventional serology in the detection of brucella abortus antibody from three laboratories. Overall agreement by Chi squared analysis is 5%. There are significant differences between laboratories and a significant number of sero negative suspect sera (from 20% - 60%) were positive by ratio-immunoassay test. We suspect that conventional serology under-reports the incidence of antibody to brucella abortus. (author)

  19. Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay

    Directory of Open Access Journals (Sweden)

    Orazio Ruzzenente

    2016-12-01

    Full Text Available Objectives: This study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT immunoassay. Design and methods: This analytical evaluation encompassed the calculation of the limit of blank (LOB, limit of detection (LOD, functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. Results: The LOB, LOD and functional sensitivity were 0.0010 ng/mL, 0.0016 ng/mL and 0.008 ng/mL, respectively. The total analytical imprecision was found to be 2.1% and the linearity was excellent (r=1.00 in the range of concentrations between 0.006–75.5 ng/mL. The correlation coefficient with Vidas BRAHMS PCT was 0.995 and the equation of the Passing and Bablok regression analysis was [Lumipulse G BRAHMS PCT]=0.76×[Vidas BRAHMS PCT]+0.04. The mean overall bias of Lumipulse G BRAHMS PCT versus Vidas BRAHMS PCT was −3.03 ng/mL (95% confidence interval [CI]: −4.32 to −1.74 ng/mL, whereas the mean bias in samples with PCT concentration between 0–10 ng/mL was −0.49 ng/mL (95% CI: −0.77 to −0.24 ng/mL. The diagnostic agreement was 100% at 0.5 ng/mL, 97% at 2.0 ng/mL and 95% at 10 ng/mL, respectively. Conclusions: These results attest that Lumipulse G BRAHMS PCT exhibits excellent analytical performance, among the best of the methods currently available on the diagnostic market. However, the significant bias compared to the Vidas BRAHMS PCT suggests that the methods cannot be used interchangeably. Keywords: Sepsis, Infection, Procalcitonin, Immunoassay

  20. Catch and measure-mass spectrometry-based immunoassays in biomarker research.

    Science.gov (United States)

    Weiß, Frederik; van den Berg, Bart H J; Planatscher, Hannes; Pynn, Christopher J; Joos, Thomas O; Poetz, Oliver

    2014-05-01

    Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

  1. Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.

    Science.gov (United States)

    Wang, Ling; Zhang, Junxian; Bai, Haili; Li, Xuan; Lv, Pintian; Guo, Ailing

    2014-07-01

    In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43%. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC · HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764%. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity.

  2. Data mining strategies to improve multiplex microbead immunoassay tolerance in a mouse model of infectious diseases.

    Directory of Open Access Journals (Sweden)

    Akshay Mani

    Full Text Available Multiplex methodologies, especially those with high-throughput capabilities generate large volumes of data. Accumulation of such data (e.g., genomics, proteomics, metabolomics etc. is fast becoming more common and thus requires the development and implementation of effective data mining strategies designed for biological and clinical applications. Multiplex microbead immunoassay (MMIA, on xMAP or MagPix platform (Luminex, which is amenable to automation, offers a major advantage over conventional methods such as Western blot or ELISA, for increasing the efficiencies in serodiagnosis of infectious diseases. MMIA allows detection of antibodies and/or antigens efficiently for a wide range of infectious agents simultaneously in host blood samples, in one reaction vessel. In the process, MMIA generates large volumes of data. In this report we demonstrate the application of data mining tools on how the inherent large volume data can improve the assay tolerance (measured in terms of sensitivity and specificity by analysis of experimental data accumulated over a span of two years. The combination of prior knowledge with machine learning tools provides an efficient approach to improve the diagnostic power of the assay in a continuous basis. Furthermore, this study provides an in-depth knowledge base to study pathological trends of infectious agents in mouse colonies on a multivariate scale. Data mining techniques using serodetection of infections in mice, developed in this study, can be used as a general model for more complex applications in epidemiology and clinical translational research.

  3. Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation.

    Science.gov (United States)

    Dittmer, W U; de Kievit, P; Prins, M W J; Vissers, J L M; Mersch, M E C; Martens, M F W C

    2008-09-30

    A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions as well as the bound/free separation are entirely controlled by magnetic forces induced by electromagnets above and below the sensor chip. During the assay, particles conjugated with tracer antibodies are actuated through the sample for target capture, and rapidly brought to the sensor surface where they bind to immobilized capture antibodies. Weakly or unbound labels are removed with a magnetic force oriented away from the GMR sensor surface. For the measurement of parathyroid hormone (PTH), a detection limit in the 10 pM range is obtained with a total assay time of 15 min when 300 nm particles are used. The same sensitivity can be achieved in 5 min when 500 nm particles are used. If 500 nm particles are employed in a 15-minute assay, then 0.8 pM of PTH is detectable. The low sample volume, high analytical performance and high speed of the test coupled with the compact GMR biosensor make the system especially suitable for sensitive testing outside of laboratory environments.

  4. Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow.

    Science.gov (United States)

    Taylor, Angela E; Keevil, Brian; Huhtaniemi, Ilpo T

    2015-08-01

    The recent onslaught of mass spectrometry (MS) to measurements of steroid hormones, including demands that they should be the only acceptable method, has confused clinicians and scientists who have relied for more than 40 years on a variety of immunoassay (IA) methods in steroid hormone measurements. There is little doubt that MS methods with their superior specificity will be the future method of choice in many clinical and research applications of steroid hormone measurement. However, the majority of steroid measurements are currently, and will continue to be, carried out using various types of IAs for several reasons, including their technical ease, cost and availability of commercial reagents. Speedy replacement of all IAs with MS is an unrealistic and unnecessary goal, because the availability of MS measurements is limited by cost, need of expensive equipment, technical demands and lack of commercial applications. Furthermore, IAs have multiple well-known advantages that vindicate their continuing use. The purpose of this article is to elucidate the advantages and limitations of the MS and IA techniques from two angles, i.e. promotion of MS and defence of IA. The purpose of the text is to give the reader an unbiased view about the current state and future trends of steroid analysis and to help him/her choose the correct assay method to serve his/her diagnostic and research needs. © 2015 European Society of Endocrinology.

  5. Application of a nitrocellulose immunoassay for quantitation of proteins secreted in cultured media

    International Nuclear Information System (INIS)

    LaDuca, F.M.; Dang, C.V.; Bell, W.R.

    1986-01-01

    A macro immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglas. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125 I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cmp) and protein concentration was found. Intra- and inter-test reproducibility was excellent. By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally define medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer

  6. Thermometric enzyme linked immunosorbent assay: TELISA.

    Science.gov (United States)

    Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

    1977-08-11

    A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

  7. The microassay on a card: A rugged, portable immunoassay

    Science.gov (United States)

    Kidwell, David

    1991-01-01

    The Microassay on a Card (MAC) is a portable, hand-held, non-instrumental immunoassay that can test for the presence of a wide variety of substances in the environment. The MAC is a simple device to use. A drop of test solution is placed on one side of the card and within five minutes a color is developed on the other side in proportion to the amount of substance in the test solution, with sensitivity approaching 10 ng/ml. The MAC is self-contained and self-timed; no reagents or timing is necessary. The MAC may be configured with multiple wells to provide simultaneous testing for multiple species. As envisioned, the MAC will be employed first as an on-site screen for drugs of abuse in urine or saliva. If the MAC can be used as a screen of saliva for drugs of abuse, it could be applied to driving while intoxicated, use of drugs on the job, or testing of the identity of seized materials. With appropriate modifications, the MAC also could be used to test for environmental toxins or pollutants.

  8. Basic and clinical investigation of T3 immunoassay kit

    International Nuclear Information System (INIS)

    Konishi, Junji; Nakajima, Akiko; Morita, Rikushi; Endo, Keigo; Ikekubo, Katsuji

    1976-01-01

    T 3 immunoassay kit was investigated basically and clinically. A good result was obtained at the prescribed incubation temperature and for 16 hours of incubation time. Moreover, it was thought to be possible that incubation time could be shortened to 1 - 4 hours at 37 0 C. Specificity of antibody was good. Recovery of added T 3 was 100+-5 (S.D.) % on an average and parallel of dilution curve of high T 3 serum was also good. Variation coefficient of accuracy of this kit was 1.5 - 2.1 % and that of reproducibility was 1.3 - 6.6 %. Mild hemolysis did not affect measurement value. Serum T 3 level in normals, untreated patients with Basedow's disease and patients with primary hypothyroidism was 142+-21 ng/100 ml, 452+-156 ng/100 ml and 67+-17 ng/100 ml, respectively. Serum T 3 level in patients with Hashimoto's disease was distributed to a wide extent, but that of patients with goiter and simple goiter ranged within normal range. On the other side, serum T 3 level of normal pregnant woman was high and that of patients with anorexia nervosa showed low level. From the above mentioned results, it was concluded that this kit was simple in method and good in sensitivity, specificity and reproducibility and it was also useful for clinical applications. (M. Tsunoda)

  9. Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development.

    Science.gov (United States)

    Fox, M; Gray, G; Kavanagh, K; Lewis, C; Doyle, S

    2004-02-01

    Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC)(50): 4-5 microg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 microg/ml free gliotoxin (30 microM) and helvolic acid (17 microM), respectively, inhibited antibody binding to cognate toxin-BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.

  10. Graphene-based chemiluminescence resonance energy transfer for homogeneous immunoassay.

    Science.gov (United States)

    Lee, Joon Seok; Joung, Hyou-Arm; Kim, Min-Gon; Park, Chan Beum

    2012-04-24

    We report on chemiluminescence resonance energy transfer (CRET) between graphene nanosheets and chemiluminescent donors. In contrast to fluorescence resonance energy transfer, CRET occurs via nonradiative dipole-dipole transfer of energy from a chemiluminescent donor to a suitable acceptor molecule without an external excitation source. We designed a graphene-based CRET platform for homogeneous immunoassay of C-reactive protein (CRP), a key marker for human inflammation and cardiovascular diseases, using a luminol/hydrogen peroxide chemiluminescence (CL) reaction catalyzed by horseradish peroxidase. According to our results, anti-CRP antibody conjugated to graphene nanosheets enabled the capture of CRP at the concentration above 1.6 ng mL(-1). In the CRET platform, graphene played a key role as an energy acceptor, which was more efficient than graphene oxide, while luminol served as a donor to graphene, triggering the CRET phenomenon between luminol and graphene. The graphene-based CRET platform was successfully applied to the detection of CRP in human serum samples in the range observed during acute inflammatory stress.

  11. Significance of isolated reactive treponemal chemiluminescence immunoassay results.

    Science.gov (United States)

    Hunter, Michael G; Robertson, Peter W; Post, Jeffrey J

    2013-05-01

    Isolated reactive serum treponemal chemiluminescence immunoassay (CIA) specimens cause clinical uncertainty. Sera were screened by CIA, and reactive samples underwent reflex testing with rapid plasma reagin (RPR), Treponema pallidum particle agglutination (TPPA), and fluorescent treponemal antibody absorption (FTA Abs) assays. Samples reactive only on the CIA were deemed "isolated" reactive CIA samples. We undertook detailed review of a subset of subjects with isolated reactive CIA specimens. Of 28 261 specimens, 1171 (4.1%) were reactive on CIA, of which 133 (11.3%) had isolated CIA reactivity. Most subjects (66 of 82 [80.5%]) with isolated reactive CIA specimens were from high-prevalence populations. We found evidence of CIA, TPPA, and FTA Abs seroreversion. The median chemiluminescent signal-to-cutoff ratio was similar for isolated reactive CIA sera and sera that were reactive on either FTA Abs or TPPA assays (2.19 vs 2.32; P = .15) but lower than for sera reactive on both FTA Abs and TPPA assays (12.37; P < .001) or for sera reactive on RPR assays (25.53; P < .001). A total of 11 of 20 patients (55%) with an isolated reactive CIA specimen who underwent medical record review had previous or subsequent evidence of syphilis infection. Isolated reactive CIA specimens may represent true T. pallidum infection and may be found after seroreversion of traditional treponemal assays.

  12. Serum gastrin: interests and limitations of radio-immunoassay

    International Nuclear Information System (INIS)

    Rougier, P.; Linhart, N.; Bok, B.

    1980-01-01

    Radio-immunoassay of serum gastrin may now be carried out in all laboratories of radio-immunology. Comparison of two commercial kits A: Schwartz-Mann and B: CEA-SORIN according to criteria of specificity, sensitivity and reproducibility within and between systems, shows that they both permit the detection of pathological hypergastrinemias (Zollinger Ellison and atropic gastritis). Both kits have an identical intrasystem coefficient of variation (10 p. cent and 7 p. cent fort A, 5 p. cent and 8 p. cent for B) on the other hand, the inter-system coefficient of variation is better for kit B (22.4 p. cent and 37 p. cent for kit A, and 11.5 p. cent and 14.2 p. cent for kit B). The normal values for each kit are quite different: 97 . 64 pg.ml -1 for A and 51 . 23 pg.ml -1 for B preventing one from comparing estimations carried out with two different kits [fr

  13. Establishment of carcinoembryonic antigen working standard for immunoassay

    International Nuclear Information System (INIS)

    Xu Ligen; Sun Youxiang; Jiao Yan

    2001-01-01

    The author is to prepare the working standard of carcinoembryonic antigen (CEA) for immunoassay and determine its potency. CEA solution of 320 μg/L was prepared from purified CEA solution of 4.6 mg/L and 1% human albumin solution buffered with 50 mmol/L sodium phosphate, pH7.4. This solution was distributed in an aliquot of 0.5 mL (160 ng per ampoule) and lyophilized. The potency of CEA working standard, in terms of present standard of CEA RIA and IRMA kits made by Chinese manufacturers and in terms of 1st IRP CEA HUMAN 73/601 supplied by WHO, has been determined. Mean immunological potency of the working standard is 163 ng per ampoule with confident limit of 159-168 ng per ampoule at 95% probability level. Test of parallelism of dose-response curve for the working standard to that for 1st IRP CEA HUMAN 73/601 has been passed. CEA working standard is suitable to the kits standard for CEA radioimmunoassay and immunoradiometric assay

  14. Designing novel nano-immunoassays: antibody orientation versus sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Puertas, S; Moros, M; Fernandez-Pacheco, R; Ibarra, M R; Grazu, V; De la Fuente, J M, E-mail: vgrazu@unizar.e, E-mail: jmfuente@unizar.e [Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, Campus RIo Ebro, EdifIcio I-D, Mariano Esquillor, s/n, 50018 Zaragoza (Spain)

    2010-12-01

    There is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly sensitive biosensors. Their use as labels of biological recognition events and their detection by means of some magnetic method constitute a very promising strategy for quantitative high-sensitive lateral-flow assays. In this paper, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral-flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on MNPs allows more successful recognition of the hCG hormone. Although we have used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. It has huge potential as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc.

  15. Immobilized enzyme studies in a microscale bioreactor.

    Science.gov (United States)

    Jones, Francis; Forrest, Scott; Palmer, Jim; Lu, Zonghuan; Elmore, John; Elmore, Bill B

    2004-01-01

    Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.

  16. Magnetic techniques for the detection and determination of xenobiotics and cells in water

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Horská, Kateřina; Popisková, K.; Šafaříková, Miroslava

    2012-01-01

    Roč. 404, č. 4 (2012), s. 1257-1273 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GAP503/11/2263 Institutional support: RVO:67179843 Keywords : bioanalytical methods * enzymes * immunoassays/ELISA * organic compounds * water * preconcentration Subject RIV: CE - Biochemistry Impact factor: 3.659, year: 2012

  17. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  18. Comparison of multiplex reverse transcription-PCR-enzyme ...

    African Journals Online (AJOL)

    Comparison of multiplex reverse transcription-PCR-enzyme hybridization assay with immunofluorescence techniques for the detection of four viral respiratory pathogens in pediatric community acquired pneumonia.

  19. Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay.

    Science.gov (United States)

    Ruzzenente, Orazio; Salvagno, Gian Luca; Gelati, Matteo; Lippi, Giuseppe

    2016-12-01

    This study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT) immunoassay. This analytical evaluation encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. The LOB, LOD and functional sensitivity were 0.0010 ng/mL, 0.0016 ng/mL and 0.008 ng/mL, respectively. The total analytical imprecision was found to be 2.1% and the linearity was excellent (r=1.00) in the range of concentrations between 0.006-75.5 ng/mL. The correlation coefficient with Vidas BRAHMS PCT was 0.995 and the equation of the Passing and Bablok regression analysis was [Lumipulse G BRAHMS PCT]=0.76×[Vidas BRAHMS PCT]+0.04. The mean overall bias of Lumipulse G BRAHMS PCT versus Vidas BRAHMS PCT was -3.03 ng/mL (95% confidence interval [CI]: -4.32 to -1.74 ng/mL), whereas the mean bias in samples with PCT concentration between 0-10 ng/mL was -0.49 ng/mL (95% CI: -0.77 to -0.24 ng/mL). The diagnostic agreement was 100% at 0.5 ng/mL, 97% at 2.0 ng/mL and 95% at 10 ng/mL, respectively. These results attest that Lumipulse G BRAHMS PCT exhibits excellent analytical performance, among the best of the methods currently available on the diagnostic market. However, the significant bias compared to the Vidas BRAHMS PCT suggests that the methods cannot be used interchangeably.

  20. Magnetic Particle-Based Immunoassay of Phosphorylated p53 Using Protein-Cage Templated Lead Phosphate and Carbon Nanospheres for Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Aiqiong; Bao, Yuanwu; Ge, Xiaoxiao; Shin, Yongsoon; Du, Dan; Lin, Yuehe

    2012-11-20

    Phosphorylated p53 at serin 15 (phospho-p53-15) is a potential biomarker of Gamma-radiation exposure. In this paper, we described a new magnetic particles (MPs)-based electrochemical immunoassay of human phospho-p53-15 using carbon nanospheres (CNS) and protein-cage templated lead phosphate nanoparticles for signal amplification. Greatly enhanced sensitivity was achieved by three aspects: 1) The protein-cage nanoparticle (PCN) and p53-15 signal antibody (p53-15 Ab2) are linked to CNS (PCNof each apoferritin; 3) MPs capture a large amount of primary antibodies. Using apoferritin templated metallic phosphate instead of enzyme as label has the advantage of eliminating the addition of mediator or immunoreagents and thus makes the immunoassay system simpler. The subsequent stripping voltammetric analysis of the released lead ions were detected on a disposable screen printed electrode. The response current was proportional to the phospho-p53-15 concentration in the range of 0.02 to 20 ng mL-1 with detection limit of 0.01 ng mL-1. This method shows a good stability, reproducibility and recovery.

  1. Highly sensitive immunoassay based on E. coli with autodisplayed Z-domain

    International Nuclear Information System (INIS)

    Jose, Joachim; Park, Min; Pyun, Jae-Chul

    2010-01-01

    The Z-domain of protein A has been known to bind specifically to the F c region of antibodies (IgGs). In this work, the Z-domain of protein A was expressed on the outer membrane of Escherichia coli by using 'Autodisplay' technology as a fusion protein of autotransport domain. The E. coli with autodisplayed Z-domain was applied to the sandwich-type immunoassay as a solid-support of detection-antibodies against a target analyte. For the feasibility demonstration of the E. coli based immunoassay, C-reactive protein (CRP) assay was carried out by using E. coli with autodisplayed Z-domain. The limit of detection (LOD) and binding capacity of the E. coli based immunoassay were estimated to be far more sensitive than the conventional ELISA. Such a far higher sensitivity of E. coli based immunoassay than conventional ELISA was explained by the orientation control of immobilized antibodies and the mobility of E. coli in assay matrix. From the test results of 45 rheumatoid arthritis (RA) patients' serum and 15 healthy samples, a cut-off value was established to have optimal sensitivity and selectivity values for RA. The CRP test result of each individual sample was compared with ELISA which is the reference method for RA diagnosis. From this work, the E. coli with Z-domain was proved to be feasible for the medical diagnosis based on sandwich-type immunoassay.

  2. Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

    Science.gov (United States)

    Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

    2014-12-01

    Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

    Science.gov (United States)

    Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

    2001-09-01

    To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

  4. Floating Chip Mounting System Driven by Repulsive Force of Permanent Magnets for Multiple On-Site SPR Immunoassay Measurements

    Science.gov (United States)

    Horiuchi, Tsutomu; Tobita, Tatsuya; Miura, Toru; Iwasaki, Yuzuru; Seyama, Michiko; Inoue, Suzuyo; Takahashi, Jun-ichi; Haga, Tsuneyuki; Tamechika, Emi

    2012-01-01

    We have developed a measurement chip installation/removal mechanism for a surface plasmon resonance (SPR) immunoassay analysis instrument designed for frequent testing, which requires a rapid and easy technique for changing chips. The key components of the mechanism are refractive index matching gel coated on the rear of the SPR chip and a float that presses the chip down. The refractive index matching gel made it possible to optically couple the chip and the prism of the SPR instrument easily via elastic deformation with no air bubbles. The float has an autonomous attitude control function that keeps the chip parallel in relation to the SPR instrument by employing the repulsive force of permanent magnets between the float and a float guide located in the SPR instrument. This function is realized by balancing the upward elastic force of the gel and the downward force of the float, which experiences a leveling force from the float guide. This system makes it possible to start an SPR measurement immediately after chip installation and to remove the chip immediately after the measurement with a simple and easy method that does not require any fine adjustment. Our sensor chip, which we installed using this mounting system, successfully performed an immunoassay measurement on a model antigen (spiked human-IgG) in a model real sample (non-homogenized milk) that included many kinds of interfering foreign substances without any sample pre-treatment. The ease of the chip installation/removal operation and simple measurement procedure are suitable for frequent on-site agricultural, environmental and medical testing. PMID:23202030

  5. Enzymes for improved biomass conversion

    Science.gov (United States)

    Brunecky, Roman; Himmel, Michael E.

    2016-02-02

    Disclosed herein are enzymes and combinations of the enzymes useful for the hydrolysis of cellulose and the conversion of biomass. Methods of degrading cellulose and biomass using enzymes and cocktails of enzymes are also disclosed.

  6. An introduction to radioimmunoassay and related techniques. 3. rev. ed.

    International Nuclear Information System (INIS)

    Chard, T.

    1987-01-01

    This book is designed as an introductory text for the staff of clinical or research laboratories who conduct or intend to conduct radioimmunoassay and related immunoassay techniques, and will also be of great value to the clinicians who make use of such services. The emphasis is on general principles with practical illustrations rather than a detailed and encyclopaedic review of the literature. This 3rd revised edition places more emphasis on alternative labels (non-isotopic immunoassays), the use of labelled antibodies (immunometric assays), and the use of monoclonal antibodies. Both principles and practice are illustrated by diagrams and illustrations. (Auth.)

  7. Highly sensitive electrochemical immunoassay for human IgG using double-encoded magnetic redox-active nanoparticles

    International Nuclear Information System (INIS)

    Tang, D.; Tang, J.; Su, B.; Chen, H.; Chen, G.; Huang, J.

    2010-01-01

    A new sandwich-type electrochemical immunoassay was developed for the detection of human IgG using doubly-encoded and magnetic redox-active nanoparticles as recognition elements on the surface of a glassy carbon electrode modified with anti-IgG on nanogold particles. The recognition elements were synthesized by coating magnetic Fe3O4 nanoparticles with Prussian blue nanoparticles and then covered with peroxidase-labeled anti-IgG antibodies (POx-anti-IgG) on Prussian blue nanoparticles. The immunoelectrode displays very good electrochemical properties towards detection of IgG via using double-encoded magnetic redox-active nanoparticles as trace and hydrogen peroxide as enzyme substrate. Its limit of detection (10 pmol.L -1 ) is 10-fold better than that of using plain POx-anti-IgG secondary antibodies. The method was applied to the detection of IgG in serum samples, and an excellent correspondence with the reference values was found. (author)

  8. Negative interference by rheumatoid factor in alpha-fetoprotein chemiluminescent microparticle immunoassay.

    Science.gov (United States)

    Wang, Hui; Bi, Xiaohui; Xu, Lei; Li, Yirong

    2017-01-01

    Background Rheumatoid factor causes positive interference in multiple immunoassays. Recently, negative interference has also been found in immunoassays in the presence of rheumatoid factor. The chemiluminescent microparticle immunoassay is widely used to determine serum alpha-fetoprotein. However, it is not clear whether the presence of rheumatoid factor in the serum causes interference in the chemiluminescent microparticle immunoassay of alpha-fetoprotein. Methods Serum alpha-fetoprotein was determined using the ARCHITECT alpha-fetoprotein assay. The estimation of alpha-fetoprotein recovery was carried out in samples prepared by diluting high-concentration alpha-fetoprotein serum with rheumatoid factor-positive or rheumatoid factor-negative serum. Paramagnetic microparticles coated with hepatitis B surface antigen-anti-HBs complexes were used to remove rheumatoid factor from the serum. Results The average recovery of alpha-fetoprotein was 88.4% and 93.8% in the rheumatoid factor-positive and rheumatoid factor-negative serum samples, respectively. The recovery of alpha-fetoprotein was significantly lower in the rheumatoid factor-positive serum samples than in the rheumatoid factor-negative serum samples. In two of five rheumatoid factor-positive samples, a large difference was found (9.8%) between the average alpha-fetoprotein recoveries in the serially diluted and initial recoveries. Fourteen rheumatoid factor-positive serum samples were pretreated with hepatitis B surface antigen-anti-HBs complex-coated paramagnetic microparticles. The alpha-fetoprotein concentrations measured in the pretreated samples increased significantly. Conclusions It was concluded that the alpha-fetoprotein chemiluminescent microparticle immunoassay is susceptible to interference by rheumatoid factor, leading to significantly lower results. Eliminating the incidence of negative interference from rheumatoid factor should be an important goal for immunoassay providers. In the meantime

  9. BIOTIN INTERFERENCE WITH ROUTINE CLINICAL IMMUNOASSAYS: UNDERSTAND THE CAUSES AND MITIGATE THE RISKS.

    Science.gov (United States)

    Samarasinghe, Shanika; Meah, Farah; Singh, Vinita; Basit, Arshi; Emanuele, Nicholas; Emanuele, Mary Ann; Mazhari, Alaleh; Holmes, Earle W

    2017-08-01

    The objectives of this report are to review the mechanisms of biotin interference with streptavidin/biotin-based immunoassays, identify automated immunoassay systems vulnerable to biotin interference, describe how to estimate and minimize the risk of biotin interference in vulnerable assays, and review the literature pertaining to biotin interference in endocrine function tests. The data in the manufacturer's "Instructions for Use" for each of the methods utilized by seven immunoassay system were evaluated. We also conducted a systematic search of PubMed/MEDLINE for articles containing terms associated with biotin interference. Available original reports and case series were reviewed. Abstracts from recent scientific meetings were also identified and reviewed. The recent, marked, increase in the use of over-the-counter, high-dose biotin supplements has been accompanied by a steady increase in the number of reports of analytical interference by exogenous biotin in the immunoassays used to evaluate endocrine function. Since immunoassay methods of similar design are also used for the diagnosis and management of anemia, malignancies, autoimmune and infectious diseases, cardiac damage, etc., biotin-related analytical interference is a problem that touches every area of internal medicine. It is important for healthcare personnel to become more aware of immunoassay methods that are vulnerable to biotin interference and to consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases where a lab result does not correlate with the clinical scenario. FDA = U.S. Food & Drug Administration FT3 = free tri-iodothyronine FT4 = free thyroxine IFUs = instructions for use LH = luteinizing hormone PTH = parathyroid hormone SA/B = streptavidin/biotin TFT = thyroid function test TSH = thyroid-stimulating hormone.

  10. Development of an Ultrasensitive Immunoassay for Detecting Tartrazine

    OpenAIRE

    Li, Zhuokun; Song, Shanshan; Xu, Liguang; Kuang, Hua; Guo, Shidong; Xu, Chuanlai

    2013-01-01

    We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most spe...

  11. Immobilized enzymes and cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucke, C; Wiseman, A

    1981-04-04

    This article reviews the current state of the art of enzyme and cell immobilization and suggests advances which might be made during the 1980's. Current uses of immobilized enzymes include the use of glucoamylase in the production of glucose syrups from starch and glucose isomerase in the production of high fructose corn syrup. Possibilities for future uses of immobilized enzymes and cells include the utilization of whey and the production of ethanol.

  12. Profiling the orphan enzymes

    Science.gov (United States)

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  13. Activity assays and immunoassays for plasma Renin and prorenin: information provided and precautions necessary for accurate measurement

    DEFF Research Database (Denmark)

    Campbell, Duncan J; Nussberger, Juerg; Stowasser, Michael

    2009-01-01

    into focus the differences in information provided by activity assays and immunoassays for renin and prorenin measurement and has drawn attention to the need for precautions to ensure their accurate measurement. CONTENT: Renin activity assays and immunoassays provide related but different information...... provided by these assays and of the precautions necessary to ensure their accuracy....

  14. Serum, plasma, and dried blood spot high sensitivity C-reactive protein enzyme immunoassay for population research

    OpenAIRE

    Brindle, Eleanor; Fujita, Masako; Shofer, Jane; O’Connor, Kathleen A.

    2010-01-01

    C-reactive protein (CRP) is used as a biomarker of morbidity and mortality risk in studies of population health, and is essential to interpretation of several micronutrient biomarkers. There is thus need for a robust high sensitivity CRP (hsCRP) measurement method for large-scale, non-clinical studies. We developed an efficient, inexpensive assay suitable for quantifying CRP across the physiological range using any blood specimen type. The ELISA uses readily available monoclonal antibodies to...

  15. Comparison of enzyme immunoassays for antibodies to Haemophilus ducreyi in a community outbreak of chancroid in the United States.

    Science.gov (United States)

    Chen, C Y; Mertz, K J; Spinola, S M; Morse, S A

    1997-06-01

    The performance of two EIAs (adsorption EIA and lipooligosaccharide [LOS] EIA) that detect antibodies to Haemophilus ducreyi was evaluated with serum specimens obtained from 163 patients (96 with genital ulcer disease [GUD]). Paired serum specimens (initial and follow-up) were obtained from 52 of the GUD patients. By use of initial serum specimens from 82 GUD patients whose etiologic agents for their ulcers had been identified, the adsorption EIA had a sensitivity and specificity for chancroid of 53% and 71%, while the LOS EIA had a sensitivity and specificity of 48% and 89%, respectively. Sensitivity and specificity of the adsorption EIA increased to 78% and 84%, respectively, when the results of follow-up serum specimens were used to calculate optimal performance. The proportion of patients testing positive for H. ducreyi who had anti-H. ducreyi IgG antibodies, as determined by adsorption EIA, increased with the duration of infection, thus limiting the role of EIAs in the diagnosis of chancroid.

  16. Enzyme-linked immunoassay for plasma-free metanephrines in the biochemical diagnosis of phaeochromocytoma in adults is not ideal.

    LENUS (Irish Health Repository)

    2012-02-01

    Abstract Background: The aim of the study was to define the analytical and diagnostic performance of the Labor Diagnostica Nord (LDN) 2-Met plasma ELISA assay for fractionated plasma metanephrines in the biochemical diagnosis of phaeochromocytoma. Methods: The stated manufacturer\\'s performance characteristics were assessed. Clinical utility was evaluated against liquid chromatography tandem mass spectrometry (LC-MS\\/MS) using bias, sensitivity and specificity outcomes. Samples (n=73) were collected from patients in whom phaeochromocytoma had been excluded (n=60) based on low probability of disease, repeat negative testing for urinary fractionated catecholamines and metanephrines, lack of radiological and histological evidence of a tumour and from a group (n=13) in whom the tumour had been histologically confirmed. Blood collected into k(2)EDTA tubes was processed within 30 min. Separated plasma was aliquoted (x2) and frozen at -40 degrees C prior to analyses. One aliquot was analysed for plasma metanephrines using the LDN 2-Met ELISA and the other by LC-MS\\/MS. Results: The mean bias of -32% for normetanephrine (ELISA) when compared to the reference method (LC-MS\\/MS) makes under-diagnosis of phaeochromocytoma likely. The sensitivity of the assay (100%) was equal to the reference method, but specificity (88.3%) lower than the reference method (95%), making it less than optimum for the biochemical diagnosis of phaeochromocytoma. Conclusions: Plasma-free metanephrines as measured by Labor Diagnostica Nord (LDN) 2-Met ELISA do not display test characteristics that would support their introduction or continuation as part of a screening protocol for the biochemical detection of phaeochromocytoma unless the calibration problem identified is corrected and other more accurate and analytically specific methods remain unavailable.

  17. Evaluation of Virological Microparticle Enzyme Immunoassay According to the ISO 15189: Real-Life Experience in a University Hospital.

    Science.gov (United States)

    Lafontaine, Sophie; Prin-Mathieu, Christine; Velay, Aurélie; Agulles, Odette; Schvoerer, Evelyne; Jeulin, Hélène

    2016-01-01

    The International Standard ISO 15189 based on the ISO 9001:2008 emphasizes specific requirements for quality and ability of medical laboratories. The accreditation of medical laboratories according to ISO 15189 includes the validation of biological methods, which depends on collection of bibliographic data and experimental proofs. Moreover, these results must be compared to provider data sheets and independent scientific data. In the immunodiagnostic field, independent published data are deeply lacking. The aim of our work was to share experience of method validation for virological immune markers on the widely used Architect i2000sr. After risk analysis, intra- and inter-assay variability, and inter-sample contamination were evaluated for each method, and sensitivity was investigated for antigen detection tests. A comparison between the two Architect i2000sr available in our laboratory was also performed. All tested methods were consistent with the manufacturer data (from the data sheet). No inter-sample contamination was observed. Both devices are broadly equivalent and can be used indifferently or as a backup solution of the other. To our knowledge, those results are the first independent complete data on the reliability of the Architect i2000sr in real-life experience. These data are needed to the accreditation of our platform and potentially useful for the accreditation of other laboratories using the same equipment.

  18. Proximity hybridization-regulated catalytic DNA hairpin assembly for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles

    International Nuclear Information System (INIS)

    Zhou, Fuyi; Yao, Yao; Luo, Jianjun; Zhang, Xing; Zhang, Yu; Yin, Dengyang; Gao, Fenglei; Wang, Po

    2017-01-01

    Novel hybridization proximity-regulated catalytic DNA hairpin assembly strategy has been proposed for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles as signal label. The DNA template-synthesized Pd nanoparticles were characterized with atomic force microscopic and X-ray photoelectron spectroscopy. The highly efficient electrocatalysis by DNA template synthesized Pd nanoparticles for NaBH 4 oxidation produced an intense detection signal. The label-free electrochemical method achieved the detection of carcinoembryonic antigen (CEA) with a linear range from 10 −15 to 10 −11  g mL −1 and a detection limit of 0.43 × 10 −15  g mL −1 . Through introducing a supersandwich reaction to increase the DNA length, the electrochemical signal was further amplified, leading to a detection limit of 0.52 × 10 −16  g mL −1 . And it rendered satisfactory analytical performance for the determination of CEA in serum samples. Furthermore, it exhibited good reproducibility and stability; meanwhile, it also showed excellent specificity due to the specific recognition of antigen by antibody. Therefore, the DNA template synthesized Pd nanoparticles based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level. - Graphical abstract: A novel label-free and enzyme-free electrochemical immunoassay based on proximity hybridization-regulated catalytic DNA hairpin assemblies for recycling of the CEA. - Highlights: • A novel enzyme-free electrochemical immunosensor was developed for detection of CEA. • The signal amplification was based on catalytic DNA hairpin assembly and DNA-template-synthesized Pd nanoparticles. • The biosensor could detect CEA down to 0.52 × 10 −16  g mL −1 level with a dynamic range spanning 5 orders of magnitude.

  19. Enzyme-driven mechanisms in biocorrosion

    OpenAIRE

    Basséguy, Régine

    2007-01-01

    Objectives (abstract of presentation): Recent works carried out in our team concerning enzymes and biocorrosion are presented at the meeting. For aerobic conditions, the direct catalysis of the reduction of oxygen on steel by enzymes or porphyrin was proved and a local electrochemical analysis technique (SVET) was developed to visualize the localization of the catalysis. On anaerobic conditions, the influence of phosphate species and other weak acids on the water reduction on steel was shown....

  20. Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals

    International Nuclear Information System (INIS)

    Zhao Hongwei; Nan Tiegui; Tan Guiyu; Gao Wei; Cao Zhen; Sun Shuo; Li Zhaohu; Li, Qing X.; Wang Baomin

    2011-01-01

    Highlights: · Two highly sensitive immunoassays for determination of Cu(II) at sub ppb levels. · The heterologous competitive enzyme linked immunosorbent assay for heavy metals. · Haptenated protein directly conjugated with HRP can reduce the loss of HRP activity. - Abstract: Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)-ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)-EDTA-BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)-EDTA-BSA-horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator-protein complexes such as EDTA-BSA and EDTA-BSA-HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules.

  1. Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Hongwei [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822 (United States); Nan Tiegui; Tan Guiyu; Gao Wei; Cao Zhen; Sun Shuo; Li Zhaohu [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China); Li, Qing X., E-mail: qingl@hawaii.edu [Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822 (United States); Wang Baomin, E-mail: wbaomin@263.com [College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193 (China)

    2011-09-19

    Highlights: {center_dot} Two highly sensitive immunoassays for determination of Cu(II) at sub ppb levels. {center_dot} The heterologous competitive enzyme linked immunosorbent assay for heavy metals. {center_dot} Haptenated protein directly conjugated with HRP can reduce the loss of HRP activity. - Abstract: Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)-ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)-EDTA-BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)-EDTA-BSA-horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator-protein complexes such as EDTA-BSA and EDTA-BSA-HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules.

  2. Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: an anticipated analytical tool for food safety.

    Science.gov (United States)

    Hervás, Miriam; López, Miguel Angel; Escarpa, Alberto

    2009-10-27

    In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 microg L(-1) and EC(50) 0.079 microg L(-1) were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

  3. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  4. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  5. Targeted enzyme prodrug therapies.

    Science.gov (United States)

    Schellmann, N; Deckert, P M; Bachran, D; Fuchs, H; Bachran, C

    2010-09-01

    The cure of cancer is still a formidable challenge in medical science. Long-known modalities including surgery, chemotherapy and radiotherapy are successful in a number of cases; however, invasive, metastasized and inaccessible tumors still pose an unresolved and ongoing problem. Targeted therapies designed to locate, detect and specifically kill tumor cells have been developed in the past three decades as an alternative to treat troublesome cancers. Most of these therapies are either based on antibody-dependent cellular cytotoxicity, targeted delivery of cytotoxic drugs or tumor site-specific activation of prodrugs. The latter is a two-step procedure. In the first step, a selected enzyme is accumulated in the tumor by guiding the enzyme or its gene to the neoplastic cells. In the second step, a harmless prodrug is applied and specifically converted by this enzyme into a cytotoxic drug only at the tumor site. A number of targeting systems, enzymes and prodrugs were investigated and improved since the concept was first envisioned in 1974. This review presents a concise overview on the history and latest developments in targeted therapies for cancer treatment. We cover the relevant technologies such as antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) as well as related therapies such as clostridial- (CDEPT) and polymer-directed enzyme prodrug therapy (PDEPT) with emphasis on prodrug-converting enzymes, prodrugs and drugs.

  6. Enzymes in Fermented Fish.

    Science.gov (United States)

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  7. Enzyme Histochemistry for Functional Histology in Invertebrates.

    Science.gov (United States)

    Cima, Francesca

    2017-01-01

    In invertebrates, enzyme histochemistry has recently found a renaissance regarding its applications in morphology and ecology. Many enzyme activities are useful for the morphofunctional characterization of cells, as biomarkers of biological and pathologic processes, and as markers of the response to environmental stressors. Here, the adjustments to classic techniques, including the most common enzymes used for digestion, absorption, transport, and oxidation, as well as techniques for azo-coupling, metal salt substitution and oxidative coupling polymerization, are presented in detail for various terrestrial and aquatic invertebrates. This chapter also provides strategies to solve the problems regarding anesthesia, small body size, the presence of an exo- or endoskeleton and the search for the best fixative in relation to the internal fluid osmolarity. These techniques have the aim of obtaining good results for both the pre- and post-embedding labeling of specimens, tissue blocks, sections, and hemolymph smears using both light and transmission electron microscopy.

  8. Immunoassays in clinical chemistry (principles of immunoradiometric assays)

    International Nuclear Information System (INIS)

    Chapman, R.S.

    1998-01-01

    The use of antibodies as reagents in clinical chemistry for the quantitation of a wide range of analytes has now become widely established. Initially antibodies were employed in precipitation techniques, usually for the analysis of serum proteins, in solution or in the form of antibody containing gels, e.g. immunoprecipitation, immunodiffusion, and immunoelectrophoresis. Further developments have led to the highly sensitive techniques of radioimmunoassay and recently immunometric assay for the measurement of drugs, tumour markers and hormones. In general, those techniques without the addition of a label e.g. immunoprecipitation, immunodiffusion and immunoturbidimetry are the older techniques used for the measurement of serum proteins. These techniques are relatively insensitive, measuring at the g/L. level, and in the case of immunodiffusion are generally slow. Automation coupled with the development of chemistries to enhance precipitation has, however, reduced measurement times to minutes in modern laboratories. Nevertheless these methods have detection limits of the order of 1 g/L

  9. An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli

    International Nuclear Information System (INIS)

    Tang, J.C.T.; Li, S.H.

    1984-01-01

    Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

  10. Radio-immunoassays for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2)

    DEFF Research Database (Denmark)

    Orskov, C; Holst, J J

    1987-01-01

    Gene-sequencing studies have shown that the glucagon precursor contains two additional glucagon-like sequences, the so-called glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). We developed radio-immunoassays against synthetic peptides corresponding to these sequences. Antisera were raised in rabb...

  11. Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder

    NARCIS (Netherlands)

    Haasnoot, W.; Olieman, K.; Cazemier, G.; Verheijen, R.

    2001-01-01

    The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders.

  12. Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

    NARCIS (Netherlands)

    Haasnoot, W.; Cazemier, G.; Koets, M.; Amerongen, van A.

    2003-01-01

    The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted

  13. Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication

    Czech Academy of Sciences Publication Activity Database

    Valeková, Ivona; Kupcová Skalníková, Helena; Jarkovská, Karla; Motlík, Jan; Kovářová, Hana

    -, č. 1212 (2015), s. 39-63 ISSN 1940 -6029 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : multiplex immunoassays * antibody microarray * luminex xMAP Subject RIV: EB - Genetics ; Molecular Biology

  14. Serum sample containing endogenous antibodies interfering with multiple hormone immunoassays. Laboratory strategies to detect interference

    Directory of Open Access Journals (Sweden)

    Elena García-González

    2016-04-01

    Full Text Available Objectives: Endogenous antibodies (EA may interfere with immunoassays, causing erroneous results for hormone analyses. As (in most cases this interference arises from the assay format and most immunoassays, even from different manufacturers, are constructed in a similar way, it is possible for a single type of EA to interfere with different immunoassays. Here we describe the case of a patient whose serum sample contains EA that interfere several hormones tests. We also discuss the strategies deployed to detect interference. Subjects and methods: Over a period of four years, a 30-year-old man was subjected to a plethora of laboratory and imaging diagnostic procedures as a consequence of elevated hormone results, mainly of pituitary origin, which did not correlate with the overall clinical picture. Results: Once analytical interference was suspected, the best laboratory approaches to investigate it were sample reanalysis on an alternative platform and sample incubation with antibody blocking tubes. Construction of an in-house ‘nonsense’ sandwich assay was also a valuable strategy to confirm interference. In contrast, serial sample dilutions were of no value in our case, while polyethylene glycol (PEG precipitation gave inconclusive results, probably due to the use of inappropriate PEG concentrations for several of the tests assayed. Conclusions: Clinicians and laboratorians must be aware of the drawbacks of immunometric assays, and alert to the possibility of EA interference when results do not fit the clinical pattern. Keywords: Endogenous antibodies, Immunoassay, Interference, Pituitary hormones, Case report

  15. Automation on an Open-Access Platform of Alzheimer's Disease Biomarker Immunoassays.

    Science.gov (United States)

    Gille, Benjamin; Dedeene, Lieselot; Stoops, Erik; Demeyer, Leentje; Francois, Cindy; Lefever, Stefanie; De Schaepdryver, Maxim; Brix, Britta; Vandenberghe, Rik; Tournoy, Jos; Vanderstichele, Hugo; Poesen, Koen

    2018-04-01

    The lack of (inter-)laboratory standardization has hampered the application of universal cutoff values for Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers and their transfer to general clinical practice. The automation of the AD biomarker immunoassays is suggested to generate more robust results than using manual testing. Open-access platforms will facilitate the integration of automation for novel biomarkers, allowing the introduction of the protein profiling concept. A feasibility study was performed on an automated open-access platform of the commercial immunoassays for the 42-amino-acid isoform of amyloid-β (Aβ 1-42 ), Aβ 1-40 , and total tau in CSF. Automated Aβ 1-42 , Aβ 1-40 , and tau immunoassays were performed within predefined acceptance criteria for bias and imprecision. Similar accuracy was obtained for ready-to-use calibrators as for reconstituted lyophilized kit calibrators. When compared with the addition of a standard curve in each test run, the use of a master calibrator curve, determined before and applied to each batch analysis as the standard curve, yielded an acceptable overall bias of -2.6% and -0.9% for Aβ 1-42 and Aβ 1-40 , respectively, with an imprecision profile of 6.2% and 8.4%, respectively. Our findings show that transfer of commercial manual immunoassays to fully automated open-access platforms is feasible, as it performs according to universal acceptance criteria.

  16. IMMUNOASSAY METHOD FOR THE DETERMINATION OF PENTACHLOROPHENOL IN SOIL AND SEDIMENT

    Science.gov (United States)

    The journal article describes the use of a prototype immunoassay method for the determination of pentacholorphenol (PCP) in soil and sediment. PCP was used as a pesticide and wood preservative and is not currently available to the general public. The paper stresses the importan...

  17. Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

    Science.gov (United States)

    The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

  18. An Inexpensive, Fast and Sensitive Quantitative Lateral Flow Magneto-Immunoassay for Total Prostate Specific Antigen

    Directory of Open Access Journals (Sweden)

    Jacqueline M. Barnett

    2014-07-01

    Full Text Available We describe the detection characteristics of a device the Resonant Coil Magnetometer (RCM to quantify paramagnetic particles (PMPs in immunochromatographic (lateral flow assays. Lateral flow assays were developed using PMPs for the measurement of total prostate specific antigen (PSA in serum samples. A detection limit of 0.8 ng/mL was achieved for total PSA using the RCM and is at clinically significant concentrations. Comparison of data obtained in a pilot study from the analysis of serum samples with commercially available immunoassays shows good agreement. The development of a quantitative magneto-immunoassay in lateral flow format for total PSA suggests the potential of the RCM to operate with many immunoassay formats. The RCM has the potential to be modified to quantify multiple analytes in this format. This research shows promise for the development of an inexpensive device capable of quantifying multiple analytes at the point-of-care using a magneto-immunoassay in lateral flow format.

  19. A review of promising new immunoassay technology for monitoring forest herbicides

    Science.gov (United States)

    Charles K. McMahon

    1993-01-01

    Rising costs of classical instrumental methods of chemical analysis coupled with an increasing need for environmental monitoring has lead to the development of highly sensitive, low-cost immunochemical methods of analysis for the detection of environmental contaminants. These methods known simply as immunoassays are chemical assays which use antibodies as reagents. A...

  20. Optimized Lateral Flow Immunoassay Reader for the Detection of Infectious Diseases in Developing Countries.

    Science.gov (United States)

    Pilavaki, Evdokia; Demosthenous, Andreas

    2017-11-20

    Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassays can be used with great success for the detection of infectious diseases. However, for the quantification of their results an electronic reader is required. This paper presents an optimized handheld electronic reader for developing countries. It features a potentially low-cost, low-power, battery-operated device with no added optical accessories. The operation of this proof of concept device is based on measuring the reflected light from the lateral flow immunoassay and translating it into the concentration of the specific analyte of interest. Characterization of the surface of the lateral flow immunoassay has been performed in order to accurately model its response to the incident light. Ray trace simulations have been performed to optimize the system and achieve maximum sensitivity by placing all the components in optimum positions. A microcontroller enables all the signal processing to be performed on the device and a Bluetooth module allows transmission of the results wirelessly to a mobile phone app. Its performance has been validated using lateral flow immunoassays with influenza A nucleoprotein in the concentration range of 0.5 ng/mL to 200 ng/mL.

  1. Optimized Lateral Flow Immunoassay Reader for the Detection of Infectious Diseases in Developing Countries

    Directory of Open Access Journals (Sweden)

    Evdokia Pilavaki

    2017-11-01

    Full Text Available Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassays can be used with great success for the detection of infectious diseases. However, for the quantification of their results an electronic reader is required. This paper presents an optimized handheld electronic reader for developing countries. It features a potentially low-cost, low-power, battery-operated device with no added optical accessories. The operation of this proof of concept device is based on measuring the reflected light from the lateral flow immunoassay and translating it into the concentration of the specific analyte of interest. Characterization of the surface of the lateral flow immunoassay has been performed in order to accurately model its response to the incident light. Ray trace simulations have been performed to optimize the system and achieve maximum sensitivity by placing all the components in optimum positions. A microcontroller enables all the signal processing to be performed on the device and a Bluetooth module allows transmission of the results wirelessly to a mobile phone app. Its performance has been validated using lateral flow immunoassays with influenza A nucleoprotein in the concentration range of 0.5 ng/mL to 200 ng/mL.

  2. Pemanfaatan Antibodi Monoklonal dalam Immunoassay untuk Deteksi Baculovirus oryctes

    Directory of Open Access Journals (Sweden)

    Susamto Somowiyarjo

    2000-12-01

    Full Text Available The application of non-precoated Indirect Enzyme-Linked Immunosorbent Assay (ELISA and Dot Immunobinding Assay (DIBA employing monoclonal antibodies (MCA against Yogyakarta isolate of Baculovirus oryctes Huger. was described. The MCA-Bv-4 having subclass of IgG2a and titer in vitro of 10^4 - 10^5 proved to be useful antibody for virus detection. The great potential of I-ELISA using MCA-Bv-4 has been it's specificity being able to discriminate between healthy an virus-infected coconut beetle (Oryctes rhinoceros L.. The assay could also differentiate between B. oryctes and Monodon baculovirus, the pathogen of shrimp disease. The best tissue for preparing virus antigen from crude extract was found to be the midgut of the beetle. Head, thorax, abdomen and tibia were not suitable for preparing the test antigen. The crude extract of beetle showed high endogenous enzyme activity to the substrate of DIBA, which precluded the detection of B. oryctes using DIBA. The MCA-Bv-4 could be used to improve the monitoring of the virus to support the program of biological control of coconut beetle using B. oryctes.

  3. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays: applications, fundamentals, and optimization

    International Nuclear Information System (INIS)

    Jeremy Daniel Driskell

    2006-01-01

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  4. Surface-Enhanced Raman Scattering (SERS) for Detection in Immunoassays. Applications, fundamentals, and optimization

    Energy Technology Data Exchange (ETDEWEB)

    Driskell, Jeremy Daniel [Iowa State Univ., Ames, IA (United States)

    2006-08-09

    Immunoassays have been utilized for the detection of biological analytes for several decades. Many formats and detection strategies have been explored, each having unique advantages and disadvantages. More recently, surface-enhanced Raman scattering (SERS) has been introduced as a readout method for immunoassays, and has shown great potential to meet many key analytical figures of merit. This technology is in its infancy and this dissertation explores the diversity of this method as well as the mechanism responsible for surface enhancement. Approaches to reduce assay times are also investigated. Implementing the knowledge gained from these studies will lead to a more sensitive immunoassay requiring less time than its predecessors. This dissertation is organized into six sections. The first section includes a literature review of the previous work that led to this dissertation. A general overview of the different approaches to immunoassays is given, outlining the strengths and weaknesses of each. Included is a detailed review of binding kinetics, which is central for decreasing assay times. Next, the theoretical underpinnings of SERS is reviewed at its current level of understanding. Past work has argued that surface plasmon resonance (SPR) of the enhancing substrate influences the SERS signal; therefore, the SPR of the extrinsic Raman labels (ERLs) utilized in our SERS-based immunoassay is discussed. Four original research chapters follow the Introduction, each presented as separate manuscripts. Chapter 2 modifies a SERS-based immunoassay previously developed in our group, extending it to the low-level detection of viral pathogens and demonstrating its versatility in terms of analyte type, Chapter 3 investigates the influence of ERL size, material composition, and separation distance between the ERLs and capture substrate on the SERS signal. This chapter links SPR with SERS enhancement factors and is consistent with many of the results from theoretical treatments

  5. Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

    Energy Technology Data Exchange (ETDEWEB)

    Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri (Turkey); Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Mus Alparslan University Faculty of Sciences, Department of Moleculer Biology, 49250-Mus (Turkey); Özdemir, Hasan, E-mail: hozdemir@atauni.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey)

    2016-04-18

    Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC{sub 50} values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

  6. Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

    Science.gov (United States)

    Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

    2016-04-01

    Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

  7. Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

    International Nuclear Information System (INIS)

    Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

    2016-01-01

    Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC_5_0 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

  8. Immunoassays for the incretin hormones GIP and GLP-1

    DEFF Research Database (Denmark)

    Deacon, Carolyn F; Holst, Jens J

    2009-01-01

    The measurement of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), using immunologically based assays is made difficult by the fact that the processing of the precursor molecules gives rise to a number of different peptides which cross......-react with antisera raised against the two hormones. For GLP-1, the picture is further complicated because of the necessity to differentiate between the intestinal and pancreatic proglucagon products. Finally, once secreted, both incretins are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4) to generate....... The use of highly specific assays using well-characterised antisera and careful sample handling is therefore required for a reliable determination of incretin hormone concentrations....

  9. Enzymic lactose hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Miller, J J; Brand, J C

    1980-01-01

    Acid or enzymic hydrolysis can be used to hydrolyze lactose. Advantages of both are compared and details of enzymic hydrolysis using yeast or fungal enzymes given. The new scheme outlined involves recycling lactase. Because lactose and lactase react to ultrafiltration (UF) membranes differently separation is possible. Milk or milk products are ultrafiltered to separate a concentrate from a lactose-rich permeate which is treated with lactase in a reactor until hydrolysis reaches a required level. The lactase can be removed by UF as it does not permeate the membrane, and it is recycled back to the reactor. Permeate from the second UF stage may or may not be recombined with the concentrate from the first stage to produce a low lactose product (analysis of a typical low-lactose dried whole milk is given). Batch or continuous processes are explained and a batch process without enzyme recovery is discussed. (Refs. 4).

  10. Indicators: Sediment Enzymes

    Science.gov (United States)

    Sediment enzymes are proteins that are produced by microorganisms living in the sediment or soil. They are indicators of key ecosystem processes and can help determine which nutrients are affecting the biological community of a waterbody.

  11. Enzyme Vs. Extremozyme -32 ...

    Indian Academy of Sciences (India)

    Enzymes are biocatalytic protein molecules that enhance the rates of ... to physical forces (hydrogen bonds, hydrophobic 1, electrostatic and Van der ... conformation. In 1995 ... surface against 14.7% in Klenow poll (some of the hydrophobic.

  12. Overproduction of ligninolytic enzymes

    Science.gov (United States)

    Elisashvili, Vladimir; Kachlishvili, Eva; Torok, Tamas

    2014-06-17

    Methods, compositions, and systems for overproducing ligninolytic enzymes from the basidiomycetous fungus are described herein. As described, the method can include incubating a fungal strain of Cerrena unicolor IBB 303 in a fermentation system having growth medium which includes lignocellulosic material and then cultivating the fungal strain in the fermentation system under conditions wherein the fungus expresses the ligninolytic enzymes. In some cases, the lignocellulosic material is mandarin peel, ethanol production residue, walnut pericarp, wheat bran, wheat straw, or banana peel.

  13. False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences.

    Science.gov (United States)

    Piketty, Marie-Liesse; Polak, Michel; Flechtner, Isabelle; Gonzales-Briceño, Laura; Souberbielle, Jean-Claude

    2017-05-01

    Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.

  14. Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Reimert, C M; Venge, P; Kharazmi, A

    1991-01-01

    Eosinophil cationic protein (ECP) is a highly basic and potent cytotoxic single-chain zinc-containing protein present in the granules of the eosinophilic granulocytes. ECP appears to be involved in defence against parasites and in the tissue damage seen in subjects with allergic and inflammatory...... disease. To investigate ECP release from in vitro activated human eosinophils and to study the involvement of eosinophils in health and disease, we have developed a sensitive and specific enzyme immunoassay. ECP was purified from normal human peripheral blood eosinophils and polyclonal antibodies to ECP...

  15. Enzymes in cleaning products: an overview of toxicological properties and risk assessment/management.

    Science.gov (United States)

    Basketter, David; Berg, Ninna; Broekhuizen, Cees; Fieldsend, Mark; Kirkwood, Sheila; Kluin, Cornelia; Mathieu, Sophie; Rodriguez, Carlos

    2012-10-01

    Enzymes used in cleaning products have an excellent safety profile, with little ability to cause adverse responses in humans. For acute toxicity, genotoxicity, sub-acute and repeated dose toxicity, enzymes are unremarkable. Reproductive toxicity and carcinogenicity are also not endpoints of concern. Exceptions are the ability of some proteases to produce irritating effects at high concentrations and more importantly, the intrinsic potential of these bacterial/fungal proteins to act as respiratory sensitizers. It is a reasonable assumption that the majority of enzyme proteins possess this hazard. However, methods for characterising the respiratory sensitisation hazard of enzymes are lacking and the information required for risk assessment and risk management, although sufficient, remains limited. Previously, most data was generated in animal models and in in vitro immunoassays that assess immunological cross-reactivity. Nevertheless, by the establishment of strict limits on airborne exposure (based on a defined minimal effect limit of 60ng active enzyme protein/m(3)) and air and health monitoring, occupational safety can be assured. Similarly, by ensuring that airborne exposure is kept similarly low, coupled with knowledge of the fate of these enzymes on skin and fabrics, it has proven possible to establish a long history of safe consumer use of enzyme containing products. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Double Antibody Method for Immunoassay of Insulin. Its Application in Population Studies

    Energy Technology Data Exchange (ETDEWEB)

    Welborn, T. A.; Stenhouse, N. S.; Curnow, D. H.; Johnstone, Clare J. [Department of Medicine, Raine Foundation Medical Statistics Unit and Department of Pathology, University of Western Australia, Perth (Australia)

    1970-02-15

    The performance of a double-antibody Immunoassay technique over a period of two years is described, during which 3300 samples from population studies were analysed for insulin concentration. The method requires safeguards to ensure that complete separation of ''free'' from ''antibody-bound'' hormone is achieved, thus labelled gamma-globulin is used in the precise preliminary standardization of precipitating antibody. The dose-response curves for crystalline insulin standards over a range of concentration 0.2 to 16,0 ng (5 to 400 micro-International-Units)/ml are consistently nonlinear, but a close fit is obtained by a cubic function obtained empirically from multiple regression analysis, which also corrects satisfactorily for ''within-assay drift'' when large numbers of samples are involved. The coefficient of variation for pooled serum standards repetitively assayed ranged from 10 to 14%. Sources of ''between-assay variability'' of the serum standards include variable integrity of the labelled insulin and the slope of the standard curve. For quality control material, the need for standards of serum as well as crystalline insulin is stressed, Serum-insulin was measured in 3300 adults of the population of Busselton, approximately 1 h after a 50 g oral glucose load. The concentrations were distributed log-normally. Females had significantly higher 'one-hour' serum-insulin levels than males, and both sexes showed a moderate rise of values with age. Multiple regression analyses revealed that the major determinant of serum-insulin was the blood-sugar level, the independent relationship being such that at ''one-hour'' blood-sugar levels exceeding 160 - 180 mg/100 ml, a decline of insulin values occurred. Body-weight had a strong positive association, and height a negative relationship, with the ''one-hour'' serum-insulin. Males with coronary heart disease and hypertension showed elevated insulin levels under 60 years of age, whereas ''ideal'' males (with obesity

  17. Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

    Science.gov (United States)

    Beck, P; Nicholas, H

    1975-03-01

    1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as

  18. IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

    Directory of Open Access Journals (Sweden)

    Tuanne Rotti ABRANTES

    2016-01-01

    Full Text Available After the report of a second case of canine visceral leishmaniasis (CVL in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA, enzyme-linked immunosorbent assay (ELISA, and rapid chromatographic immunoassay based on dual-path platform (DPP(r were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r, with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

  19. Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

    Science.gov (United States)

    Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

    2013-12-01

    Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

  20. In vitro conditions modify immunoassayability of bovine pituitary prolactin and growth hormone: insights into their secretory granule storage forms

    International Nuclear Information System (INIS)

    Lorenson, M.Y.

    1985-01-01

    The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione. Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion