Sample records for fab2 fab2 fab2

  1. Preparation of F(ab')2 fragments of immunoglobulin G. (United States)

    Killion, J J; Holtgrewe, E M


    We describe a simple protocol for the preparation of F(ab')2 fragments of immunoglobulin G, based upon the known Fc- binding properties of protein A-Sepharose. The fragment preparations of xenogeneic and allogeneic anti-IgG were noncytotoxic to intact target cells, and were able to block the cytotoxicity of intact antibody. This method should therefore be useful for functional studies not requiring biochemical homogeneity.

  2. Immunoglobulin fragments, F(ab')2, that are cytotoxic to enzyme-treated cells. (United States)

    Holtgrewe, E M; Killion, J J


    Bivalent immunoglobulin fragments of IgG, F(ab')2, prepared from normal murine sera were found to be cytotoxic to neuraminidase-treated cells. The fragments were cytotoxic to both allogenic and syngeneic targets (with respect to the source of the sera), suggesting that the antigen bound by the F(ab')2 is not related to the major histocompatibility locus of mice (H-2).

  3. Functional capacity of immunoglobulin G preparations and the F(ab')2 split product.


    Steele, R W


    Five immunoglobulin G preparations, including one 5S F(ab')2 split product, were compared for activity against common bacterial, viral, and protozoan pathogens. Standard assays were used to quantitate antibodies to tetanus, diphtheria, cytomegalovirus, herpes simplex virus types 1 and 2, rubella virus, and Toxoplasma gondii. Opsonization and killing of bacteria were examined by chemiluminescence methods using Streptococcus pneumoniae types 5, 12F, and 14 and Staphylococcus aureus. Antibodies ...

  4. Production of anti-horse antibodies induced by IgG, F(ab')2 and Fab applied repeatedly to rabbits. Effect on antivenom pharmacokinetics. (United States)

    Vázquez, Hilda; Olvera, Felipe; Alagón, Alejandro; Sevcik, Carlos


    We separated whole IgG, Fab and F(ab')2 fragments from horse plasma. We previously studied the pharmacokinetics of these immunoglobulins and fragments in rabbits and shown that Fab and F(ab')2 pharmacokinetics were well described by a three-exponential kinetics, while IgG and IgG(T) pharmacokinetics, however, deviated from the three-exponential kinetics 120 h after injecting a bolus of the immunotherapeutics; this departure was shown to be due to a surge of anti-horse antibodies occurring after 120 h, peaking at ≈260 h and decaying slowly afterward (Vázquez et al., 2010). We now describe antivenom pharmacokinetics and anti-horse IgG production in rabbits receiving three boluses (300 μg/kg, I.V.) of Fab, F(ab')2 or IgG separated by 21 days.

  5. Absorbed Doses and Risk Estimates of (211)At-MX35 F(ab')2 in Intraperitoneal Therapy of Ovarian Cancer Patients

    DEFF Research Database (Denmark)

    Cederkrantz, Elin; Andersson, Håkan; Bernhardt, Peter


    100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective......, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective...... dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation...

  6. Initial volume of a drug before it reaches the volume of distribution: pharmacokinetics of F(ab')2 antivenoms and other drugs. (United States)

    Sevcik, Carlos; Salazar, Victor; Díaz, Patricia; D'Suze, Gina


    Fast disappearance of F(ab')2 antivenoms from the plasma compartment [Sevcik et al., 2004. Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')2 extrusion mechanism from blood to tissues. Toxicon 44, 731-734; Vazquez et al., 2005. Pharmacokinetics of a F(ab')2 scorpion antivenom in healthy human volunteers. Toxicon 46, 797-805] suggests a quick time course to reach its final distribution volume. An equation was developed to describe how the volume occupied by a drug in the body grows with time. As discussed in the paper this equation is free of some shortcomings of an equation developed for the same purpose by Niazi [1976. Volume of distribution as a function of time. J. Pharm. Sci. 65, 452-454]. Fluorescence microscopy showed that the rapid initial decay in plasmatic F(ab')2 concentration may be related to uptake of F(ab')2 by vascular endothelium which, in combination with accumulation in the vascular wall connective tissue, may produce an intermediate plateau in F(ab')2 V(sl)(t), which reached its final value after 10 h. The V(sl)(t) equation predicts that the plasma concentration half-time of decay has little use to estimate how a drug distributes through the body to exert its action, and predicts that, in some instances, intermediate plateaus in the time course of V(sl)(t) exist. Data from the literature showed that the kinetic considerations for V(sl)(t) also apply to clevidipine, digoxin, digitoxin, lidocaine and thiopentone.

  7. Population Pharmacokinetics of an Indian F(ab'2 Snake Antivenom in Patients with Russell's Viper (Daboia russelii Bites.

    Directory of Open Access Journals (Sweden)

    Geoffrey K Isbister

    Full Text Available There is limited information on antivenom pharmacokinetics. This study aimed to investigate the pharmacokinetics of an Indian snake antivenom in humans with Russell's viper bites.Patient data and serial blood samples were collected from patients with Russell's viper (Daboia russelii envenoming in Sri Lanka. All patients received Indian F(ab'2 snake antivenom manufactured by VINS Bioproducts Ltd. Antivenom concentrations were measured with sandwich enzyme immunoassays. Timed antivenom concentrations were analysed using MONOLIXvs4.2. One, two and three compartment models with zero order input and first order elimination kinetics were assessed. Models were parameterized with clearance (CL, intercompartmental clearance (Q, central compartment volume (V and peripheral compartment volume (VP. Between-subject-variability (BSV on relative bioavailability (F was included to account for dose variations. Covariates effects (age, sex, weight, antivenom batch, pre-antivenom concentrations were explored by visual inspection and in model building. There were 75 patients, median age 57 years (40-70 y and 64 (85% were male. 411 antivenom concentration data points were analysed. A two compartment model with zero order input, linear elimination kinetics and a combined error model best described the data. Inclusion of BSV on F and weight as a covariate on V improved the model. Inclusion of pre-antivenom concentrations or different batches on BSV of F did not. Final model parameter estimates were CL,0.078 L h(-1, V,2.2L, Q,0.178 L h(-1 and VP,8.33L. The median half-life of distribution was 4.6 h (10-90%iles:2.6-7.1 h and half-life of elimination, 140 h (10th-90th percentilesx:95-223h.Indian F(ab'2 snake antivenom displayed biexponential disposition pharmacokinetics, with a rapid distribution half-life and more prolonged elimination half-life.

  8. Preparation and development of equine hyperimmune globulin F(ab')2 against severe acute respiratory syndrome coronavirus

    Institute of Scientific and Technical Information of China (English)

    Jia-hai LU; Bing L WONG; Nan-shan ZHONG; Zhong-min GUO; Wen-yu HAN; Guo-ling WANG; Ding-mei ZHANG; Yi-fei WANG; Sheng-yun SUN; Qin-he YANG; Huan-ying ZHENG


    Aim: The resurgence of severe acute respiratory syndrome (SARS) is still a threat because the causative agent remaining in animal reservoirs is not fully understood,and sporadic cases continue to be reported. Developing high titers of anti-SARS hyperimmune globulin to provide an alternative pathway for emergent future prevention and treatment of SARS. Methods: SARS coronavirus (CoV)F69 (AY313906)and Z2-Y3 (AY394989) were isolated and identified from 2 different Cantonese onset SARS patients. Immunogen was prepared from SARS-CoV F69 strain. Six health horses were immunized 4 times and serum was collected periodically to measure the profile of specific IgG and neutralizing antibodies using indirect enzyme-linked immunosorbent assay and a microneutralization test. Sera were collected in large amounts at the peak, where IgG was precipitated using ammonium sulphate and subsequently digested with pepsin. The product was then purified using anion-exchange chromatography to obtain F(ab')2 fragments. Results: The specific IgG and neutralizing antibody titers peaked at approximately week 7 after the first immunization, with a maximum value of 1:14210. The sera collected at the peak were then purified. Fragment of approximately 15 g F(ab')2 was obtained from 1 litre antiserum and the purity was above 90% with the titer of 1:5120, which could neutralize the other strain (SARS-CoV Z2-Y3) as well. Conclusion: This research provides a viable strategy for the prevention and treatment of SARS coronavirus infection with equine hyperimmune globulin, with the purpose of combating any resurgence of SARS.

  9. Cholestatic liver disease after rituximab and adalimumab and the possible role of cross-reacting antibodies to Fab 2 fragments.

    Directory of Open Access Journals (Sweden)

    Joerg Latus

    Full Text Available BACKGROUND: Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA-associated granulomatosis with polyangiitis (GPA who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. METHODS: Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. RESULTS: Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients' sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. CONCLUSION: This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects

  10. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid. (United States)

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John


    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

  11. Specific polyclonal F(ab')2 neutralize a large panel of highly pathogenic avian influenza A viruses (H5N1) and control infection in mice. (United States)

    Herbreteau, Cécile Hélène; Jacquot, Frédéric; Rith, Sareth; Vacher, Laurent; Nguyen, Ludovic; Carbonnelle, Caroline; Lotteau, Vincent; Jolivet, Michel; Raoul, Hervé; Buchy, Philippe; Saluzzo, Jean-François


    There is still no specific therapy for infection with the highly pathogenic avian influenza A virus (HPAI) H5N1, which caused 39 human cases with a 64% fatality rate in 2013. We prepared highly purified specific equine polyclonal immunoglobulin fragments (F(ab')2) against H5N1 and tested them for efficacy in vitro and with different administration schedules in H5N1-challenged BALB/c mice. in vitro, F(ab')2 neutralized 21 different H5N1 strains from different areas, representative of 11 different clades and sub-clades and 9 years of evolution of the virus. In vivo mouse experiments identified that the most efficient administration protocol consists of five consecutive daily injections after infection; 10 mg/kg giving a 60% increase in survival. These data demonstrate the ability of anti-H5N1 F(ab')2 to markedly reduce the mortality and morbidity associated with infection of mice with HPAI H5N1 virus, and their potential for human therapy.

  12. Preparation and activity of conjugate of monoclonal antibody HAbl8 against hepatoma F(ab')2 fragment and staphylococcal enterotoxin A

    Institute of Scientific and Technical Information of China (English)

    Lian Jun Yang; Yan Fang Sui; Zhi Nan Chen


    AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.

  13. Glomerular filtration rate after alpha-radioimmunotherapy with 211At-MX35-F(ab')2: a long-term study of renal function in nude mice

    DEFF Research Database (Denmark)

    Back, T.; Haraldsson, B.; Hultborn, R;


    and animals bearing subcutaneous xenografts of the human ovarian cancer cell line, OVCAR-3, were used. The animals received approximately 0.4, 0.8, or 1.2 MBq in one, two, or three fractions. The mean absorbed doses to the kidneys ranged from 1.5 to 15 Gy. The renal function was studied by serial GFR...... of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals...... manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions...

  14. Secretory component delays the conversion of secretory IgA into antigen-binding competent F(ab')2: a possible implication for mucosal defense. (United States)

    Crottet, P; Corthésy, B


    Secretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at approximately 10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the alpha-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the kappa light chain was not digested at all, suggesting that the F(ab')2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the alpha-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab')2 is discussed in terms of mucosal immune defenses.

  15. Targeting human prostate cancer with (111) In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, S.; Rij, C.M. van; Franssen, G.M.; Fracasso, G.; Helfrich, W.; Eek, A.; Oyen, W.J.G.; Colombatti, M.; Boerman, O.C.


    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing sub

  16. Targeting human prostate cancer with In-111-labeled D2B IgG, F(ab ')(2) and Fab fragments in nude mice with PSMA-expressing xenografts

    NARCIS (Netherlands)

    Lutje, Susanne; van Rij, Catharina M.; Franssen, Gerben M.; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J.; Colombatti, Marco; Boerman, Otto C.


    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab)(2) and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing su

  17. Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor. (United States)

    Kalli, K R; Hsu, P H; Bartow, T J; Ahearn, J M; Matsumoto, A K; Klickstein, L B; Fearon, D T


    CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

  18. 131I-肝癌单抗片段HAb18F(ab')2注射液药盒的制备%Preparation of 131I Labeled Hepatoma Monoclonal Antibody Fragment HAb18F(ab')2 Kit

    Institute of Scientific and Technical Information of China (English)

    冯强; 米力; 边惠洁; 余晓玲; 陈志南


    选择Mather高效碘标法(NBS法)制备应用于肝癌治疗的131I-肝癌单抗片段HAb18F(ab')2注射液药盒,确定并优化了冷药盒各组分的剂型、标记条件和纯化方法.制备出的药盒抗体标记物的放化纯度大于95%,整个标记过程可在10 min内完成,10 ℃以下干燥处可保存两年以上,适于临床应用.

  19. Rapid screening of purification strategies for the capture of a human recombinant F(ab')2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS. (United States)

    Pezzini, J; Brenac Brochier, V; Barrouillet, M P; Cerruti, M; Clofent-Sanchez, G; Schapman, A; Topol, A; Robert, R; Cabanne, C; Cerruti, P; Santarelli, X


    The development of a capture step of a human recombinant F(ab')(2) produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab')(2) was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.

  20. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts. (United States)

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C


    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts.

  1. Equine Immunoglobulin and Equine Neutralizing F(ab?)2 Protect Mice from West Nile Virus Infection


    Jiannan Cui; Yongkun Zhao; Hualei Wang; Boning Qiu; Zengguo Cao; Qian Li; Yanbo Zhang; Feihu Yan; Hongli Jin; Tiecheng Wang; Weiyang Sun; Na Feng; Yuwei Gao; Jing Sun; Yanqun Wang


    West Nile virus (WNV) is prevalent in Africa, Europe, the Middle East, West Asia, and North America, and causes epidemic encephalitis. To date, no effective therapy for WNV infection has been developed; therefore, there is urgent need to find an efficient method to prevent WNV disease. In this study, we prepared and evaluated the protective efficacy of immune serum IgG and pepsin-digested F(ab′)2 fragments from horses immunized with the WNV virus-like particles (VLP) expressing the WNV M and ...

  2. Polyvalent horse F(Ab`)2 snake antivenom: Development of process ...

    African Journals Online (AJOL)



    Apr 19, 2010 ... mice as animal test and the their potency expressed in terms of ED50 ... the antivenoms. All animals used in this study were maintained and ..... True Vipers: Natural History and ... Experiments on the preventive inoculation of.

  3. Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

    NARCIS (Netherlands)

    Heskamp, S.; Laarhoven, H.W.M. van; Molkenboer-Kuenen, J.D.M.; Bouwman, W.H.; Graaf, W.T. van der; Oyen, W.J.G.; Boerman, O.C.


    The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal

  4. 东亚钳蝎蝎毒的毒性及其F(ab')2抗体的制备%Toxicity of Buthus martensii Karsch venom and preparation of its F(ab')2 antibody

    Institute of Scientific and Technical Information of China (English)

    李妍; 范泉水; 张志晓; 郭平; 叶锋平; 姜晓梅; 邱薇; 张富强; 郑颖


    目的 测定东亚钳蝎(Buthus martensii Karsch,BMK)蝎毒的毒性,并制备能有效中和BMK蝎毒的F(ab')2抗体.方法 分别经腹腔和肌肉注射小鼠,测定不同产地(山西、山东、河南、辽宁)BMK蝎毒对小鼠的半数致死量(LD5o);以山西产BMK蝎毒免疫云南矮种马,获得免疫血浆,经盐析、酶解、加热等步骤制备F(ab')2抗体,采用ELISA、免疫扩散和小鼠体外中和试验分别测定超免血浆、纯化的IgG及F(ab')2抗体对各地产BMK蝎毒的抗体效价.结果 山西产BMK蝎毒的LD50最小,小鼠腹腔注射的LD50为1.67 mg/kg,肌肉注射LD50为2.06 mg/kg.制备的F(ab')2抗体的纯度为86.5%;纯化的IgG稀释至1×10-9 mg/ml,抗体ELISA效价呈阳性,较纯化前(1×10-7mg/ml)提高了100倍;纯化的IgG和制备的F(ab')2抗体与山西产BMK蝎毒比例为0.4∶1时,免疫扩散试验出现清晰的沉淀线,与其他产地BMK蝎毒比例均为0.1∶1时,出现清晰的沉淀线;F(ab')2抗体与山西产BMK蝎毒质量比为2∶1时,可保护小鼠100%存活.结论 成功利用蝎毒免疫动物制备了高纯度的F(ab')2抗体,该抗体可有效中和多个产地的BMK蝎毒.

  5. Spectral characteristics of fluorescence and circular dichroism of aflatoxin B1 reaction with its anti-idiotypic antibody (United States)

    Liu, Aiping; Yang, Hongxiu; Wang, Xiaohong; Chen, Fusheng


    Aflatoxin B1 (AFB1) is a toxic secondary metabolite and sensitive methods for its analysis have been developed. In our lab, a number of works have been carried out, including exploitation of detection methods and production of anti-idiotypic antibody (Ab2) against Fab fragment of anti-AFB1 antibody (Ab1). In this paper, Ab2 was generated upon the immunization of mice with F(ab')2 fragment, which was specific to AFB1 and obtained by pepsin digestion of Ab1. The characteristics of Ab2 was primarily investigated by indirect competitive enzyme-linked immunosorbent assay (icELISA), which indicated that Ab2, might bear an internal image of antigen AFB1 and was able to combine to F(ab')2 in competition with AFB1, and the concentration of Ab2 to cause 50% inhibition of binding (IC50) was 131.8 μg/mL. In addition, fluorescence and circular dichroism studies were designed to explore the mutual relationship among AFB1, F(ab')2 and Ab2. The fluorescence spectroscopy implied that both AFB1 and Ab2 act as a quencher upon F(ab')2, and the Ab2 could compete with AFB1 when both of Ab2 and AFB1 reacted with F(ab')2. The circular dichroism (CD) spectrum suggested that both the binding of Ab2 and AFB1 on F(ab')2 brought secondary conformation change of F(ab')2, especially in the changes of α helix and β sheet. The research performed would provide unique insight into the comprehension of interaction among AFB1, F(ab')2 and Ab2 as well as offer structural information for substitution researches of toxic antigen like AFB1.

  6. Variants of ELISA in plant virus diagnosis. (United States)

    Koenig, R; Paul, H L


    Variations of enzyme-linked immunosorbent assay (ELISA) were compared with respect to their ability to detect and to differentiate serologically related plant viruses. The broadest range of serologically related viruses was detected by an indirect ELISA on unprecoated plates. Coating the plates with F(ab')2 fragments led to narrowing of the specificity in heterologous reactions of tymo-, tombus- and tobamoviruses in indirect ELISA. With Andean potato latent virus (APLV) heterologous reactions were weaker on plates precoated with F(ab')2 fragments than on those precoated with intact antibodies. Even on plates precoated with F(ab')2 fragments the indirect ELISA detected a broader range of serologically related viruses than the direct double antibody sandwich method. Heterologous reactions in indirect ELISA procedures on plates precoated with either intact antibodies or F(ab')2 fragments were always weaker than homologous reactions independent of the concentration of coating reactants and detecting antibodies. Attempts to differentiate closely related strains of APLV or radish mosaic virus by direct ELISA using F(ab')2 fragments either for coating the plates or after labelling with alkaline phosphatase for detecting the trapped antigens failed. Under suitable conditions, the additional working step usually necessary for indirect ELISA could be avoided by using a short procedure which at low concentrations of detecting antibodies was more sensitive than the conventional procedure.

  7. Cyclic AMP-insensitive activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI. (United States)

    Ichinohe, T; Takayama, H; Ezumi, Y; Yanagi, S; Yamamura, H; Okuma, M


    Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation. Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')2 fragments of this antibody (F(ab')2-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')2-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')2-anti-p62 in a way insensitive to elevation of cAMP. In contrast, in the presence of prostacyclin, F(ab')2-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase. cAMP-insensitive activation of c-Src and Syk was also observed in collagen but not thrombin-stimulated platelets. Moreover, either F(ab')2-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.

  8. Intraperitoneal alpha-radioimmunotherapy in mice using different specific activities

    DEFF Research Database (Denmark)

    Elgqvist, Jörgen; Andersson, Håkan; Haglund, Elin;


    The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At.......The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At....

  9. Drug: D05944 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D05944 Drug Sulesomab (USAN/INN); LeukoScan (TN) Monoclonal antibody [diagnostic fo...r the detection of infectious lesions] Sulesomab is Tc-99m-antigranulocyte monoclonal antibody fragment Fab'2. CAS: 167747-19-5 PubChem: 47207602 ...

  10. Serum and plasma fibronectin binds to complement reacted immune complexes primarily via Clq

    DEFF Research Database (Denmark)

    Baatrup, G; Svehag, S E


    The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phas...

  11. Bispecific antibody fragments with CD20 X CD28 specificity allow effective autologous and allogeneic T-cell activation against malignant cells in peripheral blood and bone marrow cultures from patients with B-cell lineage leukemia and lymphoma. (United States)

    Brandl, M; Grosse-Hovest, L; Holler, E; Kolb, H J; Jung, G


    Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation, such as the TCR/CD3 complex and the costimulatory receptor CD28, are capable of mediating T-cell activation resulting in tumor cell killing. In this study, we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2, when used alone rather than in combination, markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore, these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and, in particular, of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients.

  12. Structure-guided residence time optimization of a dabigatran reversal agent. (United States)

    Schiele, Felix; van Ryn, Joanne; Litzenburger, Tobias; Ritter, Michael; Seeliger, Daniel; Nar, Herbert


    Novel oral anticoagulants are effective and safe alternatives to vitamin-K antagonists for anticoagulation therapy. However, anticoagulation therapy in general is associated with an elevated risk of bleeding. Idarucizumab is a reversal agent for the direct thrombin inhibitor, dabigatran etexilate (Pradaxa®) and is currently in Phase 3 studies. Here, we report data on the antibody fragment aDabi-Fab2, a putative backup molecule for idarucizumab. Although aDabi-Fab2 completely reversed effects of dabigatran in a rat model in vivo, we observed significantly reduced duration of action compared to idarucizumab. Rational protein engineering, based on the X-ray structure of aDabi-Fab2, led to the identification of mutant Y103W. The mutant had optimized shape complementarity to dabigatran while maintaining an energetically favored hydrogen bond. It displayed increased affinity for dabigatran, mainly driven by a slower off-rate. Interestingly, the increased residence time translated into longer duration of action in vivo. It was thus possible to further enhance the efficacy of aDabi-Fab2 based on rational design, giving it the potential to serve as a back-up candidate for idarucizumab.

  13. CD3 directed bispecific antibodies induce increased lymphocyte-endothelial cell interactions in vitro

    NARCIS (Netherlands)

    Molema, G; Tervaert, JWC; Kroesen, BJ; Helfrich, W; Meijer, DKF; de Leij, LFMH


    Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells; intravenous administration of BIS-1 F(ab')(2) to carcinoma patients in a phase I/II clinical trial, caused immunomodu

  14. Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use. (United States)

    Korimbocus, Jehanara; Dehay, Nicolas; Tordo, Noël; Cano, François; Morgeaux, Sylvie


    In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Sécurité du Médicament et des Produits de Santé where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Mortality and antibody responses of mice to three successive episodes of experimental scorpion (Centruroides limpidus limpidus) envenomation and immunological rescue. (United States)

    Padilla, Alejandro; Govezensky, Tzipe; Possani, Lourival D; Larralde, Carlos


    Mortality rates of mice and their levels of anti-venom and anti-F(ab')2 antibodies were assessed after three episodes of subcutaneous envenomations with or without treatment with horse F(ab')2. Soluble venom from the Mexican scorpion Centruroides limpidus limpidus was used for these experiments. Repetition of episodes did not induce different mortality rates in untreated mice. F(ab')2 rescued about 85% of the mice in the first two episodes and 66% in the third, without distinction of gender or ostensible side-effects: a suggestion of selection of the most resistant mice. Surviving mice produced in vitro neutralizing antibodies to the scorpion venom and also antibodies to F(ab')2, when injected alone but more so if combined: a possible immunological adjuvant or alarm effect of the venom or of the cascading physiopathology of envenomation. In the few surviving mice, both anti-venom and anti-F(ab')2 antibodies increased significantly after the first envenomation but not thereafter, showing no correlation with mortality rates: a suggestion of their clinical irrelevance, the few hard-to kill mice appeared to resist envenomation by mechanisms other than antibody response. Injection of F(ab')2 alone induced production of detectable anti-venom antibodies in a few mice and injection of venom alone induced that of anti-F(ab')2 antibodies, perhaps due to trace amounts of venom in the high affinity fraction of F(ab')2 and to anti-idiotypic antibodies or polyclonal activity in the envenomation episode, respectively.

  16. Physical functional outcome assessment of patients with major burns admitted to a UK Burn Intensive Care Unit. (United States)

    Smailes, Sarah T; Engelsman, Kayleen; Dziewulski, Peter


    Determining the discharge outcome of burn patients can be challenging and therefore a validated objective measure of functional independence would assist with this process. We developed the Functional Assessment for Burns (FAB) score to measure burn patients' functional independence. FAB scores were taken on discharge from ICU (FAB 1) and on discharge from inpatient burn care (FAB 2) in 56 patients meeting the American Burn Association criteria for major burn. We retrospectively analysed prospectively collected data to measure the progress of patients' physical functional outcomes and to evaluate the predictive validity of the FAB score for discharge outcome. Mean age was 38.6 years and median burn size 35%. Significant improvements were made in the physical functional outcomes between FAB 1 and FAB 2 scores (ppatients were discharged home, 8 of these with social care. 8 patients were transferred to another hospital for further inpatient rehabilitation. FAB 1 score (≤ 9) is strongly associated with discharge outcome (pburn patients.

  17. Synthesis of bifunctional antibodies for immunoassays. (United States)

    DeSilva, B S; Wilson, G S


    The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.

  18. Solid phase synthesis of bifunctional antibodies. (United States)

    DeSilva, B S; Wilson, G S


    Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.


    Institute of Scientific and Technical Information of China (English)

    邹颖; 卞美璐; 杨子义; 连利娟; 刘文淑; 许秀英


    In the present study,four different proteases (pepsin,papain,bromelain and ficin) were screened with a murine monoclonal antibody OC859,in order to verify whether different digestion procedures could improve yield and stability of the F(ab')2 or Fab fragments.The yields of F(ab')2 or Fab fragments from digestion with pepsin,papain,bromelain and ficin were respectively 20.3+/-2.0%,50.5%+/-5.0%,74.4+/-2.7% and 82.8+/-10.2% of the theoretical maximum.Immunoreactivity in a noncompetitive solid-phase radioimmunoassay (SPRIA) of the fragments generated by the four proteases were respectively 10+/-5%,36+/-5%,60+/-6% and 75+/-6% of the intact OC859 IgG.These results suggested that the fragmentation of OC859 with ficin gave a higher yield of superior immunoreactive fragments.

  20. Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus envenoming in Papua New Guinea.

    Directory of Open Access Journals (Sweden)

    Mariángela Vargas


    Full Text Available Snake bite is a common medical emergency in Papua New Guinea (PNG. The taipan, Oxyuranus scutellatus, inflicts a large number of bites that, in the absence of antivenom therapy, result in high mortality. Parenteral administration of antivenoms manufactured in Australia is the current treatment of choice for these envenomings. However, the price of these products is high and has increased over the last 25 years; consequently the country can no longer afford all the antivenom it needs. This situation prompted an international collaborative project aimed at generating a new, low-cost antivenom against O. scutellatus for PNG.A new monospecific equine whole IgG antivenom, obtained by caprylic acid fractionation of plasma, was prepared by immunising horses with the venom of O. scutellatus from PNG. This antivenom was compared with the currently used F(ab'(2 monospecific taipan antivenom manufactured by CSL Limited, Australia. The comparison included physicochemical properties and the preclinical assessment of the neutralisation of lethal neurotoxicity and the myotoxic, coagulant and phospholipase A(2 activities of the venom of O. scutellatus from PNG. The F(ab'(2 antivenom had a higher protein concentration than whole IgG antivenom. Both antivenoms effectively neutralised, and had similar potency, against the lethal neurotoxic effect (both by intraperitoneal and intravenous routes of injection, myotoxicity, and phospholipase A(2 activity of O. scutellatus venom. However, the whole IgG antivenom showed a higher potency than the F(ab'(2 antivenom in the neutralisation of the coagulant activity of O. scutellatus venom from PNG.The new whole IgG taipan antivenom described in this study compares favourably with the currently used F(ab'(2 antivenom, both in terms of physicochemical characteristics and neutralising potency. Therefore, it should be considered as a promising low-cost candidate for the treatment of envenomings by O. scutellatus in PNG, and is ready to be tested in clinical trials.

  1. Progress and improvement of the manufacturing process of snake antivenom

    Directory of Open Access Journals (Sweden)

    Zolfagharian H.


    Full Text Available Antivenoms have been used successfully for more than a century and up to now constitute the only effectivetreatment for snakebites .The production of antivenin started long time ago when the calmette was preparedthe antivenom in 1894.The method currently used to prepare antivenom by most of the manufacturers areoriginated from the method of Pope which was develop in 1938. Several new approaches in the production ofantivenom have been proposed to produce IgG, F(ab2, F(ab antivenin to improve their quality .Theseimprovement include complete or partial modification in the antivenom production regarding animal,immunization protocols , new adjuvants in hyperimmunization of animals , purification processes ( caprylicacid ,chromatography , diafiltration and ulterafiltration ,enzymatic digestion of IgG (pepsin, papain andfractionation of venom .When the IgG is digested enzymatically, different fragments are obtained depending on the enzyme used, that is, if papain is used, three fragments are obtained, the crystallizing fragment (Fc and two antigen-binding fragments F(ab and, if pepsin is used, one F(ab'2 fragment is obtained, while thecrystallizing fragment is digested. Fab and F(ab2 fragments conserve their capacity to specifically bind to the antigen that gave rise to them.

  2. Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

    Institute of Scientific and Technical Information of China (English)

    Jun LI; Xue-hao WANG; Xiao-ming WANG; Zhao-lai CHEN


    Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-l,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method.First, stable F(ab')2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

  3. Repeated Intraperitoneal alpha-Radioimmunotherapy of Ovarian Cancer in Mice

    DEFF Research Database (Denmark)

    Elgqvist, Jörgen; Andersson, Håkan; Jensen, Holger


    The aim of this study was to investigate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice using different fractionated treatment regimens. The study was performed using the monoclonal antibody MX35 F(ab')(2) labeled with the alpha-particle emitter (211)At. Methods....... Nude mice were intraperitoneally inoculated with ~1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later 6 groups of animals were given 400 kBq (211)At-MX35 F(ab')(2) as a single or as a repeated treatment of up to 6 times (n = 18 in each group). The fractionated treatments were given every...... seventh day. Control animals were treated with unlabeled MX35 F(ab')(2) (n = 12). Eight weeks posttreatment the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined. Results. The tumor-free fractions (TFFs) of the animals, defined as the fraction of animals...

  4. Structural Basis for Enhanced HIV-1 Neutralization by a Dimeric Immunoglobulin G Form of the Glycan-Recognizing Antibody 2G12

    Directory of Open Access Journals (Sweden)

    Yunji Wu


    Full Text Available The human immunoglobulin G (IgG 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1 envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs are intramolecularly domain exchanged, resulting in a rigid (Fab2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.

  5. Improvement of the crystallizability and expression of an RNA crystallization chaperone

    Energy Technology Data Exchange (ETDEWEB)

    Ravindran, P.; Heroux, A.; Ye, J.-D.


    Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNA-Fab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of {Delta}C209 P4-P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of Fab-RNA complex.

  6. Pre-clinical studies of toxin-specific nanobodies: evidence of in vivo efficacy to prevent fatal disturbances provoked by scorpion envenoming. (United States)

    Hmila, Issam; Cosyns, Bernard; Tounsi, Hayfa; Roosens, Bram; Caveliers, Vicky; Abderrazek, Rahma Ben; Boubaker, Samir; Muyldermans, Serge; El Ayeb, Mohamed; Bouhaouala-Zahar, Balkiss; Lahoutte, Tony


    Scorpions represent a significant threat to humans and animals in various countries throughout the world. Recently, we introduced Nanobodies (Nbs) to combat more efficiently scorpion envenoming and demonstrated the performance of NbAahIF12 and NbAahII10 to neutralize scorpion toxins of Androctonus australis hector venom. A bispecific Nb construct (NbF12-10) comprising these two Nbs is far more protective than the classic Fab'(2) based therapy and is the most efficient antivenom therapy against scorpion sting in preclinical studies. Now we investigate the biodistribution and pharmacokinetics of (99m)Tc labeled Nbs by in vivo imaging in rodents and compared these data with those of the Fab'(2) product (PAS). The pharmacodynamics of the Nbs was investigated in rats by in vivo echocardiography and it is shown that NbF12-10 prevents effectively the hemodynamic disturbances induced by a lethal dose of venom. Moreover, even a late injection of NbF12-10 restores the heart rate and brings the blood pressure to baseline values. Histology confirms that NbF12-10 prevents lung and heart lesions of treated mice after envenoming. In conjunction, in this preclinical study, we provide proof of concept that NbF12-10 prevents effectively the fatal disturbances induced by Androctonus venom, and that the Nanobody based therapeutic has a potential to substitute the classic Fab'(2) based product as immunotherapeutic in scorpion envenoming. Further clinical study using larger cohorts of animals should be considered to confirm the full protecting potential of our NbF12-10.

  7. Increasing the utility of the Functional Assessment for Burns Score: Not just for major burns. (United States)

    Smailes, Sarah T; Engelsman, Kayleen; Rodgers, Louise; Upson, Clara


    The Functional Assessment for Burns (FAB) score is established as an objective measure of physical function that predicts discharge outcome in adult patients with major burn. However, its validity in patients with minor and moderate burn is unknown. This is a multi-centre evaluation of the predictive validity of the FAB score for discharge outcome in adult inpatients with minor and moderate burns. FAB assessments were undertaken within 48 h of admission to (FAB 1), and within 48 h of discharge (FAB 2) from burn wards in 115 patients. Median age was 45 years and median burn size 4%. There were significant improvements in the patients' FAB scores (pburns.

  8. Studies towards the improvement of an anti-cocaine monoclonal antibody for treatment of acute overdose. (United States)

    Zhou, Bin; Eubanks, Lisa M; Jacob, Nicholas T; Ellis, Beverly; Roberts, Amanda J; Janda, Kim D


    There is currently no clinically-approved antidote for cocaine overdose. Efforts to develop a therapy via passive immunization have resulted in a human monoclonal antibody, GNCgzk, with a high affinity for cocaine (Kd=0.18nM). Efforts to improve the production of antibody manifolds based on this antibody are disclosed. The engineering of an HRV 3C protease cleavage site into the GNCgzk IgG has allowed for increased production of a F(ab')2 with a 20% superior capacity to reduce mortality for cocaine overdose in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. What is the optimum concentration of m-cresol in antivenoms?


    MA Abd-Elsalam; N Abdoon; MS Al-Ahaidib


    Antivenoms against snake and scorpion envenomations are usually equine in nature and composed mostly of F(ab')2; additionally, phenol and m-cresol are mainly employed for their preservation. Although there is no study on this subject, m-cresol is utilized by most manufacturers in a concentration that ranges from 0.15 to 0.35 g%. Decreasing the concentration of m-cresol to its minimal effective level may protect victims from its toxic effects and keep the antivenom stable during its shelf life...

  10. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M


    Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...... with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364...

  11. What is the optimum concentration of m-cresol in antivenoms?

    Directory of Open Access Journals (Sweden)

    MA Abd-Elsalam


    Full Text Available Antivenoms against snake and scorpion envenomations are usually equine in nature and composed mostly of F(ab'2; additionally, phenol and m-cresol are mainly employed for their preservation. Although there is no study on this subject, m-cresol is utilized by most manufacturers in a concentration that ranges from 0.15 to 0.35 g%. Decreasing the concentration of m-cresol to its minimal effective level may protect victims from its toxic effects and keep the antivenom stable during its shelf life without forming any aggregates. In the present work, different concentrations of m-cresol, ranging from 0.1 to 0.35 g%, were used with some selected batches of snake and scorpion antivenoms. A low concentration of 0.15 g% showed an acceptable preserving result that complies perfectly with antimicrobial specifications stated by the British Pharmacopoeia. Tested antivenoms (in 12 batches, when kept in a cold room for 39 months (more than their shelf life, retained their physical, chemical and microbiological activities according to the specifications of pharmacopeias. The present data demonstrated that reduction of m-cresol concentration to 0.15 g% in case of equine F(ab'2 antivenoms will improve safety of such preparations and preserve their stability during their shelf life.

  12. Liposomes Coloaded with Elacridar and Tariquidar To Modulate the P-Glycoprotein at the Blood-Brain Barrier. (United States)

    Nieto Montesinos, Rita; Béduneau, Arnaud; Lamprecht, Alf; Pellequer, Yann


    This study prepared three liposomal formulations coloaded with elacridar and tariquidar to overcome the P-glycoprotein-mediated efflux at the blood-brain barrier. Their pharmacokinetics, brain distribution, and impact on the model P-glycoprotein substrate, loperamide, were compared to those for the coadministration of free elacridar plus free tariquidar. After intravenous administration in rats, elacridar and tariquidar in conventional liposomes were rapidly cleared from the bloodstream. Their low levels in the brain did not improve the loperamide brain distribution. Although elacridar and tariquidar in PEGylated liposomes exhibited 2.6 and 1.9 longer half-lives than free elacridar and free tariquidar, respectively, neither their Kp for the brain nor the loperamide brain distribution was improved. However, the conjugation of OX26 F(ab')2 fragments to PEGylated liposomes increased the Kps for the brain of elacridar and tariquidar by 1.4- and 2.1-fold, respectively, in comparison to both free P-gp modulators. Consequently, the Kp for the brain of loperamide increased by 2.7-fold. Moreover, the plasma pharmacokinetic parameters and liver distribution of loperamide were not modified by the PEGylated OX26 F(ab')2 immunoliposomes. Thus, this formulation represents a promising tool for modulating the P-glycoprotein-mediated efflux at the blood-brain barrier and could improve the brain uptake of any P-glycoprotein substrate that is intended to treat central nervous system diseases.

  13. The role of monogamous bivalency and Fc interactions in the binding of anti-DNA antibodies to DNA antigen. (United States)

    Stearns, Nancy A; Pisetsky, David S


    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. These antibodies can bind DNA avidly by monogamous bivalency, a mechanism which requires the interaction of both Fab combining regions with antigenic determinants on the same polynucleotide. To explore further this mechanism, we tested Fab and F(ab')2 fragments prepared from IgG from patient plasmas in an ELISA with native DNA antigen, detecting antibody with a peroxidase conjugated anti-Fab reagent. These studies showed that Fab fragments, which can only bind monovalently, had negligible activity. Although bivalent F(ab')2 fragments would be predicted to bind DNA, these fragments also showed poor anti-DNA activity. Control studies showed that the fragments retained antibody activity to tetanus toxoid and an EBV antigen preparation. Together, these findings suggest that anti-DNA avidity depends on monogamous bivalency, with the antibody Fc portion also influencing DNA binding, in a mechanism which can be termed Fc-dependent monogamous bivalency.

  14. Proteomic analysis of proton beam irradiated human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Sylwia Kedracka-Krok

    Full Text Available Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH, (ii cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70, (iii cell metabolism (TIM, GAPDH, VCP, and (iv cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B. A substantial decrease (2.3 x was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.

  15. Experimental envenoming of mice with venom from the scorpion Centruroides limpidus limpidus: differences in mortality and symptoms with and without antibody therapy relating to differences in age, sex and strain of mouse. (United States)

    Padilla, Alejandro; Govezensky, Tzipe; Possani, Lourival D; Larralde, Carlos


    C57Bl/6J and BALB/cAnN inbred strains of mice differed significantly in mortality and symptoms when intoxicated subcutaneously with one LD(50) of venom from Centruroides limpidus limpidus. Higher mortality was observed in C57Bl/6J than in BALB/cAnN. Also, C57Bl/6J mice more quickly developed muscular and respiratory collapse whilst BALB/cAnN mice were hyperactive before dying. Also, the symptoms in the survivors lasted for 24 h in C57Bl/6J and for 2 h in BALB/cAnN. The age and sex of mice were also related to mortality: younger mice were more resistant than older mice and females were more susceptible than males, especially in the younger groups. Antivenom (horse F(ab')(2)) administration 5-10 min after envenoming of mice with one LD(50) rescued 60% of BALB/cAnN and 52% of C57Bl/6J mice, respectively. Results indicate that genetic background, gender and age differences are of consequence in the pathogenesis of C. limpidus scorpion envenomation in mice, and that timely treatment with active antivenom F(ab')(2) saves a significant fraction of intoxicated mice without statistically significant distinction of strains.

  16. Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Arendrup, M


    also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection...... with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364......Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...

  17. A comparison of 67Cu- and 131I-labelled forms of monoclonal antibodies SEN7 and SWA20 directed against small-cell lung cancer. (United States)

    Smith, A; Zangemeister-Wittke, U; Waibel, R; Schenker, T; Schubiger, P A; Stahel, R A


    The intact anti-SCLC monoclonal antibody (MAb) SEN7 and its F(ab')2 were labelled with the beta-emitting isotope 67Cu. Both materials retained their biological activity in vitro as determined by the Lindmo assay. In a direct comparison of in vivo distribution in a xenograph model, 131I- and 67Cu-labelled intact SEN7 showed similar absolute tumour accumulation. Blood levels were markedly lower in the case of the 67Cu-labelled antibody, resulting in improved tumour:blood ratios which reached a maximum of 13:1 compared with only 4.5:1 for 131I-SEN7. In the case of the 67Cu-labelled F(ab')2, very high accumulation of the nuclide was observed in the kidney. Levels of radio copper in liver and spleen were also found to be significantly raised when compared with radio iodine. SWA20, a MAb which had previously failed to show any selective in vivo accumulation in tumour xenografts when labelled with radio iodine showed higher and more stable tumour accumulation when labelled with 67Cu.

  18. Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab')2 Fragment of a Polyclonal Antibody by Two Different Methods. (United States)

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjattallah; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Goli, Leila Balaei; Babaei, Mahdokht; Ghods, Roya


    R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity.

  19. Data of rational process optimization for the production of a full IgG and its Fab fragment from hybridoma cells. (United States)

    Röhm, Martina; Handl, Alina; König, Maria; Mavoungou, Chrystelle; Handrick, René; Schindowski, Katharina


    This data article focuses on the production of monoclonal antibodies (mAb) and their fragments Fab and F(ab')2. Here, we present the data of an optimization protocol to improve the product yield of a hybridoma cell process using a Design of Experiment (DoE) strategy. Furthermore, the data of the evaluated conditions were used to test feeding strategies in shake flasks. They were verified in controlled 2 L fed-batch bioreactor processes. Supplementing the culture medium with human insulin-like growth factor-I (IGF-I) and Pluronic F-68, as well as a nutrient rich additive for fed-batch, resulted in improved cell growth correlating with a 7 day elongated process time and a 4.5 fold higher product titer. Finally, a rapid Fab generation protocol and the respective data are presented using different papain digestion and a camelid anti-kappa light chain VHH affinity ligand.

  20. Immunogenicity of anti-tumor necrosis factor antibodies-toward improved methods of anti-antibody measurement. (United States)

    Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan


    To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.

  1. Study on the paper cellulose immobilized papain and its exploration%纸纤维固定木瓜蛋白酶的研究及应用

    Institute of Scientific and Technical Information of China (English)

    易喻; 李慧娟; 陈建澍; 梅建凤; 朱克寅; 应国清


    To use paper cellulose as a carrier, immobilization of papain on paper cellulose was studied. Thus, papain was immobilized onto glutaraldehyde-activated paper cellulose under acid conditions. The enzymatic properties, such as proteolytic activity, heat-stability, glutaraldehyde volume fraction, enzyme amount and pH-activity profile, of the immobilized papain preparation were examined. The immobilization conditions were optimized by orthogonal experiments. The prepared products were applied to digest anti-HCG to obtain F(ab )2 fragments. The results indicated that the immobilized enzyme has an high activity at 40 ℃ when the pH of the buffer solution is 5. 0 as well as with the glutaraldehyde volume fraction is 0. 01%. Besides the enzyme concentration is 46 mg/g. The activity of the products can reach up to 19 250 U/g which are ten times higher than those came from the processes not optimized. The preparation and characterization of the F(ab')2 fragments of anti-HCG are presented in detail. The monoclonal antibody was digested with immobilized papain to produce antibody fragment. The F(ab')2 fragments were then purified by using DEAE-FF size exclusion chromatography. The F(ab')2 fragment(110 KDa) maintained potent suppressive activity and the titer can reaches 1:12 000.%利用纸纤维为载体,制备高活性的固定化木瓜蛋白酶并应用于抗HCG抗体的水解.利用戊二醛交联法,制备固定化木瓜蛋白酶,并通过一系列优化,得到最佳固定化条件,包括pH、给酶量、温度、戊二醛体积分数,将制备的固定化木瓜蛋白酶应用于抗HCG抗体水解并制备F(ab')2片段.研究表明:反应温度为40℃时,固定化酶在pH5.0,0.01 mol/L的醋酸缓冲液条件下,给酶量为46 mg/g载体,戊二醛体积分数为0.01%时,固定化酶的酶活达到最高19 250 U/g,相对于优化前1 940 U/g提高了近10倍,成功应用于抗HCG抗体水解制备F(ab')2片段:经SDS-PAGE电泳检测,F(ab’)2片段达到电泳纯.ELISA反应检测表明:片段仍保留较高的活性,效价为1:12 000.

  2. 利用亲和柱分离酶切产物制备抗体Fab片段

    Institute of Scientific and Technical Information of China (English)

    王丹; 赵美萍



  3. Role of muscarinic-3 receptor antibody in systemic sclerosis: correlation with disease duration and effects of IVIG. (United States)

    Kumar, Sumit; Singh, Jagmohan; Kedika, Ramalinga; Mendoza, Fabian; Jimenez, Sergio A; Blomain, Erik S; DiMarino, Anthony J; Cohen, Sidney; Rattan, Satish


    Gastrointestinal dysmotility in systemic sclerosis (SSc) is associated with autoantibodies against muscarinic-3 receptor (M3-R). We investigated the temporal course of the site of action of these autoantibodies at the myenteric neurons (MN) vs. the smooth muscle (SM) M3-R in relation to disease duration, and determined the role of intravenous immunoglobulin (IVIG) in reversing these changes. Immunoglobulins purified from SSc patients (SScIgG) were used to assess their differential binding to MN and SM (from rat colon) employing immunohistochemistry (IHC). Effect of SScIgG on neural and direct muscle contraction was determined by cholinergic nerve stimulation and bethanechol-induced SM contraction. Effects of IVIG and its antigen-binding fragment F(ab')2 on SScIgG binding were studied by enzyme-linked immunosorbent assay (ELISA) of rat colonic longitudinal SM myenteric plexus (LSMMP) lysate and to second extracellular loop peptide of M3-R (M3-RL2). SScIgG from all patients demonstrated significantly higher binding to MN than to SM. With progression of SSc duration, binding at MN and SM increased in a linear fashion with a correlation coefficient of 0.696 and 0.726, respectively (P < 0.05). SScIgG-mediated attenuation of neural and direct SM contraction also increased with disease duration. ELISA analysis revealed that IVIG and F(ab')2 significantly reduced SScIgG binding to LSMMP lysate and M3-RL2. Dysmotility in SSc occurs sequentially, beginning with SScIgG-induced blockage of cholinergic neurotransmission (neuropathy), which progresses to inhibition of acetylcholine action at the SM cell (myopathy). IVIG reverses this cholinergic dysfunction at the neural and myogenic receptors by anti-idiotypic neutralization of SScIgG.

  4. O-glycans direct selectin ligands to lipid rafts on leukocytes. (United States)

    Shao, Bojing; Yago, Tadayuki; Setiadi, Hendra; Wang, Ying; Mehta-D'souza, Padmaja; Fu, Jianxin; Crocker, Paul R; Rodgers, William; Xia, Lijun; McEver, Rodger P


    Palmitoylated cysteines typically target transmembrane proteins to domains enriched in cholesterol and sphingolipids (lipid rafts). P-selectin glycoprotein ligand-1 (PSGL-1), CD43, and CD44 are O-glycosylated proteins on leukocytes that associate with lipid rafts. During inflammation, they transduce signals by engaging selectins as leukocytes roll in venules, and they move to the raft-enriched uropods of polarized cells upon chemokine stimulation. It is not known how these glycoproteins associate with lipid rafts or whether this association is required for signaling or for translocation to uropods. Here, we found that loss of core 1-derived O-glycans in murine C1galt1(-/-) neutrophils blocked raft targeting of PSGL-1, CD43, and CD44, but not of other glycosylated proteins, as measured by resistance to solubilization in nonionic detergent and by copatching with a raft-resident sphingolipid on intact cells. Neuraminidase removal of sialic acids from wild-type neutrophils also blocked raft targeting. C1galt1(-/-) neutrophils or neuraminidase-treated neutrophils failed to activate tyrosine kinases when plated on immobilized anti-PSGL-1 or anti-CD44 F(ab')2. Furthermore, C1galt1(-/-) neutrophils incubated with anti-PSGL-1 F(ab')2 did not generate microparticles. In marked contrast, PSGL-1, CD43, and CD44 moved normally to the uropods of chemokine-stimulated C1galt1(-/-) neutrophils. These data define a role for core 1-derived O-glycans and terminal sialic acids in targeting glycoprotein ligands for selectins to lipid rafts of leukocytes. Preassociation of these glycoproteins with rafts is required for signaling but not for movement to uropods.

  5. Targeting BCL1 lymphoma with anti-idiotype antibodies: biodistribution kinetics of directly labeled antibodies and bispecific antibody-targeted bivalent haptens. (United States)

    Manetti, C; Rouvier, E; Gautherot, E; Loucif, E; Barbet, J; Le Doussal, J M


    The mouse BCL1 lymphoma model has been used for evaluating immunotherapy with anti-idiotype (anti-Id) antibodies, including Id immunisation, IgG therapy and bispecific (Bs) antibody-targeted cytotoxicity. Here, we provide quantitative data on the targeting of small (25 +/- 12 mg) intrasplenic BCL1 tumours, using anti-Id IgG, F(ab')2 and anti-Id x anti-hapten BsF(ab')2 covalently labelled with 125iodine, as well as noncovalent complexes of BsF(ab')2 and 125I-labelled bivalent hapten. The results are the following: 1) up to 115% of the injected dose per gram (% ID/g) of spleen can be localised in the first hour, corresponding to approximately 600% ID/g of tumour; 2) localisation is specific for cell-surface Id; 3) optimal doses can overcome circulating Id; 4) circulating Id markedly increases the catabolism of IgG, thus impairing tumour localisation; 5) bivalent reagents are internalised by the target cells; 6) iodine covalently bound to bivalent antibodies [IgG, F(ab')2] is rapidly (T(1/2): 6-9 hr) released from the tumour; in contrast, the bivalent hapten is retained for a longer time (T(1/2): 25 hr); and 7) in the absence of bivalent hapten, the monovalent BsF(ab')2 is not rapidly internalised and dissociates from tumour cell-surface Id. Our results suggest that monovalent anti-Id, lacking Fc, can efficiently be targeted to the BCL1 tumour surface. For radioimmunotherapy, the intracellular targeting of catabolism-resistant 125I-labelled bivalent hapten provides optimal tissue selectivity.

  6. Evaluation of the safety, immunogenicity, and pharmacokinetic profile of a new, highly purified, heat-treated equine rabies immunoglobulin, administered either alone or in association with a purified, Vero-cell rabies vaccine. (United States)

    Lang, J; Attanath, P; Quiambao, B; Singhasivanon, V; Chanthavanich, P; Montalban, C; Lutsch, C; Pepin-Covatta, S; Le Mener, V; Miranda, M; Sabchareon, A


    A clinical evaluation of a new, purified, heat-treated equine rabies immunoglobulin (PHT-Erig), F(ab')2 preparation, was carried out in Thailand and in the Philippines-two countries where rabies is endemic. An initial prospective, randomised, controlled trial (Study 1), compared the safety and pharmacokinetics (serum concentrations of rabies antibodies) after administration either of PHT-Erig or of a commercially-available, equine rabies immune globulin (Erig PMC). A second trial (Study 2) simulated post-exposure rabies prophylaxis by using a reference cell culture vaccine, the purified Vero-cell rabies vaccine (PVRV), administered in association with either Erig PMC or PHT-Erig. In Study 1, 27 healthy, Thai adults received a 40 IU kg(-1) dose of either Erig PMC (n = 12) or PHT-Erig (n = 15) via the intramuscular (i.m.) route; half of the dose was injected into the deltoid area and the other half into the buttocks. Serum for rabies antibody determination and F(ab')2 concentration was collected at hours (H) 0, 6 and 12, and on day (D) 2, 3, 4, 6, 8, 10, 12 and 15. Both products were safe, with no serious adverse events, and in particular, no anaphylactic reactions or serum sickness was reported. A statistical comparison of the pharmacokinetic parameters did not demonstrate bioequivalence of the two products. Nonetheless, the relative bioavailability of 93% and the similar absorption rates suggest the pharmacokinetic profiles of Erig and PHT-Erig are similar. The antibody level in either group were low throughout the 15-day study period. The geometric mean titer (GMT) values ranged from group 0.027-0.117 IU ml(-1) in the Erig group and from 0.029 to 0.072 IU ml(-1) in the PHT-Erig. There was no significant difference between the evolution of GMT values for the two groups. In Study 2, 71 healthy volunteers received 40 IU kg(-1) via the intramuscular route of either Erig PMC (n = 36) or PHT-Erig (n = 35) on D0, in association with five doses of PVRV on D0, D3, D7, D14 and D28. The safety evaluation was performed during the 28-day follow-up and serum samples for anti-rabies antibody titration were collected on D0 (before injection) D3, D7, D14 and D28. No serious reactions were reported in either group. In particular, no immediate (anaphylactic type) or delayed (serum sickness) allergic reactions were observed. Over the 28-day follow-up period, GMT profiles of the two groups were statistically equivalent. On D14, 100% of the subjects had protective antibody titers (anti-rabies antibodies > or = 0.5 IU ml(-1), which is the WHO-recommended level of seroconversion), and Erig PMC and PHT-Erig were indistinguishable according to the clinical definition chosen. On D28, the GMT values were 33.2 IU ml(-1) (95% CI, 23.8-46.1 IU ml(-1)) in the Erig PMC/PVRV group and 31.4 IU ml(-1) (95% confidence interval, CI, 23.4-42.2 IU ml(-1)) in the PHT-Erig/PVRV group, showing evidence of adequate vaccine-induced antibody responses in both groups. The increased purity, the heat-treatment step introduced in the manufacturing process of PHT-Erig, and the good clinical results substantiate the use of this new generation, purified equine F(ab')2 preparation in the post-exposure prophylaxis of rabies.

  7. Facilitated monocyte-macrophage uptake and tissue distribution of superparmagnetic iron-oxide nanoparticles.

    Directory of Open Access Journals (Sweden)

    Arnaud Beduneau

    Full Text Available BACKGROUND: We posit that the same mononuclear phagocytes (MP that serve as target cells and vehicles for a host of microbial infections can be used to improve diagnostics and drug delivery. We also theorize that physical and biological processes such as particle shape, size, coating and opsonization that affect MP clearance of debris and microbes can be harnessed to facilitate uptake of nanoparticles (NP and tissue delivery. METHODS: Monocytes and monocyte-derived macrophages (MDM were used as vehicles of superparamagnetic iron oxide (SPIO NP and immunoglobulin (IgG or albumin coated SPIO for studies of uptake and distribution. IgG coated SPIO was synthesized by covalent linkage and uptake into monocytes and MDM investigated related to size, time, temperature, concentration, and coatings. SPIO and IgG SPIO were infused intravenously into naïve mice. T(2 measures using magnetic resonance imaging (MRI were used to monitor tissue distribution in animals. RESULTS: Oxidation of dextran on the SPIO surface generated reactive aldehyde groups and permitted covalent linkage to amino groups of murine and human IgG and F(ab'(2 fragments and for Alexa Fluor(R 488 hydroxylamine to form a Schiff base. This labile intermediate was immediately reduced with sodium cyanoborohydride in order to stabilize the NP conjugate. Optical density measurements of the oxidized IgG, F(ab'(2, and/or Alexa Fluor(R 488 SPIO demonstrated approximately 50% coupling yield. IgG-SPIO was found stable at 4 degrees C for a period of 1 month during which size and polydispersity index varied little from 175 nm and 200 nm, respectively. In vitro, NP accumulated readily within monocyte and MDM cytoplasm after IgG-SPIO exposure; whereas, the uptake of native SPIO in monocytes and MDM was 10-fold less. No changes in cell viability were noted for the SPIO-containing monocytes and MDM. Cell morphology was not changed as observed by transmission electron microscopy. Compared to unconjugated SPIO, intravenous injection of IgG-SPIO afforded enhanced and sustained lymphoid tissue distribution over 24 hours as demonstrated by MRI. CONCLUSIONS: Facilitated uptake of coated SPIO in monocytes and MDM was achieved. Uptake was linked to particle size and was time and concentration dependent. The ability of SPIO to be rapidly taken up and distributed into lymphoid tissues also demonstrates feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy.

  8. Preparasi Imunoglobulin G Kelinci sebagai Antigen Penginduksi Antibodi Spesifik Terhadap Virus Avian Influenza H5N1 Strain Legok

    Directory of Open Access Journals (Sweden)

    Ketut Karuni Nyanakumari Natih


    Full Text Available The aim of this research was to prepare rabbit Immunoglobulin G as anti-idiotype antibody (Ab2 ofAvian Influenza Virus (AIV H5N1. A polyclonal antibody was collected from guinea pigs immunized withinactivated AI vaccine H5N1of Legok strain. Antibody of H5N1 AI in serum was detected by Agar gelprecipitation test (AGPT and an Inhibition Hemmaglutination test (IHT. The highest titre of antibodywas obtained one week after the third immunization. Serum of guinea pigs containing IgG was purifiedusing the Montage Antibody purification kit & spin column with Prosep A media (Millipore. The AI H5N1IgG concentration was 8 mg/ml. AI H5N1 IgG, was then digested with pepsin to obtain F(ab2 fraction andwas called Ab1. The concentration of IgG and F(ab2 and purity of IgG were determined by UVspectrophotometer which showed Ab1 concentration 1 mg/ml. Molecular weight was estimated by sodiumdodecyl sulfate- polyacrilamide gel electrophoresis (SDS-PAGE. Ab2 was produced by immunization ofrabbit with Ab1. The first immunization was carried out by subcutaneous injection with 500 ?g of Ab1emulsified in Complete Freund Adjuvant. The immunization was repeated with the same dose of Ab1emulsified in Incomplete Freund Adjuvan at 1 week intervals. One week after the second immunization,rabbit’s serum was harvested and IgG was purified using the Montage Antibody purification kit & spincolumn with Prosep A media (Millipore. The rabbit IgG, called Ab2, was an anti-idiotypic antibody againstAIV-H5N1. In AGPT, a precipitation line appeared between Ab1 and Ab2. A partial reaction appearedbetween Ab2 and the AI H5N1 antigen was also detected. The results indicated that Ab2 is a possiblecandidate of imunogen for protection against an AI virus H5N1 infection.

  9. Characterization of botulinum neurotoxin type A neutralizing monoclonal antibodies and influence of their half-lives on therapeutic activity.

    Directory of Open Access Journals (Sweden)

    Christelle Mazuet

    Full Text Available Botulinum toxins, i.e. BoNT/A to/G, include the most toxic substances known. Since botulism is a potentially fatal neuroparalytic disease with possible use as a biowarfare weapon (Centers for Disease Control and Prevention category A bioterrorism agent, intensive efforts are being made to develop vaccines or neutralizing antibodies. The use of active fragments from non-human immunoglobulins (F(ab'(2, Fab', scFv, chemically modified or not, may avoid side effects, but also largely modify the in vivo half-life and effectiveness of these reagents. We evaluated the neutralizing activity of several monoclonal anti-BoNT/A antibodies (mAbs. F(ab'(2 fragments, native or treated with polyethyleneglycol (PEG, were prepared from selected mAbs to determine their half-life and neutralizing activity as compared with the initial mAbs. We compared the protective efficiency of the different biochemical forms of anti-toxin mAbs providing the same neutralizing activity. Among fourteen tested mAbs, twelve exhibited neutralizing activity. Fragments from two of the best mAbs (TA12 and TA17, recognizing different epitopes, were produced. These two mAbs neutralized the A1 subtype of the toxin more efficiently than the A2 or A3 subtypes. Since mAb TA12 and its fragments both exhibited the greatest neutralizing activity, they were further evaluated in the therapeutic experiments. These showed that, in a mouse model, a 2- to 4-h interval between toxin and antitoxin injection allows the treatment to remain effective, but also suggested an absence of correlation between the half-life of the antitoxins and the length of time before treatment after botulinum toxin A contamination. These experiments demonstrate that PEG treatment has a strong impact on the half-life of the fragments, without affecting the effectiveness of neutralization, which was maintained after preparation of the fragments. These reagents may be useful for rapid treatment after botulinum toxin A contamination.

  10. Accidente ofídico en animales de pastoreo: acercamiento epidemiológico, clínico y de manejo

    Directory of Open Access Journals (Sweden)

    Sebastián Estrada-Gómez


    Full Text Available En animales de pastoreo, el reporte de accidentes ofídicos es muy bajo y, además, presenta un amplio subregistro. A pesar de esto, es de gran utilidad conocer la incidencia de estos accidentes, pues son de gran importancia por las pérdidas económicas que pueden causar en el sector pecuario. Los accidentes por serpientes venenosas, tanto de la familia Viperidae como Elapidae, en caballos y ganado vacuno son frecuentes en muchos lugares del mundo, debido al aumento en la destinación de las tierras para la actividad ganadera. Colombia no es ajena a ello; sin embargo, el registro de este tipo de accidentalidad es muy bajo. En los diferentes reportes de accidentes ofídicos en animales, se ha observado que las principales manifestaciones sistémicas están caracterizadas por complicaciones cardiacas, hemodinámicas y neurotóxicas. Igualmente, se observan efectos locales, los cuales se caracterizan por presencia de hemorragias locales, edema y necrosis. El tratamiento específico utilizado en los accidentes por serpientes es la seroterapia, que consta de inmunoglobulinas G (Sueros IgG completas o fraccionadas (Faboterápicos Fab o Fab2, específicas para un género o especie.

  11. Efficacy of North American crotalid antivenom against the African viper Bitis gabonica (Gaboon viper). (United States)

    Meggs, William J; Wiley, Christopher N; Brewer, Kori L; Hack, Jason B


    Envenomations by exotic snakes occur from zoological collections and private individual collectors. Antivenoms to these snakes may not be readily available. The objective of this study is to determine the efficacy of North American crotalid antivenin in treating mice envenomated with venom of the African viper Bitis gabonica (Gaboon viper). The subjects of the study were Swiss Webster mice weighing approximately 30 g. The study was conducted in the University research laboratory. B. gabonica venom was obtained from Venom Supplies Pty Ltd (Tanunda, South Australia) and reconstituted in sterile water. North American Crotalid Fab2 antivenin (Anavyp, Instituto Bioclon, Mexico) was donated by the manufacturer. The experimental groups were: Group I received two times an intraperitoneal LD(50) dose of venom, 2.58 mg/kg. Group II received the same dose after incubation for 1 h with 10 mg of antivenin. Time to onset of toxicity defined as respiratory rate Mexico) demonstrated efficacy in increasing time to onset of distress in mice poisoned with B. gabonica (Gaboon viper) venom. Based on this result, treatment of humans envenomated with B. gabonica with North American Croatlid antivenin could be considered for severe envenomations if specific B. gabonica antivenin is unavailable.

  12. A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

    DEFF Research Database (Denmark)

    Owens, T


    Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2...... to plastic resulted in weak T cell activation, and these T cells did not induce B cell responses. Haptenated B cell populations, although recognized by E9.D4, were not activated. Separation of T and B cells by a 0.4-micron membrane prevented T-dependent B cell activation, although Th cell-derived B cell...

  13. CTCF genomic binding sites in Drosophila and the organisation of the bithorax complex.

    Directory of Open Access Journals (Sweden)

    Eimear E Holohan


    Full Text Available Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C. We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; "Fab-2," "Fab-3," and "Fab-4." With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation.

  14. Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies. (United States)

    Klaus, Tomasz; Bzowska, Monika; Kulesza, Małgorzata; Kabat, Agnieszka Martyna; Jemioła-Rzemińska, Małgorzata; Czaplicki, Dominik; Makuch, Krzysztof; Jucha, Jarosław; Karabasz, Alicja; Bereta, Joanna


    Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab')2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.

  15. Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons (United States)

    Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.


    Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

  16. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies. (United States)

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey


    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  17. Silver nanoparticle enhanced immunoassays: one step real time kinetic assay for insulin in serum. (United States)

    Lochner, Nina; Lobmaier, Christina; Wirth, Michael; Leitner, Alfred; Pittner, Fritz; Gabor, Franz


    Silver nanoparticle enhanced fluorescence is introduced as an alternative method to surface plasmon resonance techniques for real time monitoring of biorecognitive interactions or immunoassays. This method relies on the phenomenon that an electromagnetic near field is generated upon illumination on the surface of silver nanoparticles. The interaction of this field with nearby fluorophores results in fluorescence enhancement. Thus, fluorophores in the bulk solution can be discriminated from surface bound fluorophores. Anti-insulin-antibodies were immobilized on the surface of silver colloids in the following order: A ready to use microplate was prepared by bottom up coating with layers of aminosilane, silver nanoparticles, Fc-recognizing F(ab)(2)-fragments and anti-insulin-antibodies. At equilibrium conditions fluorescein-labeled insulin could only be detected in the presence of the colloid; the detection limit was 250 nM, and a fourfold increase in fluorescence was observed upon real time monitoring. The competitive assay of labeled and unlabeled insulin revealed a working range of 10-200 nM insulin in serum. The rapid single step immunoassay is easy to perform even in microplate format, its sensitivity is comparable to ELISA techniques, and offers broad application for real time monitoring of molecular recognitive processes.

  18. Thermal stability of pepsin: A predictive thermodynamic model of a multi-domain protein

    Directory of Open Access Journals (Sweden)

    Ali Asghar Rastegari


    Full Text Available Pepsin is generally used in the preparation of F(ab2 fragments from antibodies. The antibodies that are one of the largest and fastest growing categories of bio- pharmaceutical candidates. Differential scanning calorimetric is principally suitable method to follow the energetics of a multi-domain, fragment to perform a more exhaustive description of the thermodynamics in an associating system. The thermodynamical models of analysis include the construction of a simultaneous fitting of a theoretical expression. The expression depending on the equilibrium unfolding data from multimeric proteins that have a two-state monomer. The aim of the present study is considering the DSC data in connection with pepsin going through reversible thermal denaturation. Afterwards, we calculate the homology modeling identification of pepsin in complex multi-domain families with varied domain architectures. In order to analyze the DSC data, the thermal denaturation of multimer proteins were considered, the “two independent two-state sequential transitions with domains dissociation model” was introduced by using of the effective ΔG concept. The reversible unfolding of the protein description was followed by the two-state transition quantities which is a slower irreversible process of aggregation. The protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. These evaluations are also applicable for high throughput investigation of protein stability.

  19. Development of an enzyme-linked immunosorbent assay system for detecting β'-component (Onk k 5), a major IgE-binding protein in salmon roe. (United States)

    Shimizu, Yutaka; Oda, Hiroshi; Seiki, Kohsuke; Saeki, Hiroki


    A novel enzyme-linked immunosorbent assay (ELISA) system has been established for selective detection of chum salmon (Oncorhynchus keta) yolk protein (SYP). Rabbit and rat polyclonal Immunoglobulin G antibodies to β'-component (the major allergic protein in fish roe; anti-β) were applied for designing the ELISA system. The sandwich ELISA using rabbit anti-β for the capture antibody and horseradish peroxidase-labeled F(ab')2 fragment of rat anti-β for the detection antibody obtained high sensitivity and narrow specificity for SYP. Protein extraction using sodium dodecyl sulfate and 2-mercaptoethanol ensured strict specificity of the ELISA, and components of three popular processed foods had no effect on the ELISA response. The limits of determination and quantification of SYP were estimated to be 0.78 μg/g and 2.60 μg/g of food sample, respectively. In conclusion, the developed ELISA system has a probability to be applied for the detection of contaminated chum salmon roe in processed food.

  20. DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin. (United States)

    Werner, P A; Galbraith, R M; Arnaud, P


    Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

  1. Mechanism of Action of Anti-TNF Therapy in Inflammatory Bowel Disease. (United States)

    Levin, Alon D; Wildenberg, Manon E; van den Brink, Gijs R


    Several anti-tumour necrosis factor [TNF] blocking strategies have been evaluated in patients with Crohn's disease. Compounds that have been tested included the full monoclonal IgG1 antibodies infliximab and adalimumab, the pegylated anti-TNF F[ab']2 fragment certolizumab, an IgG4 anti-TNF CDP571 with reduced affinity for the Fc receptor, the soluble TNF receptor I onercept, and the TNF receptor II-Fc fusion protein etanercept. The endpoints of these studies suggest that not all methods of blocking TNF are equal. Here we will review the differences in the clinical, biochemical, and endoscopic endpoints of the major clinical studies. Collectively the data suggest that only IgG1 monoclonal antibodies have the ability to induce complete clinical, biochemical, and endoscopic remission. We discuss the potential multiple modes of action that may contribute to the response to full IgG1 anti-TNFs, focusing on the rapid induction of lamina propria T cell apoptosis and Fc receptor-dependent induction of M2-type wound-healing macrophages. We discuss how novel insights into the mechanism of action of anti-TNFs in Crohn's disease may contribute to the development of novel anti-TNFs with improved efficacy.

  2. Intracellular uptake and catabolism of anti-IgM antibodies and bi-specific antibody-targeted hapten by B-lymphoma cells. (United States)

    Manetti, C; Le Doussal, J M; Rouvier, E; Gruaz-Guyon, A; Barbet, J


    The efficiency of radioimmunotherapy with iodine-labelled antibodies is often limited by intracellular internalisation and catabolism after initial binding to the cellular targets. We have developed a technique called affinity enhancement system (AES) which uses bi-specific antibodies to target radiolabelled bivalent haptens to cells. This targeting method has been applied successfully to tumour imaging in colorectal cancer patients and is now considered for therapy. We have investigated the potential of this technique to target iodine radioisotopes by comparing it to targeting with covalently iodine-labelled antibodies in a rapidly internalising antigenic system, the surface IgM of a B-lymphoma cell line. A 5-fold increase in the intracellular retention time of activity as compared to 125I-labelled F(ab')2 or IgG was observed. The radiolabelled hapten did not undergo any catabolism after internalisation. Resistance to cellular proteases and failure of recognition of the hapten by amino acid transporter systems may be potential explanations for these observations. This should make non-covalent targeting, particularly the AES, a method of choice to target modulating antigens for the therapy of malignant hemopathies.

  3. A new ELISA for determination of potency in snake antivenoms. (United States)

    Rial, A; Morais, V; Rossi, S; Massaldi, H


    A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.

  4. Modulation of innate immune responses by influenza-specific ovine polyclonal antibodies used for prophylaxis.

    Directory of Open Access Journals (Sweden)

    Catherine Rinaldi

    Full Text Available In the event of a novel influenza A virus pandemic, prophylaxis mediated by antibodies provides an adjunct control option to vaccines and antivirals. This strategy is particularly pertinent to unvaccinated populations at risk during the lag time to produce and distribute an effective vaccine. Therefore, development of effective prophylactic therapies is of high importance. Although previous approaches have used systemic delivery of monoclonal antibodies or convalescent sera, available supply is a serious limitation. Here, we have investigated intranasal delivery of influenza-specific ovine polyclonal IgG antibodies for their efficacy against homologous influenza virus challenge in a mouse model. Both influenza-specific IgG and F(ab'2 reduced clinical scores, body weight loss and lung viral loads in mice treated 1 hour before virus exposure. Full protection from disease was also observed when antibody was delivered up to 3 days prior to virus infection. Furthermore, effective prophylaxis was independent of a strong innate immune response. This strategy presents a further option for prophylactic intervention against influenza A virus using ruminants to generate a bulk supply that could potentially be used in a pandemic setting, to slow virus transmission and reduce morbidity associated with a high cytokine phenotype.

  5. Implication of Hsc70, PDI and integrin αvβ3 involvement during entry of the murine rotavirus ECwt into small-intestinal villi of suckling mice. (United States)

    Santana, Ana Y; Guerrero, Carlos A; Acosta, Orlando


    In the present study, a homologous rotavirus, ECwt, infecting small intestinal villi isolated from ICR and BALB/c mice were used as a model for identifying cell-surface molecules involved in rotavirus entry. Small-intestinal villi were treated with anti-Hsc70, anti-PDI, anti-integrin β3 or anti-ERp57 antibodies or their corresponding F(ab')2 fragments before inoculation with rotavirus ECwt, RRV or Wa. Pretreatment of villi decreased virus infectivity by about 50-100 % depending of the rotavirus strain, antibody structure and detection assay used. Similar results were obtained by treating viral inocula with purified proteins Hsc70, PDI or integrin β3 before inoculation of untreated villi. Rotavirus infection of villi proved to be sensitive to membrane-impermeant thiol/disulfide inhibitors such as DTNB and bacitracin, suggesting the involvement of a redox reaction in infection. The present results suggest that PDI, Hsc70 and integrin β3 are used by both homologous and heterologous rotaviruses during infection of isolated mouse villi.

  6. Assessment of 188Re marked anti MHC class Ⅱ antibody by peripheral blood mononuclear cells stimulated by donor alloantigen

    Institute of Scientific and Technical Information of China (English)

    DING Guo-ping; CAO Li-ping; LIU Jie; LIU Da-ren; QUE Ri-sheng; ZHU Lin-hua; ZHOU Yi-ming; MAO Ke-jie; HU Jun-an


    Background Previous studies showed that anti MHC-Ⅱ monoclone antibody (MAb) only had partial inhibiting effect of alloreactive mixed lymphocyte reaction (MLR) in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope 188Re marked MHC-Ⅱ antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-Ⅱ antibody.Methods 188Re was incorporated to 2E9/13F(ab')2 which is against swine MHC class Ⅱ antigen (MAb-188Re). Porcine peripheral blood mononuclear (PBMC) cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-Ⅱ MAb or MAb-188Re.Results The proliferative response of recipient PBMCs in mixed lymphocyte reaction (MLR) to donor alloantigen showed that the stimulation index of MAb-188Re group was significantly lower than the MHC-Ⅱ MAb group and control (P<0.05). mRNA expression of interleukin 2, interferon Y and tumor necrosis factor α (type 1 cytokines) was lower in MAb-188Re group than the MHC-Ⅱ MAb group, while interleukin 10 (type 2 cytokines) was higher in MAb-188Re group in the first 24 hours.Conclusion MAb-188Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.

  7. Treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis with high-dose intravenous immunoglobulin. (United States)

    Richter, C; Schnabel, A; Csernok, E; De Groot, K; Reinhold-Keller, E; Gross, W L


    In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG), 30 g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin, ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab')2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on anti-PR3 activity. An inhibition of anti-PR3 activity by 25-70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality.

  8. Black widow spider (Latrodectus mactans) antivenom in clinical practice. (United States)

    Monte, Andrew A


    Black widow spider (Latrodectus mactans) envenomation has been recognized since antiquity. The syndrome, latrodectism, is characterized by painful muscle rigidity and autonomic disturbances such as tachycardia, hypertension, and diaphoresis. Symptoms typically last for 1-3 days. Treatment has ranged from local folk remedies to administration of specific antivenom. Opioid analgesics combined with muscle relaxants, such as benzodiazepines, are only effective at symptomatic and temporary control. Antivenom is by far the most efficacious therapy available based on symptom resolution, need for subsequent therapy, and hospital admission rates. Fear of allergic type reactions from antivenom administration has limited its use in the United States. A new purified F(ab)2 fragment Latrodectus mactans antivenom, Analatro®, is currently undergoing clinical trials. The product is expected to have similar efficacy and be associated with fewer adverse reactions when compared to the currently available partially purified whole IgG Merck product. This shift in the risk-benefit analysis may ultimately lead to more antivenom administration in significantly envenomated patients.

  9. Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab. (United States)

    Golay, Josée; Da Roit, Fabio; Bologna, Luca; Ferrara, Claudia; Leusen, Jeanette H; Rambaldi, Alessandro; Klein, Christian; Introna, Martino


    Obinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer-mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcγR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab')2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B.

  10. The Effect of CD3-Specific Monoclonal Antibody on Treating Experimental Autoimmune Myasthenia Gravis

    Institute of Scientific and Technical Information of China (English)

    Ruonan Xu; Jianan Wang; Guojiang Chen; Gencheng Han; Renxi Wang; Beffen Shen; Yan Li


    CD3-specific monoclonal antibody was the first one used for clinical practice in field of transplantation. Recently,renewed interests have elicited in its capacity to prevent autoimmune diabetes by inducing immune tolerance. In this study, we tested whether this antibody can also be used to treat another kind of autoimmune disease myasthenia gravis (MG) and explored the possible mechanisms. MG is caused by an autoimmune damage mediated by antibody- and complement-mediated destruction of AChR at the neuromuscular junction. We found that administration of CD3-specific antibody (Fab)2 to an animal model with experimental autoimmune myasthenia gravis (EAMG) (B6 mice received 3 times of AChR/CFA immunization) could not significantly improve the clinical signs and clinical score. When the possible mechanisms were tested, we found that CD3 antibody treatment slightly down-regulated the T-cell response to AChR, modestly up-regulation the muscle strength. And no significant difference in the titers of IgG2b was found between CD3 antibody treated and control groups. These data indicated that CD3-specific antibody was not suitable for treating MG, an antibody- and complementmediated autoimmune disease, after this disease has been established. The role of CD3-specific antibody in treating this kind of disease remains to be determined.

  11. A sensitive enzyme immunoassay for human epidermal growth factor. Determination of hEGF in human serum and urine and pharmacokinetics in mouse. (United States)

    Hayashi, T; Hashimoto, K; Sakamoto, S


    A sensitive enzyme immunoassay for human epidermal growth factor (hEGF) is described. The anti-hEGF antibody was prepared by immunizing rabbits with hEGF, which was synthesized by Escherichia coli using the genetic engineering technique. The present assay system was based on the sandwiching of an antigen between anti-hEGF F(ab')2 precoated on a 96-well polystyrene plate and beta-D-galactosidase-labeled anti-hEGF Fab'. The range of measurable hEGF by this assay was 0.1-100 pg/well. Recoveries of hEGF added to serum and urine ranged between 94 and 108%. The intra- and inter-assay coefficients of variation were less than 6 and 8%, respectively. The results obtained by this assay method correlated well with those obtained by the radioimmunoassay method. By using this assay, the time course of serum hEGF levels in mice after the various administrations were also examined.

  12. 双功能螯合剂BAT与鼠IgG1 Fab'片段的点特异性交联方法的研究%Site-Specific Conjugation of Bifunctional Chelator BAT to Mouse IgG1 Fab' Fragment

    Institute of Scientific and Technical Information of China (English)

    李君; 王学浩; 成峰; 王晓明; 陈兆雷; 沈喜平


    目的报道一种用双功能螯合剂BAT与鼠单克隆抗体Fab'片段行点特异性交联的新方法.方法通过位于Fab'铰链区、远离抗原结合位点的活性巯基(-SH)直接与BAT相交联.用简单的两步法制备B43单抗的Fab'片段.首先单抗用胃蛋白酶处理,产生稳定的F(ab')2 片段,然后 F(ab')2经半胱氨酸还原后产生Fab'片段,最后Fab'片段直接与BAT交联.结果每个Fab'分子平均含1.8个-SH基团,74%的Fab'片段可与BAT生成交联物,平均每个Fab'分子携带1.28个BAT.经检测,F(ab')2、Fab' 及 Fab'-BAT 均保持着良好的免疫活性.结论该交联法简单、高效,为双功能螯合剂BAT与鼠单抗Fab'片段的交联提供了一个新途径.

  13. A reduced immunization scheme to obtain an experimental anti-Loxosceles laeta ("violinist spider") venom. (United States)

    de Roodt, Adolfo Rafael; Litwin, Silvana; Dokmetjian, José Christian; Vidal, Juan Carlos


    Bites by Loxosceles (L.) laeta spiders can produce severe envenomation in humans. The only specific treatment is the early administration of antivenom. The production of anti-Loxosceles antivenom is hampered by the extremely low venom yield by these spiders and by the difficulties in maintaining a large breeder of Loxosceles. We developed an experimental equinum L. laeta antivenom, using as immunogen venom glands homogenates from spiders captured in Argentina. Horses immunized with venom gland homogenate (1.0 mg total protein per horse) by the subcutaneous route were bled after completion of the immunization scheme. Plasma was fractionated by ammonium sulfate precipitation and treated with pepsin to obtain F(ab')2 fragments. The protein composition of the experimental antivenom was assessed by SDS-PAGE, and its immunochemical reactivity was compared with those of other anti-Loxosceles antivenoms available for therapeutic use in Argentina by ELISA and Western blot. The experimental, homologous anti-L. laeta antivenom appeared to be more efficient in neutralizing the lethal potency in mice and the necrotizing activity in rabbits than of the heterologous antivenom.

  14. Blockage of complement regulators in the conjunctiva and within the eye leads to massive inflammation and iritis. (United States)

    Bardenstein, D S; Cheyer, C J; Lee, C; Cocuzzi, E; Mizuno, M; Okada, N; Medof, M E


    The open environment of the eye is continuously subject to an influx of foreign agents that can activate complement. Decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 are regulators that protect self-cells from autologous complement activation on their surfaces. They are expressed in the eye at unusually high levels but their physiological importance in this site is unstudied. In the rat, a structural analogue termed 5I2 antigen (5I2 Ag) has actions overlapping DAF and MCP. In this investigation, we injected F(ab')2 fragments of 5I2 mAb into the conjunctiva and aqueous humor, in the latter case with and without concomitant blockage of CD59. Massive neutrophilic infiltration of the stroma and iris resulted upon blocking 5I2 Ag activity. Frank necrosis of the iris occurred upon concomitant intraocular blockage of CD59. C3b was identified immunohistochemically, and minimal effects were seen in complement-depleted animals and in those treated with non-relevant antibody. The finding that blockage of 5I2 Ag function in periocular tissues and within the eye causes intense conjunctival inflammation and iritis demonstrates the importance of intrinsic complement regulators in protecting ocular tissues from spontaneous or bystander attack by autologous complement.

  15. Delayed anti-CD3 therapy results in depletion of alloreactive T cells and the dominance of Foxp3+ CD4+ graft infiltrating cells. (United States)

    Goto, R; You, S; Zaitsu, M; Chatenoud, L; Wood, K J


    The engineered Fc-nonbinding (crystallizable fragment-nonbinding) CD3 antibody has lower mitogenicity and a precise therapeutic window for disease remission in patients with type 1 diabetes. Before anti-CD3 can be considered for use in transplantation, the most effective timing of treatment relative to transplantation needs to be elucidated. In this study anti-CD3F(ab')2 fragments or saline were administered intravenously for 5 consecutive days (early: d1-3 or delayed: d3-7) to mice transplanted with a cardiac allograft (H2(b)-to-H2(k); d0). Survival of allografts was prolonged in mice treated with the early protocol (MST = 48 days), but most were rejected by d100. In contrast, in mice treated with the delayed protocol allografts continued to survive long term. The delayed protocol significantly inhibited donor alloreactivity at d30 as compared to the early protocol. A marked increase in Foxp3(+) T cells (50.3 ± 1.6%) infiltrating the allografts in mice treated with the delayed protocol was observed (p d100. We conclude that the timing of treatment with anti-CD3 therapy is critical for inducing long-term graft survival. Delaying administration effectively inhibits the alloreactivity and promotes the dominance of intragraft Foxp3(+) T cells allowing long-term graft acceptance. © 2013 The Authors. American Journal of Transplantation Published by Wiley Periodicals Inc.

  16. Silencing Bruton's tyrosine kinase in alveolar neutrophils protects mice from LPS/immune complex-induced acute lung injury. (United States)

    Krupa, Agnieszka; Fol, Marek; Rahman, Moshiur; Stokes, Karen Y; Florence, Jon M; Leskov, Igor L; Khoretonenko, Mikhail V; Matthay, Michael A; Liu, Kathleen D; Calfee, Carolyn S; Tvinnereim, Amy; Rosenfield, Gabriel R; Kurdowska, Anna K


    Previous observations made by our laboratory indicate that Bruton's tyrosine kinase (Btk) may play an important role in the pathophysiology of local inflammation in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We have shown that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study the function of Btk in vivo we have developed a unique two-hit model of ALI: LPS/immune complex (IC)-induced ALI. Furthermore, we conjugated F(ab)2 fragments of anti-neutrophil antibodies (Ly6G1A8) with specific siRNA for Btk to silence Btk specifically in alveolar neutrophils. It should be stressed that we are the first group to perform noninvasive transfections of neutrophils, both in vitro and in vivo. Importantly, our present findings indicate that silencing Btk in alveolar neutrophils has a dramatic protective effect in mice with LPS/IC-induced ALI, and that Btk regulates neutrophil survival and clearance of apoptotic neutrophils in this model. In conclusion, we put forward a hypothesis that Btk-targeted neutrophil specific therapy is a valid goal of research geared toward restoring homeostasis in lungs of patients with ALI/ARDS. Copyright © 2014 the American Physiological Society.

  17. Thrombin Increases Expression of Fibronectin Antigen on the Platelet Surface (United States)

    Ginsberg, Mark H.; Painter, Richard G.; Forsyth, Jane; Birdwell, Charles; Plow, Edward F.


    Fibronectins (fn) are adhesive glycoproteins which bind to collagen and to fibrin and appear to be important in cellular adhesion to other cells or surfaces. Fn-related antigen is present in human platelets, suggesting a possible role for fn in the adhesive properties of platelets. We have studied the localization of fn in resting and thrombin-stimulated platelets by immunofluorescence and quantitative binding of radiolabeled antibody. In resting fixed platelets, variable light surface staining for fn was observed. When these cells were made permeable to antibody with detergent, staining for fn was markedly enhanced and was present in a punctate distribution, suggesting intracellular localization. Stimulation with thrombin, which is associated with increased platelet adhesiveness, resulted in increased staining for fn antigen on intact platelets. These stimulated cells did not leak 51Cr nor did they stain for F-actin, thus documenting that the increased fn staining was not due to loss of plasma membrane integrity. The thrombin-induced increase in accessible platelet fn antigen was confirmed by quantitative antibody binding studies in which thrombin-stimulated platelets specifically bound 15 times as much radiolabeled F(ab')2 anti-fn as did resting cells. Thus, thrombin stimulation results in increased expression of fn antigen on the platelet surface. Here it may participate in interactions with fibrin, connective tissue, or other cells.

  18. Evolution of endotoxin contamination during production of a therapeutic serum. (United States)

    Massaldi, Hugo; Morais, Victor


    A comparative bench-scale study of endotoxin contamination is presented for two common processes of immunoglobulin purification from equine plasma: ammonium sulphate fractionation of F(ab')2 fragments and caprylic acid precipitation of non-IgG proteins. To this end, both processes were carried out under normal sterile conditions, using sanitized material and equipment and optimal water quality in a clean but open environment. Stream samples, taken at different stages from each process, were analyzed for endotoxin content by the Limulus Amebocyte Lysate (LAL) test. It was found that exogenous contamination preferentially came from endotoxins already present in reagents and/or raw materials, whereas contamination from the environment was minimal. Endogenous endotoxin accumulation, concomitant with the concentration of proteins during processing, was found to be an important factor. With classic technology, blood extraction and sterilizing filtration are critical points for both processes. It is concluded that sterility is not a sufficient condition to obtain an endotoxin-free product. Only with proper sanitization of material, and by applying the caprylic acid purification process with a starting plasma below 4-5 EU/mL, would it be possible to achieve a final product within the norm.

  19. Architecture of Human IgM in Complex with P. falciparum Erythrocyte Membrane Protein 1

    Directory of Open Access Journals (Sweden)

    Reetesh Raj Akhouri


    Full Text Available Plasmodium falciparum virulence is associated with sequestration of infected erythrocytes. Microvascular binding mediated by PfEMP1 in complex with non-immune immunoglobulin M (IgM is common among parasites that cause both severe childhood malaria and pregnancy-associated malaria. Here, we present cryo-molecular electron tomography structures of human IgM, PfEMP1 and their complex. Three-dimensional reconstructions of IgM reveal that it has a dome-like core, randomly oriented Fab2s units, and the overall shape of a turtle. PfEMP1 is a C- shaped molecule with a flexible N terminus followed by an arc-shaped backbone and a bulky C terminus that interacts with IgM. Our data demonstrate that the PfEMP1 binding pockets on IgM overlap with those of C1q, and the bulkiness of PfEMP1 limits the capacity of IgM to interact with PfEMP1. We suggest that P. falciparum exploits IgM to cluster PfEMP1 into an organized matrix to augment its affinity to host cell receptors.

  20. Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv). (United States)

    Zhou, N; Paemen, L; Opdenakker, G; Froyen, G


    The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

  1. Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Nielsen, EH; Andersen, Ove


    Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy....... Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high...... affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate...

  2. IVIg inhibits reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia independent of anti-idiotype reactivity. (United States)

    Crow, A R; Song, S; Semple, J W; Freedman, J; Lazarus, A H


    Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab')2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti-idiotype interactions are not required.

  3. A nationwide study of Vipera berus bites during one year-epidemiology and morbidity of 231 cases. (United States)

    Karlson-Stiber, Christine; Salmonson, Heléne; Persson, Hans


    To describe clinical course, influence of treatment, and epidemiology of Vipera berus envenomation in a defined population, and to compare the results with those of a similar, nationwide study in 1975. Design. Retrospective case review study. Case records regarding all patients treated in Swedish hospitals during 1995 for bites by the common European adder, V. berus, were studied. A severity grading was applied. Possible dropout was fewer than 10 patients. A total of 231 inpatients were treated for V. berus bites in Sweden in 1995. Children less than 10 years old were overrepresented and there was a slight predominance for males. Maximum severity of envenomation was none in 11%, minor in 47%, moderate in 29%, and severe in 13% of the cases. A few patients with initially minor or moderate symptoms eventually met the criteria of severe envenomation. Less commonly reported features were pulmonary edema, generalized plasma leakage, seizures, deep venous thrombosis, compartment syndrome, numbness and paraesthesia, and myocardial infarction. Treatment included antivenom in 42 patients [ovine Fab in 30 and equine F(ab')2 in 12 cases]. Systemic symptoms resolved during or shortly after the antivenom infusion. Extensive edema involving the trunk occurred in 5% of the cases in 1995, whereas 14% of the patients had extensive swelling in 1975. Incidence and other epidemiological data were similar to those 20 years ago, whereas the clinical course was more benign. It seems reasonable to believe that this is due to the introduction of effective antivenoms.

  4. Binding of complement proteins C1q and C4bp to serum amyloid P component (SAP) in solid contra liquid phase

    DEFF Research Database (Denmark)

    Sørensen, Inge Juul; Nielsen, EH; Andersen, Ove;


    Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy....... Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high...... affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate...

  5. Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy. (United States)

    Hébert, J; Bernier, D; Mourad, W


    Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.

  6. Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

    Directory of Open Access Journals (Sweden)

    Justin M Scheer

    Full Text Available The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab'(2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

  7. Elastic-contractile model proteins: Physical chemistry, protein function and drug design and delivery. (United States)

    Urry, Dan W; Urry, Kelley D; Szaflarski, Witold; Nowicki, Michal


    This review presents the structure and physico-chemical properties of ECMPs, elastic-contractile model proteins using sparse design modifications of elastic (GVGVP)(n); it describes the capacity of ECMP to perform the energy conversions that sustain living organisms; it arrives at the hydration thermodynamics of ECMP in terms of the change in Gibbs free energy of hydrophobic association, ΔG(HA), and the apolar-polar repulsive free energy of hydration, ΔG(ap); it applies ΔG(HA), ΔG(ap), and the nature of elasticity to describe the function of basic diverse proteins, namely - the F₁-motor of ATP synthase, Complex III of mitochondria, the KscA potassium-channel, and the molecular chaperonin, GroEL/ES; it applies ΔG(HA) and ΔG(ap) to describe the function of ABC exporter proteins that confer multi-drug resistance (MDR) on micro-organisms and human carcinomas and suggests drug modifications with which to overcome MDR. Using ECMP, means are demonstrated, for quantifying drug hydrophobicity with which to combat MDR and for preparing ECMP drug delivery nanoparticles, ECMPddnp, decorated with synthetic antigen-binding fragments, Fab1 and Fab2, with which to target specific up-regulated receptors, characteristic of human carcinoma cells, for binding and localized drug release. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. [Evaluation of the diagnostic usefulness of CA125 immunoscintigraphy for ovarian carcinoma follow-up after treatment: contribution of this technique in Grenoble University Medical Center]. (United States)

    Vuillez, J P; Levrot, E; Mousseau, M; Buffaz, P D; Bolla, M; Payan, R; Comet, M; Schaerer, R


    Immunoscintigraphy using indium-111-labeled OC125 monoclonal antibody F(ab')2 fragments is a technic complementary of morphological imaging (i.e. ultrasonography and computed tomography). It allows early detection of recurrences of ovarian carcinomas. We performed immunoscintigraphy 30 times in 26 patients who previously underwent radical treatment for ovarian carcinoma, and were suspected to have a recurrence. Our purposes were appreciation of diagnostic accuracy of the method, and above all its impact on clinical decisions and evolution of the patients. There were, after reevaluation of the results, 18 true positives, 7 true negatives, 3 false negatives and 2 false positive cases (sensitivity 85.7%, specificity 77.8%). Bayesian analysis showed positive and negative predictive values of 86% and 87% when probability of recurrence a priori was 50%, and 80% and 58% when probability of recurrence a priori was 70%. The result of immunoscintigraphy contributed to clinical decisions in 24 cases out of 30, and led to a correct decision for the patient in 21 cases. Conversely, for the 6 cases in which the result has not been considered, to take this result into account would have been beneficial in 4 cases, but harmful in 2. Finally, survival tended to be longer when immunoscintigraphy was negative, which could be associated with a better prognosis. We conclude that OC125-immunoscintigraphy may be useful for ovarian carcinoma follow-up and may contribute to a better therapeutic strategy.

  9. Science Letters: Demonstration of a new biosensing concept for immunodiagnostic applications based on change in surface conductance of antibodies after biomolecular interactions

    Institute of Scientific and Technical Information of China (English)

    VASHIST Sandeep Kumar; KAUR Inderpreet; BAJPAI Ram Prakash; BHARADWAJ Lalit Mohan; TEWARI Rupinder; RAITERI Roberto


    We report an important observation that the surface conductivity of antibody layer immobilized on polylysine-coated glass substrate decreases upon the formation of complex with their specific antigens. This change in conductivity has been observed for both monoclonal and polyclonal antibodies. The conductance of monoclonal mouse IgG immobilized on polylysine-coated glass substrate changed from 1.02×l0-8 Ω-1 to 1.41×l0-11 Ω-1 at 10 V when complex is formed due to the specific biomolecular interactions with rabbit anti-mouse IgG F(ab')2. Similar behavior was observed when the same set up was tested in two clinical assays: (1) anti-Leishmania antigen polyclonal antibodies taken from Kala Azar positive patient serum interacting with Leishmania promastigote antigen, and (2) anti-p21 polyclonal antibodies interacting with p21 antigen. The proposed concept can represent a new immunodiagnostic technique and may have wide ranging applications in biosensors and nanobiotechnology too.

  10. Characterization of self-assembled monolayers (SAMs) on silicon substrate comparative with polymer substrate for Escherichia coli O157:H7 detection (United States)

    Moldovan, Carmen; Mihailescu, Carmen; Stan, Dana; Ruta, Lavinia; Iosub, Rodica; Gavrila, Raluca; Purica, Munizer; Vasilica, Schiopu


    This article presents the characterization of two substrates, silicon and polymer coated with gold, that are functionalized by mixed self-assembled monolayers (SAMs) in order to efficiently immobilize the anti- Escherichia coli O157:H7 polyclonal purified antibody. A biosurface functionalized by SAMs (self-assembled monolayers) technique has been developed. Immobilization of goat anti- E. coli O157:H7 antibody was performed by covalently bonding of thiolate mixed self-assembled monolayers (SAMs) realized on two substrates: polymer coated with gold and silicon coated with gold. The F(ab') 2 fragments of the antibodies have been used for eliminating nonspecific bindings between the Fc portions of antibodies and the Fc receptor on cells. The properties of the monolayers and the biofilm formatted with attached antibody molecules were analyzed at each step using infrared spectroscopy (FTIR-ATR), atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In our study the gold-coated silicon substrates approach yielded the best results. These experimental results revealed the necessity to investigate each stage of the immobilization process taking into account in the same time the factors that influence the chemistry of the surface and the further interactions as well and also provide a solid basis for further studies aiming at elaborating sensitive and specific immunosensor or a microarray for the detection of E. coli O157:H7.

  11. Epidermal polymeric immunoglobulin receptors: leads from intraepidermal neutrophilic IgA dermatosis-type IgA pemphigus. (United States)

    Tsuchisaka, Atsunari; Ishii, Norito; Hamada, Takahiro; Teye, Kwesi; Sogame, Ryosuke; Koga, Hiroshi; Tsuruta, Daisuke; Ohata, Chika; Furumura, Minao; Hashimoto, Takashi


    In this study, we attempted to identify unknown autoantigen for intraepidermal neutrophilic IgA dermatosis-type IgA pemphigus by novel IgA-specific immunoprecipitation. Mass-spectrometry study identified polymeric immunoglobulin receptor (PIGR) as the candidate protein, and we confirmed that PIGR expressed in both epidermis and cultured keratinocytes. Eukaryotic recombinant protein of PIGR expressed in COS7 cells was reacted with both patient and normal sera, indicating that PIGR binds physiologically to IgA. To detect antigen-specific binding by IgA autoantibodies, we performed several experiments using deglycosylated PIGR and F(ab)2 fragments from patient sera. However, these analyses suggested that patient IgA bound physiologically, but not immunologically, to PIGR. Nevertheless, our study provided two important insights. Newly developed IgA-immunoprecipitation system should be a useful tool in the future study of identification of antigens for IgA autoantibodies. Detection of epidermal PIGR in this study confirmed previous results and indicated possible immunological role of PIGR in epidermis.

  12. Exposure levels of anti-LINGO-1 Li81 antibody in the central nervous system and dose-efficacy relationships in rat spinal cord remyelination models after systemic administration. (United States)

    Pepinsky, R Blake; Shao, Zhaohui; Ji, Benxiu; Wang, Qin; Meng, Gym; Walus, Lee; Lee, Xinhua; Hu, Yinghui; Graff, Christilyn; Garber, Ellen; Meier, Werner; Mi, Sha


    LINGO-1 (leucine-rich repeat and Ig domain containing NOGO receptor interacting protein-1) is a negative regulator of myelination and repair of damaged axons in the central nervous system (CNS). Blocking LINGO-1 function leads to robust remyelination. The anti-LINGO-1 Li81 antibody is currently being evaluated in clinical trials for multiple sclerosis (MS) and is the first MS therapy that directly targets myelin repair. LINGO-1 is selectively expressed in brain and spinal cord but not in peripheral tissues. Perhaps the greatest concern for Li81 therapy is the limited access of the drug to the CNS. Here, we measured Li81 concentrations in brain, spinal cord, and cerebral spinal fluid in rats after systemic administration and correlated them with dose-efficacy responses in rat lysolecithin and experimental autoimmune encephalomyelitis spinal cord models of remyelination. Remyelination was dose-dependent, and levels of Li81 in spinal cord that promoted myelination correlated well with affinity measurements for the binding of Li81 to LINGO-1. Observed Li81 concentrations in the CNS of 0.1 to 0.4% of blood levels are consistent with values reported for other antibodies. To understand the features of the antibody that affect CNS penetration, we also evaluated the pharmacokinetics of Li81 Fab2, Fab, and poly(ethylene glycol)-modified Fab. The reagents all showed similar CNS exposure despite large differences in their sizes, serum half-lives, and volumes of distribution, and area under the curve (AUC) measurements in the CNS directly correlated with AUC measurements in serum. These studies demonstrate that exposure levels achieved by passive diffusion of the Li81 monoclonal antibody into the CNS are sufficient and lead to robust remyelination.

  13. Remodeling of Membrane Lipids in Iron-starved Chlamydomonas* (United States)

    Urzica, Eugen I.; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M. Dudley; Casero, David; Gallaher, Sean D.; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S.


    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24–48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii. PMID:23983122

  14. Remodeling of membrane lipids in iron-starved Chlamydomonas. (United States)

    Urzica, Eugen I; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M Dudley; Casero, David; Gallaher, Sean D; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S


    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

  15. Glycidol modulation of the immune responses in female B6C3F1 mice. (United States)

    Guo, T L; McCay, J A; Brown, R D; Musgrove, D L; Butterworth, L; Munson, A E; Germolec, D R; White, K L


    The immunotoxic potential of glycidol was evaluated in female B6C3F1 mice using a battery of functional assays and three host resistance models. Glycidol was administered to the animals by oral gavage as a solution in sterile distilled water daily for 14 days at doses of 25, 125 and 250 mg/kg. In tier I, we observed that glycidol exposure produced a dose-related decrease in splenocyte IgM antibody-forming cell response to sheep red blood cells (sRBC); the spleen natural killer (NK) cell activity was also decreased. A decrease in B cell proliferative responses to anti-IgM F(ab')2 and/or interleukin-4 (IL-4) was observed while the splenocyte proliferative responses to T cell mitogen ConA and B cell mitogen LPS were not affected. The splenocyte proliferative response to allogeneic cells as evaluated in the mixed leukocyte reaction (MLR) to DBA/2 spleen cells was not affected. In tier II, we found that exposure to glycidol decreased the number and percentage of B cells and the absolute number of CD4+ T cells in the spleen while the number of total T cells, CD8+ T cells and CD4+CD8+ T cells was not affected. The cytotoxic T lymphocyte (CTL) response to mitomycin C-treated P815 mastocytoma was not affected; the cytotoxic activity of peritoneal macrophages was not suppressed. Moreover, the host resistance to Listeria monocytogenes was not affected although a slight increase in host resistance to Streptococcus pneumoniae was observed. However, exposure to glycidol decreased host resistance to the B16F10 melanoma tumor model with the maximal tumor formation in lung observed in the high dose group. Overall, these dada support the finding that glycidol is an immunosuppressive agent in female B6C3F1 mice.

  16. Human schistosomiasis mansoni: studies on in vitro granuloma modulation

    Directory of Open Access Journals (Sweden)

    Juçara C. Parra


    Full Text Available Infection with Schistosoma mansoni induces humoral and T cell mediated responses and leads to delayed hipersensitivity that results in granulomatous inflamatory disease around the parasite eggs. Regulation of these responses resulting in a reduction in this anti-egg inflamatory disease is appsrently determined by idiotypic repertoires of the patient, associated with genetic background and multiple external factors. We have previously reported on idiotype/anti-idiotype-receptor transactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive idiotypes develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We repport here on experiments wich extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA idiotypes and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA idiotype reactive T cells were capable of regulating autologous in vitro granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the present of intact (F(ab'2 immunoglobulin molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA sensitized individuals. These data support the hypothesis that anti-SEA cross reactive idiotypes are important in regulating granulomatous hypersensitivy in chronic intestinal schistosomiasis patients and these cross-reactive idiotypes appear to play a major role in cell-cell interactions which result in the regulation of anti-SEA cellular immune responses.

  17. Protein content of antivenoms and relationship with their immunochemical reactivity and neutralization assays. (United States)

    de Roodt, A R; Clement, H; Dolab, J A; Litwin, S; Hajos, S E; Boyer, L; Alagón, A


    Therapy for snakebites relies on the application of antivenoms, which may be produced with different immunogenic mixtures of venom and possess different pharmaceutical characteristics. For these reasons, immunological cross-reactivity and heterologous neutralization were analyzed relative to the protein content of three antivenoms used in the Americas. The antivenoms studied were composed of equine F(ab')2 fragments from animals immunized with Crotalinae venoms. The antivenoms were tested against venoms of seven pit viper species from Argentina, seven from Mexico, one from Costa Rica, and one from Colombia. Immunoblotting showed high cross-reactivity of all major protein bands with all the antivenoms tested. ELISA results also showed high cross-reactivity among the different venoms and antivenoms, and a high heterologous neutralization was observed. The results can be interpreted in different ways depending on whether the reactivity is considered in terms of the volume of antivenom used or by the amount of protein contained in this volume of antivenom. The antivenoms with high immunochemical reactivity and neutralizing capacity were those with higher protein content per vial; but when doses were adjusted by protein content, antivenoms of apparently lower neutralizing capacity and immunochemical reactivity showed at least similar potency and reactivity although volumetrically at higher doses. Protein content relative to neutralization potency of different products must be taken into account when antivenoms are compared, in addition to the volume required for therapeutic effect. These results show the importance of obtaining high-affinity and high-avidity antibodies to achieve good neutralization using low protein concentration and low-volume antivenoms.

  18. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages. (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco


    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  19. Fast and efficient proteolysis by reusable pepsin-encapsulated magnetic sol-gel material for mass spectrometry-based proteomics applications. (United States)

    Kayili, H Mehmet; Salih, Bekir


    Hydrophobic silicon-based material having magnetic properties was fairly synthesized by a classical sol-gel approach. Pepsin enzyme was encapsulated in the sol-gel material and the enzyme activity was evaluated in consequence of the digestion of some common proteins such as α- and β-casein, cytochrome c, myoglobin, and bovine serum albumin (BSA) both in a single protein batch and in the protein mixture. The optimum digestion time of the studied proteins using pepsin-encapsulated magnetic sol-gel material was found to be 20min. To produce the magnetic sol-gel material for convenient and easy proteomics applications, Fe3O4 was doped inside sol-gel material during the gelation step. It was observed that the activity of encapsulated pepsin was not affected by the amount of Fe3O4. Poly(ethylene glycol) was also inserted in sol-gel bulk to obtain suitable roughness and increase the hydrophilicity of the material surface to let protein molecules reach to the sol-gel material easily. The digestion of the protein mixture and non-fat bovine milk was performed with the pepsin-encapsulated magnetic sol-gel material and the digested solutions were analyzed using SDS-PAGE, MALDI-TOF-MS and LC-MS/MS for the protein identification. Reusability of the pepsin-encapsulated sol-gel material was examined and it was determined that they could be used at least 20 times. Finally, IgG digestions with a fast incubation time period were carried out using pepsin-encapsulated sol-gel material for generation of (Fab)2 product to evaluate the kinetic performance of the material.

  20. Human platelets express CAR with localization at the sites of intercellular interaction

    Directory of Open Access Journals (Sweden)

    Othman Maha


    Full Text Available Abstract Adenovirus has a wide tissue tropism. The virus attaches to the surface of cells via the fiber protein knob binding to the Coxsackie and Adenovirus receptor known as CAR. Virus entry inside cells is facilitated by integrins αVβ3 and αVβ5. Mice platelets are shown to be the predominant Ad binding blood cell type and the virus is documented inside platelets. CAR was identified on human platelets in one study yet contradicted in another. The presence of CAR appears to be the most reasonable initial step for virus entry into platelets and is a key to the understanding of platelet adenovirus interaction. This study aimed to re investigate the presence of CAR on human platelets. Platelets were tested by indirect immune-fluorescence using rabbit H-300 polyclonal anti-CAR antibody and goat anti-rabbit IgG F(ab'2 Texas Red antibodies, alongside with CAR positive and negative controls. Platelets were found to express CAR on their surface and in contrast to the previous study only 3.5 ± 1.9% of the tested platelets did express CAR. In addition, CAR was seen within intracellular aggregates localized at the sites of cell-cell contacts indicating that CAR expression might be upregulated in response to platelet stimulation. We confirm the presence of CAR on human platelets, we provide explanation to some of the discrepancies in this regards and we add that this receptor is localized at the sites of intercellular interaction.

  1. Exclusion of polymeric immunoglobulins and selective immunoglobulin Y transport that recognizes its Fc region in avian ovarian follicles. (United States)

    Kitaguchi, Kohji; Osada, Kenichi; Horio, Fumihiko; Murai, Atsushi


    In avian species, blood immunoglobulin (Ig) Y, the equivalent to mammalian IgG, is selectively incorporated into ovarian follicles, but other classes, IgA and IgM, are much less abundant in the follicles. Several mammalian Igs, including IgG and IgA, are also incorporated into ovarian follicles when administered to birds. To clarify the Ig structure required for incorporation into ovarian follicles, Ig uptakes were determined after the intravenous injection of chicken and human Igs. Three chicken Igs (cIgY, cIgA and cIgM) and two human IgAs (monomeric hIgA and polymeric hIgA) were labeled with digoxigenin, and their uptakes into quail (Coturnix japonica) egg yolks were determined by ELISA and SDS-PAGE. The uptake of cIgY was the highest among the three cIgs (22% of injected cIgY was recovered from egg yolks). Chicken IgA was efficiently incorporated into egg yolk when it formed a monomeric state. Pentameric IgM was untransportable into egg yolk. We also found that the uptake of monomeric hIgA was much more efficient than that of polymeric hIgA. These results suggest that the retention of the monomeric form contributes to the efficient transport of Igs into ovarian follicles. On the other hand, Ig uptakes among monomeric Igs nevertheless differed; for example, a time-course analysis showed that the rate of monomeric cIgY uptake was approximately eight times faster than that of monomeric hIgA. The injection of cIgY fragments Fc, Fab and F(ab')(2) resulted in the largest uptake of Fc fragment, with the same level as that of cIgY. These results suggest the presence of a selective IgY transport system that recognizes its Fc region in avian ovarian follicles.

  2. Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

    Directory of Open Access Journals (Sweden)

    Ahmad Bakur Mahmoud


    Full Text Available The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR. Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional", or unlicensed ("hypofunctional". Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab'2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

  3. Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines. (United States)

    De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L


    Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab')2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn.

  4. T cells play an essential role in anti-F1 mediated rapid protection against bubonic plague. (United States)

    Levy, Yinon; Flashner, Yehuda; Tidhar, Avital; Zauberman, Ayelet; Aftalion, Moshe; Lazar, Shirley; Gur, David; Shafferman, Avigdor; Mamroud, Emanuelle


    Plague, which is initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated. Antibiotics are an effective plague therapy, but antibiotic-resistant Y. pestis strains have been reported and therefore alternative countermeasures are needed. In the present study, we assessed the potential of an F1 plus LcrV-based vaccine to provide protection shortly pre- or post-exposure to a lethal Y. pestis infection. Mice vaccinated up to one day before or even several hours after subcutaneous challenge were effectively protected. Mice immunized one or three days pre-challenge were protected even though their anti-F1 and anti-LcrV titers were below detection levels at the day of challenge. Moreover, using B-cell deficient μMT mice, we found that rapidly induced protective immunity requires the integrity of the humoral immune system. Analysis of the individual contributions of vaccine components to protection revealed that rF1 is responsible for the observed rapid antibody-mediated immunity. Applying anti-F1 passive therapy in the mouse model of bubonic plague demonstrated that anti-F1 F(ab')(2) can delay mortality, but it cannot provide long-lasting protection, as do intact anti-F1 molecules. Fc-dependent immune components, such as the complement system and (to a lesser extent) neutrophils, were found to contribute to mouse survival. Interestingly, T cells but not B cells were found to be essential for the recovery of infected animals following passive anti-F1 mediated therapy. These data extend our understanding of the immune mechanisms required for the development of a rapid and effective post-exposure therapy against plague. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET. (United States)

    Schuhmacher, J; Klivényi, G; Kaul, S; Henze, M; Matys, R; Hauser, H; Clorius, J


    We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter 68Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab')(2) fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab') fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma. Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 x 10(7) M(-1)) while the binding capacity of cells was high (8.4 x 10(6) BS-MAbs per cell). Tumor uptake of the 67Ga labeled chelate in pretargeted animals was to 5.8 +/- 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with 125I-labeled native 12H12 at 24h and 48h postinjection. No difference in the tumor uptake of both the 68Ga and 67Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the 68Ga chelate, clearly visualized all tumors.

  6. Structural Analysis of Variable Domain Glycosylation of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Reveals the Presence of Highly Sialylated Glycans. (United States)

    Hafkenscheid, Lise; Bondt, Albert; Scherer, Hans U; Huizinga, Tom W J; Wuhrer, Manfred; Toes, René E M; Rombouts, Yoann


    Recently, we showed the unexpectedly high abundance of N-linked glycans on the Fab-domain of Anti-Citrullinated Protein Antibodies (ACPA). As N-linked glycans can mediate a variety of biological functions, we now aimed at investigating the structural composition of the Fab-glycans of ACPA-IgG to better understand their mediated biological effects. ACPA-IgG and noncitrulline specific (control) IgG from plasma and/or synovial fluid of nine ACPA positive rheumatoid arthritis patients were affinity purified. The N-linked glycosylation of total, Fc and F(ab')2 fragments, as well as heavy and light chains of ACPA-IgG and control IgG were analyzed by UHPLC and MALDI-TOF mass spectrometry. The Fc-glycosylation of ACPA-IgG and IgG was analyzed at the glycopeptide level using LC-MS. The structural analyses revealed that ACPA-IgG molecules contain highly sialylated glycans in their Fab-domain. Importantly, Fab-glycans were estimated to be present on over 90% of ACPA-IgG, which is five times higher than in control IgG isolated from the same patients. This feature was more prominent on ACPA isolated from synovial fluid compared with peripheral blood. These observations provide the first evidence pointing to the ability of ACPA-IgG to mediate novel immunological activities, for example through binding specific lectins via hyper-sialylated Fab-glycans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Pathogenic relevance of IgG and IgM antibodies against desmoglein 3 in blister formation in pemphigus vulgaris. (United States)

    Tsunoda, Kazuyuki; Ota, Takayuki; Saito, Masataka; Hata, Tsuyoshi; Shimizu, Atsushi; Ishiko, Akira; Yamada, Taketo; Nakagawa, Taneaki; Kowalczyk, Andrew P; Amagai, Masayuki


    Pemphigus vulgaris is an autoimmune disease caused by IgG antibodies against desmoglein 3 (Dsg3). Previously, we isolated a pathogenic mAb against Dsg3, AK23 IgG, which induces a pemphigus vulgaris-like phenotype characterized by blister formation. In the present study, we generated a transgenic mouse expressing AK23 IgM to examine B-cell tolerance and the pathogenic role of IgM. Autoreactive transgenic B cells were found in the spleen and lymph nodes, whereas anti-Dsg3 AK23 IgM was detected in the cardiovascular circulation. The transgenic mice did not develop an obvious pemphigus vulgaris phenotype, however, even though an excess of AK23 IgM was passively transferred to neonatal mice. Similarly, when hybridoma cells producing AK23 IgM were inoculated into adult mice, no blistering was observed. Immunoelectron microscopy revealed IgM binding at the edges of desmosomes or interdesmosomal cell membranes, but not in the desmosome core, where AK23 IgG binding has been frequently detected. Furthermore, in an in vitro dissociation assay using cultured keratinocytes, AK23 IgG and AK23 IgM F(ab')(2) fragments, but not AK23 IgM, induced fragmentation of epidermal sheets. Together, these observations indicate that antibodies must gain access to Dsg3 integrated within desmosomes to induce the loss of keratinocyte cell-cell adhesion. These findings provide an important framework for improved understanding of B-cell tolerance and the pathophysiology of blister formation in pemphigus. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Evidence for a role of eosinophils in blister formation in bullous pemphigoid. (United States)

    de Graauw, E; Sitaru, C; Horn, M; Borradori, L; Yousefi, S; Simon, H-U; Simon, D


    Bullous pemphigoid (BP) is an autoimmune bullous disease of the skin characterized by subepidermal blister formation due to tissue-bound and circulating autoantibodies to the hemidesmosomal antigens BP180 and BP230. Although eosinophils and their toxic mediators are found abundantly in BP lesions, their role in blister formation has remained unclear. To investigate the role of eosinophils in the pathogenesis of BP with a specific focus on blister formation and to define conditions inducing dermal-epidermal separation (DES). In an ex vivo human model of BP, normal human skin cryosections were incubated with purified human peripheral blood eosinophils with or without activation in the presence or absence of BP autoantibodies, brefeldin A, diphenyleneiodonium, DNase or blocking F(ab')2 fragments to CD16, CD18, CD32 and CD64. Dermal-epidermal separation was assessed by light microscopy studies and quantified using Fiji software. Following activation with IL-5 and in the presence of BP autoantibodies, eosinophils induced separation along the dermal-epidermal junction of ex vivo skin. Dermal-epidermal separation was significantly reduced by blocking any of the following: Fcγ receptor binding (P = 0.048), eosinophil adhesion (P = 0.046), reactive oxygen species (ROS) production (P = 0.002), degranulation (P blister formation in the presence of BP autoantibodies. Dermal-epidermal separation by IL-5-activated eosinophils depends on adhesion and Fcγ receptor activation, requires elevated ROS production and degranulation and involves EET formation. Thus, targeting eosinophils may be a promising therapeutic approach for BP. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. A Targeting Microbubble for Ultrasound Molecular Imaging.

    Directory of Open Access Journals (Sweden)

    James Shue-Min Yeh

    Full Text Available Microbubbles conjugated with targeting ligands are used as contrast agents for ultrasound molecular imaging. However, they often contain immunogenic (streptavidin, which impedes application in humans. Although targeting bubbles not employing the biotin-(streptavidin conjugation chemistry have been explored, only a few reached the stage of ultrasound imaging in vivo, none were reported/evaluated to show all three of the following properties desired for clinical applications: (i low degree of non-specific bubble retention in more than one non-reticuloendothelial tissue; (ii effective for real-time imaging; and (iii effective for acoustic quantification of molecular targets to a high degree of quantification. Furthermore, disclosures of the compositions and methodologies enabling reproduction of the bubbles are often withheld.To develop and evaluate a targeting microbubble based on maleimide-thiol conjugation chemistry for ultrasound molecular imaging.Microbubbles with a previously unreported generic (non-targeting components composition were grafted with anti-E-selectin F(ab'2 using maleimide-thiol conjugation, to produce E-selectin targeting microbubbles. The resulting targeting bubbles showed high specificity to E-selectin in vitro and in vivo. Non-specific bubble retention was minimal in at least three non-reticuloendothelial tissues with inflammation (mouse heart, kidneys, cremaster. The bubbles were effective for real-time ultrasound imaging of E-selectin expression in the inflamed mouse heart and kidneys, using a clinical ultrasound scanner. The acoustic signal intensity of the targeted bubbles retained in the heart correlated strongly with the level of E-selectin expression (|r|≥0.8, demonstrating a high degree of non-invasive molecular quantification.Targeting microbubbles for ultrasound molecular imaging, based on maleimide-thiol conjugation chemistry and the generic composition described, may possess properties (i-(iii desired for clinical applications.

  10. The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2). (United States)

    Del Valle, Juana M; Engel, Pablo; Martín, Margarita


    CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.

  11. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies (United States)

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon


    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  12. A new and robust method of tethering IgG surrogate antigens on lipid bilayer membranes to facilitate the TIRFM based live cell and single molecule imaging experiments.

    Directory of Open Access Journals (Sweden)

    Shaosen Zhang

    Full Text Available Our understanding of cell-cell interactions has been significantly improved in the past years with the help of Total Internal Reflection Fluorescence Microscope (TIRFM in combination with an antigen presenting system supported by planar lipid bilayer (PLB membranes, which are used to mimic the extensive receptor and ligand interactions within cell-cell contact interface. In TIRFM experiments, it is a challenge to uniformly present ligand molecules in monomeric format on the surface of PLB membranes. Here, we introduce a new and robust method of tethering IgG surrogate antigen ligands on the surface of Ni(2+-containing PLB membranes. In this method, we use a modified D domain from staphylococcal protein A molecule that is fused with an N-terminus polyhistidine tag (H12-D-domain to tether IgG surrogate antigens on Ni(2+-containing PLB membranes. We systematically assessed the specificity and capability of H12-D-domain construct to capture IgG molecules from different species through live cell and single molecule TIRFM imaging. We find that these IgG surrogate antigens tethered by H12-D-domain show better lateral mobility and are more uniformly distributed on PLB membranes than the ones tethered by streptavidin. Neither IgM molecules, nor Fab or F(ab'2 fragments of IgG molecules can be tethered on PLB membranes by H12-D-domain construct. These tethered IgG surrogate antigens strongly induce the formation and accumulation of signaling active antigen receptor microclusters within the immunological synapse in B or T lymphocyte cells. Thus our method provides a new and robust method to tether IgG surrogate antigens or other molecules fused with IgG Fc portion on PLB membranes for TIRFM based molecule imaging experiments.

  13. T cell activation and retargeting using staphylococcal enterotoxin B and bispecific antibody: an effective in vivo antitumor strategy. (United States)

    Porter, L E; Nelson, H; Ethem Gecim, I; Rice, D C; Thibault, C; Chapoval, A I


    The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with staphylococcal enterotoxin B (SEB) and retargeting with antitumor x anti-CD3 F(ab')2 bispecific antibodies (bsAb). All studies were performed in C3H/HeN mice using syngeneic tumor cell lines. For survival studies, mice were injected intravenously on day 0 with CL62 (a p97-transfected clone of the K1735 murine melanoma tumor). Day-3 treatments included saline (control), SEB (50 gamma g intraperitoneal) with or without bsAb (5 micrograms i.v.). Cured mice, surviving beyond 60 days, were rechallenged with subcutaneous CL62, K1735, or a nonmelanoma control, AG104. SEB activation studies were performed with pulmonary tumor-infiltrating lymphocytes isolated from 10-day established CL62 tumors. Maximal tumor-infiltrating lymphocyte cytotoxicity was demonstrated 24 h following SEB injection, therefore bsAb treatments were administered 24 h after SEB. When survival was examined at 60 days, there were significantly more survivors in the group receiving SEB plus bsAb (70%) compared to the group receiving SEB alone (30%), and the controls (0%) (P = 0.02 and P < 0.01, respectively). Mice cured of CL62 using SEB alone or with bsAb demonstrated equal immunity to CL62, however, mice treated with SEB plus bsAb were more often immune to the p97-parental cell line, K1735(P = 0.001). Ag104 consistently grew in all mice. Results of these studies demonstrate that SEB plus bsAb can be effective, not only in curing tumors but also in providing protective immunity against targeted and non-targeted tumor antigens.

  14. Synthesis and stability test of radioimmunoconjugate 177Lu-DOTA-F(ab′2-trastuzumab for theranostic agent of HER2 positive breast cancer

    Directory of Open Access Journals (Sweden)

    Sandra Hermanto


    Full Text Available The use of trastuzumab as intact IgG labeling radionuclide for HER2 positive breast cancer theranostic agent is not ideal because it is slowly eliminated from the blood and normal tissues resulting in low tumor/blood (T/B and tumor/normal tissue (T/NT ratios. To overcome this limitation, we developed the trastuzumab F(ab′2 fragments and radiolabeling of the fragments by β and γ-particle of Lutetium-177. F(ab2 fragments were produced by digestion of trastuzumab IgG (Herceptin with pepsin for 18 h at 37 °C. The F(ab′2 fragment fractionated in PD-10 column, followed by the conjugation with 2-(4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA as a metal chelator and radiolabeling with 177LuCl3. Molecular weight of fragments was calculated by LCMS (Liquid Chromatography Mass Spectroscopy and the radiochemical purity was evaluated by ITLC-SG (Instan Thin Layer Chromatography. Our study showed that the purity of F(ab′2 fragment generated by PD-10 fractions was >98% and the molecular weight of F(ab′2 was 98.35 kDa. The average numbers of pSCN-Bn-DOTA chelates per antibody fragment were 5.03 ± 1.5 and the optimum conjugation reactions was performed at molar ratio 20:1 (chelator to antibody. The stability test of the radioimmunoconjugate in the human serum albumin (HSA at 37 °C showed the radiochemical purity was 91.96 ± 0.26% after 96 h storage. This indicated that the radioimmunoconjugate is relatively stable when applied to the human body's physiological condition.

  15. Phagocytosis of gram-negative bacteria by a unique CD14-dependent mechanism. (United States)

    Schiff, D E; Kline, L; Soldau, K; Lee, J D; Pugin, J; Tobias, P S; Ulevitch, R J


    THP-1-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI)-anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated)-opsonized Escherichia coli (opEc). Both the GPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coli opsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolyl-maleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab')2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 beta-chain) or anti-Fcgamma receptor mAb.

  16. Structural characterization of antibody drug conjugate by a combination of intact, middle-up and bottom-up techniques using sheathless capillary electrophoresis - Tandem mass spectrometry as nanoESI infusion platform and separation method. (United States)

    Said, Nassur; Gahoual, Rabah; Kuhn, Lauriane; Beck, Alain; François, Yannis-Nicolas; Leize-Wagner, Emmanuelle


    Antibody-drug conjugates (ADCs) represent a fast growing class of biotherapeutic products. Their production leads to a distribution of species exhibiting different number of conjugated drugs overlaying the inherent complexity resulting from the monoclonal antibody format, such as glycoforms. ADCs require an additional level of characterization compared to first generation of biotherapeutics obtained through multiple analytical techniques for complete structure assessment. We report the development of complementary approaches implementing sheathless capillary electrophoresis-mass spectrometry (sheathless CE-MS) to characterize the different aspects defining the structure of brentuximab vedotin. Native MS using sheathless CE-MS instrument as a nanoESI infusion platform enabled accurate mass measurements and estimation of the average drug to antibody ratio alongside to drug load distribution. Middle-up analysis performed after limited IdeS proteolysis allowed to study independently the light chain, Fab and F(ab')2 subunits incorporating 1, 0 to 4 and 0 to 8 payloads respectively. Finally, a CZE-ESI-MS/MS methodology was developed in order to be compatible with hydrophobic drug composing ADCs. From a single injection, complete sequence coverage could be achieved. Using the same dataset, glycosylation and drug-loaded peptides could be simultaneously identified revealing robust information regarding their respective localization and abundance. Drug-loaded peptide fragmentation mass spectra study demonstrated drug specific fragments reinforcing identification confidence, undescribed so far. Results reveal the method ability to characterize ADCs primary structure in a comprehensive manner while reducing tremendously the number of experiments required. Data generated showed that sheathless CZE-ESI-MS/MS characteristics position the methodology developed as a relevant alternative for comprehensive multilevel characterization of these complex biomolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. A novel mechanism of thrombosis in antiphospholipid antibody syndrome. (United States)

    Vlachoyiannopoulos, Panayiotis G; Routsias, John G


    Antiphospholipid antibody syndrome (APS) is an autoimmune thrombophilia mediated by autoantibodies directed against phospholipid-binding plasma proteins, mainly β2 Glycoprotein I (β2GPI)-a plasma apolipoprotein and prothrombin (PT). A subgroup of these antibodies termed "Lupus Anticoagulant" (LA) elongate in vitro the clotting times, this elongation not corrected by adding normal plasma in the detection system. The exact mechanism by which these autoantibodies induce thrombosis is not well understood. Resistance to natural anticoagulants such as protein C, impaired fibrinolysis, activation of endothelial cells to a pro-coagulant phenotype and activation of platelets, are among the mechanisms partially supported by experimental evidence. Artificially dimerized β2GPI binds tightly to platelet membrane activating them. We search for mechanisms of natural dimerization of β2GPI by proteins of the platelet membranes and found that platelet factor 4 (PF4) assembled in homotetramers binds two molecules of β2GPI and this complex is recognized by anti-β2GPI antibodies, the whole complexes being thrombogenic in terms of activating platelets as confirmed by p38MAP kinase phosphorylation and thromboxane B2 production. Of note PF4/heparin complexes are also immunogenic triggering the production of anti-PF4/heparin antibodies which activate also platelets (the so-called "heparin-induced thrombocytopenia and thrombosis syndrome", HITT). The anti-β2GPI antibodies activate platelets by their F(ab)2, while the anti-PF4/heparin by their Fc fragments. Thus PF4 is a common denominator in the pathogenesis of APS and HITT which share also clinical characteristics such as thrombocytopenia and thrombosis.

  18. [Glycoprotein IIb-IIIa antagonist Monafram in primary angioplasty of patients with acute coronary syndrome without st segment elevation]. (United States)

    Pevzner, D V; Staroverov, I I; Samko, A N; Frolova, N S; Mazurov, A V; Ruda, M Ya


    Glycoprotein (GP) llb-llla anagonist monafram is the F(ab)2 fragments of anti GP llb llla monoclonal antibody FraMon (CRC64). Efficacy and safety of monafram in primary coronary angioplasty of patients with acute coronary syndrome without ST segment elevation (non ST ACS) was evaluated in this study. Monafram was introduced intravenously to 284 patients just before angioplasty at standard dosage - 0.25 mg/kg as single i.v. bolus. Control group included 203 patients. All patients received aspirin (loading dose 300 mg and then 75 mg daily) and more than 90% - clopidogrel (loading dose 300-600 mg and then 75 mg daily). Within 30 days of follow up period monafram decreased by more than 2.5 fold the total amount of unfavorable outcomes (cardiovascular death, myocardial infarction and indications for repeat revascularization due to angina recurrence) - from 19.2% to 7.4% (p<0.001). The rate of indications for revascularization was most strongly decreased - by more than 7 times - from 7.9% to 1.1% (p<0.001). The number of myocardial infarctions was reduced by more than 2 times - from 8.4% to 3.9% (p=0.057). The amount of lethal outcomes did not differ between two groups (2.9% and 2.4% in the control and monafram groups, respectively). In the control group 8.9% patients received monafram during primary angioplasty due to urgent indications. Monafram did not cause any allergic reaction in all tested patients. Major bleeding was registered in one (less than 0.5%) and deep thrombocytopenia (<20000 platelets per 1 ul) - in 3 (1.1%) out of 284 patients. The data obtained indicated that monafram decreased the number of thrombotic complications in non ST ACS patients undergoing angioplasty upon the dual antiplatelet therapy (aspirin+clopidogrel) and without significant increase of dangerous side effects.

  19. Synergistic reversal of type 1 diabetes in NOD mice with anti-CD3 and interleukin-1 blockade: evidence of improved immune regulation. (United States)

    Ablamunits, Vitaly; Henegariu, Octavian; Hansen, Jakob Bondo; Opare-Addo, Lynn; Preston-Hurlburt, Paula; Santamaria, Pere; Mandrup-Poulsen, Thomas; Herold, Kevan C


    Inflammatory cytokines are involved in autoimmune diabetes: among the most prominent is interleukin (IL)-1β. We postulated that blockade of IL-1β would modulate the effects of anti-CD3 monoclonal antibody (mAb) in treating diabetes in NOD mice. To test this, we treated hyperglycemic NOD mice with F(ab')(2) fragments of anti-CD3 mAb with or without IL-1 receptor antagonist (IL-1RA), or anti-IL-1β mAb. We studied the reversal of diabetes and effects of treatment on the immune system. Mice that received a combination of anti-CD3 mAb with IL-1RA showed a more rapid rate of remission of diabetes than mice treated with anti-CD3 mAb or IL-1RA alone. Combination-treated mice had increased IL-5, IL-4, and interferon (IFN)-γ levels in circulation. There were reduced pathogenic NOD-relevant V7 peptide-V7(+) T cells in the pancreatic lymph nodes. Their splenocytes secreted more IL-10, had increased arginase expression in macrophages and dendritic cells, and had delayed adoptive transfer of diabetes. After 1 month, there were increased concentrations of IgG1 isotype antibodies and reduced intrapancreatic expression of IFN-γ, IL-6, and IL-17 despite normal splenocyte cytokine secretion. These studies indicate that the combination of anti-CD3 mAb with IL-1RA is synergistic in reversal of diabetes through a combination of mechanisms. The combination causes persistent remission from islet inflammation.

  20. Enhanced antitumor effect of anti-tissue factor antibody-conjugated epirubicin-incorporating micelles in xenograft models. (United States)

    Yamamoto, Yoshiyuki; Hyodo, Ichinosuke; Koga, Yoshikatsu; Tsumura, Ryo; Sato, Ryuta; Obonai, Toshihumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Yasunaga, Masahiro; Harada, Mitsunori; Kato, Yasuki; Ohtsu, Atsushi; Matsumura, Yasuhiro


    For the creation of a successful antibody-drug conjugate (ADC), both scientific and clinical evidence has indicated that highly toxic anticancer agents (ACA) should be conjugated to a monoclonal antibody (mAb) to administer a reasonable amount of ADC to patients without compromising the affinity of the mAb. For ordinary ACA, the conjugation of a mAb to ACA-loaded micellar nanoparticles is clinically applicable. Tissue factor (TF) is often overexpressed in various cancer cells and tumor vascular endothelium. Accordingly, anti-TF-NC-6300, consisting of epirubicin-incorporating micelles (NC-6300) conjugated with the F(ab')2 of anti-TF mAb was developed. The in vitro and in vivo efficacy and pharmacokinetics of anti-TF-NC-6300 were compared to NC-6300 using two human pancreatic cancer cell lines, BxPC3 (high TF expression) and SUIT2 (low TF expression), and a gastric cancer cell line, 44As3 (high TF expression). The intracellular uptake of epirubicin was faster and greater in BxPC3 cells treated with anti-TF-NC-6300, compared with NC-6300. Anti-TF-NC-6300 showed a superior antitumor activity in BxPC3 and 44As3 xenografts, compared with NC-6300, while the activities of both micelles were similar in the SUIT2 xenograft. A higher tumor accumulation of anti-TF-NC-6300 compared to NC-6300 was seen, regardless of the TF expression levels. However, anti-TF-NC-6300 appeared to be localized to the tumor cells with high TF expression. These results indicated that the enhanced antitumor effect of anti-TF-NC6300 may be independent of the tumor accumulation but may depend on the selective intratumor localization and the preferential internalization of anti-TF-NC-6300 into high TF tumor cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  1. Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma. (United States)

    Ely, P; Wallace, P K; Givan, A L; Graziano, R F; Guyre, P M; Fanger, M W


    To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.

  2. The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity. (United States)

    Tóth, Gábor; Szöőr, Árpád; Simon, László; Yarden, Yosef; Szöllősi, János; Vereb, György


    Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition.

  3. Characterization of the co-elution of host cell proteins with monoclonal antibodies during protein A purification. (United States)

    Zhang, Qingchun; Goetze, Andrew M; Cui, Huanchun; Wylie, Jenna; Tillotson, Ben; Hewig, Art; Hall, Michael P; Flynn, Gregory C


    Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co-purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D-LC-HDMS(E) approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (∼10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co-purification with individual Fc and (Fab')2 antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708-717, 2016. © 2016 American Institute of Chemical Engineers.

  4. Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

    Institute of Scientific and Technical Information of China (English)


    Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. Methods The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F(ab)2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

  5. Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells

    Directory of Open Access Journals (Sweden)

    Parr Rebecca D


    Full Text Available Abstract Background Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM, the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types. Methods Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls. Results Only full-length (FL, endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells. Conclusions The intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.

  6. The correlation studies on the quantities of the fetal RBCs in the pregnancies with type O be quantified by flow cytometer (FCM) and the incidence of hemolytic disease of newborn( HDN)%流式细胞术检测O型孕妇血液中胎儿微量A(B)红细胞与新生儿溶血病发病率的相关性研究

    Institute of Scientific and Technical Information of China (English)

    周英; 吕文彬; 李健; 陈雪; 王乃红; 迭敏; 方军


    目的 运用流式细胞术产前非侵入性检测ABO血型不合导致新生儿溶血病,探讨该方法在临床的实用性.方法 运用已建立的间接标记法[一抗为IgG抗-A(B),二抗为FITC-抗人IgG(γ),F(ab’)2]染色,采用流式细胞术检测O型孕妇血液中胎儿微量A(B)红细胞.结果 流式细胞术与血清学方法检测ABO血型不合导致新生儿溶血病两种方法结果差异有统计学意义.追踪检测O型孕妇体内胎儿微量A(B)红细胞所占比例数据显示,较早进行产前诊断能在一定程度上帮助预防、诊断HDN. 结论 建立了流式细胞术产前非侵入性检测ABO血型不合导致新生儿溶血病的方法,孕妇在产前常规作流式细胞术检测,为ABO血型不合导致新生儿溶血病提供了一种可靠的诊断依据.%Objective To use the noninvasive method of flow cytometer( FCM) detecting the hemolytic disease of new-bom (HDN) due to the mismatching of ABO blood group system, exploring the practical of this method. Methods Using the indirect method(IgG anti-A(B) as the first antibody,FITC- anti- IgG F(ab')2 as the second antibody) to dyeing the cells,and the trace quantities of the fetal RBCs in the pregnancies with type 0 was quantified by flow cytometer ( FCM). Results Difference between the two methods of flow cytometer( FCM) and serological test was significant . The detected data in the investigation shows that it could be used to prevent hemolytic disease of newborn(HDN) ,if the ante-natal detecting be carried out much earlier.Conclusion Establishing the noninvasive method of flow cytometer( FCM) to detect the hemolytic disease of newborn (HDN). It may be used for diagnosis of hemolytic disease of newbom(HDN)due to the mismatching of ABO blood group system in ante-natal detecting.

  7. Cytometric evaluation of intracellular IFN-γ and IL-4 levels in thyroid follicular cells from patients with autoimmune thyroid diseases

    Directory of Open Access Journals (Sweden)

    Bossowski Artur


    Full Text Available Abstract Background In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD. The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18, Hashimoto's thyroiditis (HT; n = 18 and non-toxic multinodular goiter (NTMG; n = 18. Methods Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab'2 labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL. Results In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p Conclusions We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.

  8. Unusual Water-mediated Antigenic Recognition of the Proinflammatory Cytokine Interleukin-18

    Energy Technology Data Exchange (ETDEWEB)

    Argiriadi, Maria A.; Xiang, Tao; Wu, Chengbin; Ghayur, Tariq; Borhani, David W.; (Abbott)


    The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 {angstrom} resolution; the 125-2H Fab (2.3 {angstrom}); and the ABT-325 Fab (1.5 {angstrom}). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (>10 {angstrom}) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966-971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific antibody

  9. Circulating IgG autoantibodies to IgE in atopic syndromes. (United States)

    Quinti, I; Brozek, C; Wood, N; Geha, R S; Leung, D Y


    Sera from nonatopic healthy donors and patients with hyper-IgE syndrome, allergic respiratory disease, i.e., allergic rhinitis and asthma, and atopic dermatitis were assayed for the presence of IgG and IgM antibodies to IgE. The assay used was based on an ELISA method that measured the binding of IgG or IgM in test sera to myeloma IgE (PS)-coated microtiter wells. The levels of IgG anti-IgE but not of IgM anti-IgE were elevated in patient sera of all three categories tested. The same sera failed to demonstrate increased levels of IgG anti-IgM or IgG anti-IgA. Significant IgG anti-IgE activity remained after absorption of patient sera over pooled human IgG F(ab')2 Sepharose. The IgG anti-IgE activity appeared to be directed toward the Fc portion of IgE because absorption of positive sera over IgE (ADZ) Sepharose but not over myeloma IgG Sepharose completely removed their reactivity with IgE (PS) and because sera from atopic individuals but not from normal subjects contained IgG anti-IgE activity against the protein backbone of the Fc portion of IgE synthesized from a fragment of the cloned gene of human myeloma IgE (ND) heavy chain. Regression analysis demonstrated a weak but significant correlation (r = 0.31; p less than 0.05) between serum IgE levels and IgG anti-IgE activity. Fractionation of sera from the three patient categories by gel filtration over Sepharose 6B revealed that IgG anti-IgE activity was present both as monomeric IgG and in IgE containing immune complexes (IC). Intermediate molecular size IC (between 7S and 19S) were present in all three patient groups.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Functional analysis of alpha 1 beta 1 integrin in human natural killer cells. (United States)

    Pérez-Villar, J J; Melero, I; Gismondi, A; Santoni, A; López-Botet, M


    Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

  11. Utility of epirubicin-incorporating micelles tagged with anti-tissue factor antibody clone with no anticoagulant effect. (United States)

    Sugaya, Akinori; Hyodo, Ichinosuke; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Takashima, Hiroki; Sato, Ryuta; Tsumura, Ryo; Furuya, Fumiaki; Yasunaga, Masahiro; Harada, Mitsunori; Tanaka, Ryosuke; Matsumura, Yasuhiro


    Tissue factor (TF), an initiator of the extrinsic blood coagulation cascade, is overexpressed in different types of cancer. Tissue factor overexpression is also known as a poor prognostic factor in pancreatic cancer. We recently developed anti-TF antibody (clone1849)-conjugated epirubicin-incorporating micelles (NC-6300), and reported that this anti-TF1849-NC-6300 showed enhanced antitumor activity against TF-high expressed human pancreatic cancer cells, when compared with NC-6300 alone. However, clone 1849 antibody inhibited TF-associated blood coagulation activity. We studied another anti-TF antibody, clone 1859, which had no effect on blood coagulation and prepared anti-TF1859-NC-6300. In addition, to determine the optimum size of the antibody fragment to conjugate with NC-6300, three forms of the 1859 antibody (whole IgG, F[ab']2 , and Fab') were conjugated to NC-6300. The antitumor effect of each anti-TF1859-NC-6300 was studied in vitro and in vivo, using two human pancreatic cancer cell lines, BxPC3 with high-expressed TF, and SUIT2 with low levels of TF. In vitro, all forms of anti-TF1859-NC-6300 showed higher cytocidal effects than NC-6300 in BxPC3, whereas this enhanced effect was not observed in SUIT2. Likewise, all forms of anti-TF1859-NC-6300 significantly suppressed tumor growth when compared to NC-6300 in the BxPC3, but not in the SUIT2, xenograft model. Among the three forms of conjugates, anti-TF1859-IgG-NC-6300 had a higher antitumor tendency in TF-high expressed cells. Thus, we have confirmed an enhanced antitumor effect of anti-TF1859-NC-6300 in a TF-high expressing tumor; anti-TF1859-IgG-NC-6300 could be used to simplify the manufacturing process of the antibody-micelle conjugation for future clinical studies. © 2015 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. Inhibition of IL-10 production by maternal antibodies against Group B Streptococcus GAPDH confers immunity to offspring by favoring neutrophil recruitment.

    Directory of Open Access Journals (Sweden)

    Pedro Madureira


    Full Text Available Group B Streptococcus (GBS is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10 by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab'(2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10(-/- mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood, this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen.

  13. Preparation and identification of the fragment of rabbit's polyclonal antibodies against human ouabain%抗哇巴因多克隆抗体F(ab)2 片段的研制及鉴定

    Institute of Scientific and Technical Information of China (English)

    张明娟; 吕卓人; 袁育康; 贺浪冲; 王颢


    Objective Anti-ouabain polyclonal antibody was digested with pepsin to prepare F fragment in order to find the base of reshaped antibody. Methods Anti-ouabain polyclonal antibody was prepared by immunizing rabbits. Then anti-ouabain polyclonal antibody was digested under different conditions. The reactants were analyzed and purified by High Performance Size Ex-clude Chromatography (HPSEC). The immune activities were detected and identified by Enzyme Linked Immunosorbent Assay (ELISA). Results The proper reactive conditions for preparation of anti-ouabain polyclonal antibody F(ab)2 fragment with pepsin were established. The eligible dose of pepsin, by which 100mg anti-onabain polyclonal antibody was digested better, was 2mg. The eligible digesuve time was 18h. The value of pH of reactive system was 3.0. Conclusion The active F fragment of anti-ouabain polyclonal antibody can be obtained with pepsin. The method is simple and feasible.%目的用胃蛋白酶酶解抗哇巴因多克隆抗体,制备出片段,为改型抗体的研究提 供依据。方法免疫家兔制备出抗哇巴因多克隆抗体,并用胃蛋白酶分别在不同的酶解条件下消化抗体,继之用高效液相排阻色谱法(HPSEC)对其进行分析、纯化,采用酶联免疫吸附法(ELISA)检测、鉴定其活性。结果用胃蛋白酶2mg/100mg抗哇巴因多克隆抗体,消化18h,反应体系的pH3.0时,可获得片段。结论用胃蛋白酶酶解抗哇巴因多克隆抗体是研制出有活性的片段的有效方法。

  14. A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA. (United States)

    Rouwendal, Gerard Ja; van der Lee, Miranda M; Meyer, Saskia; Reiding, Karli R; Schouten, Jan; de Roo, Guy; Egging, David F; Leusen, Jeanette Hw; Boross, Peter; Wuhrer, Manfred; Verheijden, Gijs F; Dokter, Wim H; Timmers, Marco; Ubink, Ruud


    Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

  15. Anti-interleukin-1 alpha autoantibodies in humans: Characterization, isotype distribution, and receptor-binding inhibition--higher frequency in Schnitzler's syndrome (urticaria and macroglobulinemia)

    Energy Technology Data Exchange (ETDEWEB)

    Saurat, J.H.; Schifferli, J.; Steiger, G.; Dayer, J.M.; Didierjean, L. (Department of Dermatology, University Hospital, Geneva (Switzerland))


    Since autoantibodies (Abs) to cytokines may modify their biologic activities, high-affinity binding factors for interleukin-1 alpha (IL-1 alpha BF) were characterized in human sera. IL-1 alpha BF was identified as IgG (1) by sucrose density-gradient centrifugation followed by immunodiffusion autoradiography, (2) by ligand-blotting method, (3) by ligand binding to affinity-immobilized serum IgG, and (4) by IgG affinity purification followed by sucrose density-gradient centrifugation. IL-1 alpha binding activity resided in the F(ab)2 fragment. The apparent equilibrium constant was in the range of IgG found after immunization with conventional antigens (i.e., 10(-9) to 10(-10) mol/L). Anti-IL-1 alpha IgG auto-Abs represented only an extremely small fraction of total IgG (less than 1/10(-5)). Some sera with IL-1 alpha BF and purified IgG thereof were able to inhibit by 96% to 98% the binding of human recombinant IL-1 alpha to its receptor on murine thymoma EL4-6.1 cells, whereas other sera did not. When 125I-labeled anti-IL-1 alpha IgG complexes were injected into rats, they prolonged the plasma half-life of 125I-labeled IL-1 alpha several fold and altered its tissue distribution. The predominant class was IgG (12/19), mainly IgG4 (9/19), but in five of the sera, anti-IL-1 alpha IgA was also detected. In a screening of 271 sera, IL-1 alpha BF was detected in 17/98 normal subjects and was not more frequent in several control groups of patients, except in patients with Schnitzler's syndrome (fever, chronic urticaria, bone pain, and monoclonal IgM paraprotein) (6/9; p less than 0.005). The pathologic significance of these auto-Abs remains to be determined.

  16. Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA Gold 4 epitope and designed for radioimmunotherapy (RIT of colorectal cancers

    Directory of Open Access Journals (Sweden)

    Pugnière Martine


    Full Text Available Abstract Background Human monoclonal antibodies (MAbs are needed for colon cancer radioimmunotherapy (RIT to allow for repeated injections. Carcinoembryonic antigen (CEA being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs. Methods XenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice. Results Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g, close to that of 131I-X4 (21.7 ± 7.2%ID/g. At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g. At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. Conclusion Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab'2 fragments, or as a bispecific antibody.

  17. Enhanced immunogenicity of pneumococcal surface adhesin A (PsaA in mice via fusion to recombinant human B lymphocyte stimulator (BLyS

    Directory of Open Access Journals (Sweden)

    Mambula Salamatu S


    Full Text Available Abstract Background B lymphocyte stimulator (BLyS is a member of the tumor necrosis factor superfamily of ligands that mediates its action through three known receptors. BLyS has been shown to enhance the production of antibodies against heterologous antigens when present at elevated concentrations, supporting an immunostimulatory role for BLyS in vivo. Methods We constructed a fusion protein consisting of human BLyS and Pneumococcal Surface Adhesin A (PsaA and used this molecule to immunize mice. The immunostimulatory attributes mediated by BLyS in vivo were evaluated by characterizing immune responses directed against PsaA. Results The PsaA-BLyS fusion protein was able to act as a co-stimulant for murine spleen cell proliferation induced with F(ab'2 fragments of anti-IgM in vitro in a fashion similar to recombinant BLyS, and immunization of mice with the PsaA-BLyS fusion protein resulted in dramatically elevated serum antibodies specific for PsaA. Mice immunized with PsaA admixed with recombinant BLyS exhibited only modest elevations in PsaA-specific responses following two immunizations, while mice immunized twice with PsaA alone exhibited undetectable PsaA-specific serum antibody responses. Sera obtained from PsaA-BLyS immunized mice exhibited high titers of IgG1, IgG2a, IgG2b, and IgG3, but no IgA, while mice immunized with PsaA admixed with BLyS exhibited only elevated titers of IgG1 following two immunizations. Splenocytes from PsaA-BLyS immunized mice exhibited elevated levels of secretion of IL-2, IL-4 and IL-5, and a very modest but consistent elevation of IFN-γ following in vitro stimulation with PsaA. In contrast, mice immunized with either PsaA admixed with BLyS or PsaA alone exhibited modestly elevated to absent PsaA-specific recall responses for the same cytokines. Mice deficient for one of the three receptors for BLyS designated Transmembrane activator, calcium modulator, and cyclophilin ligand [CAML] interactor (TACI exhibited attenuated PsaA-specific serum antibody responses following immunization with PsaA-BLyS relative to wild-type littermates. TACI-deficient mice also exhibited decreased responsiveness to a standard pneumococcal conjugate vaccine. Conclusion This study identifies covalent attachment of BLyS as a highly effective adjuvant strategy that may yield improved vaccines. In addition, this is the first report demonstrating an unexpected role for TACI in the elicitation of antibodies by the PsaA-BLyS fusion protein. Reviewers This article was reviewed by Jonathan Yewdell, Rachel Gerstein, and Michael Cancro (nominated by Andy Caton.

  18. 抗肿瘤坏死因子α多克隆抗体对滑石粉和多西环素胸膜固定术效果的影响%The effects of intrapleural anti-TNFα antibody on chemical pleurodesis induced by talc slurry or doxycycline

    Institute of Scientific and Technical Information of China (English)


    Objective To determine whether the intrapleural injection of TNFα neutralizing Fab antibody fragment (anti-TNFα Fab) would prevent the pleurodesis that occurs after the intrapleural injection of talc or doxycycline. Methods In a randomized blinded placebo controlled study, 34 New Zealand white rabbits were given 400 mg/kg talc or 10 mg/kg doxycycline intrapleurally as a sclerosant via a chest tube. Half of the rabbits in each group were also given polyclonal affinity purified anti-TNFα Fab 2 000 units or saline as placebo immediately before and 12 hours after the injection of the sclerosants. Chest tube were aspirated at 12 hours intervals until their removal at 4 days. Rabbits were sacrificed at 28 days. The pleural fluid volume, cell counts, LDH and pleurodesis scores were compared among groups. Results Both talc and doxycycline produced an exudative pleural effusion. The pleural fluid volume and LDH levels were significantly greater in the doxycycline group than talc group. However, the pleural fluid leukocyte counts in talc group were higher than doxycycline group. The administration of anti-TNFα Fab had no significant effect on pleural fluid volume, LDH or leukocyte counts in either groups. However, the intrapleural administration of anti-TNFα Fab results in a significant decrease in the pleurodesis score for the talc group than doxycycline group (1.8±0.9 vs 3.2±0.8). In contrast the pleurodesis score was virtually identical in the doxycycline group with (3.5±0.5) and without (3.4±0.7) anti-TNFα Fab. Conclusion The intrapleural administration of anti-TNFα Fab diminishes the pleurodesis induced by talc but not that which results from doxycycline. These findings suggest that different mechanisms are involed with the two different sclerosants.%目的 研究胸膜腔内注入抗肿瘤坏死因子α(TNFα)多克隆抗体是否能够阻断滑石粉或多西环素引起的胸膜粘连。方法 34只新西兰白兔分成2组,18


    Directory of Open Access Journals (Sweden)

    R. A. Vinogradov


    (p>0.05. MMSE3 showed a significant improvement in the test results in each group compared to MMSE1 and MMSE2. MMSE3 results were significantly better (p<0.01 in group 1 than in group 2. MoCA revealed most significant differences in group 1 results with a significant decrease in cognitive dysfunction both in MoCA1, MoCA2, MoCA3 either within the group (p<0.05 or compared to MoCA3 data between CE group and TBA group (p<0.01. FAB showed that the FAB2 test amounted to 16.2 points in the group with CE and 14.6 points in the group with TBA of ICAs (p<0.05. A significant improvement in the performance of mnestic functions was noted when examining patients in the dynamics of FAB3: 17.3 and 15.6 points (p<0.05, respectively. According to Hamilton Depresion Rating Scale and Beck Depression Inventory, both groups of patients showed a moderate level of anxiety and depression (12.4 and 15.8 according to Hamilton Depression Rating Scale (p<0.05, 12.3 and 14.4 according to Beck Depression Inventory (p>0.05. During the second test, the depressive mood of patients was reduced (on the Hamilton scale 8.4 and 13.8 points (p<0.05 according to Hamilton Depression Rating Scale, 9.1 and 11.2 points (p<0.05 according to Beck Depression Inventory. Upon the third testing of patients there was an improvement in the psychological background in both groups. In the group with CE, the level reached 5.8 according to Hamilton Scale, which was more statistically significant (p<0.001 compared to the group of TBA of ICAs with 11.0 points. The results obtained during Beck tests had significant differences (p<0.05 both within the groups in dynamics, and between group 1 and 2. Conclusion When comparing HMFs in patients who underwent a different surgical approach (CE vs TBA of ICAs in the treatment of obliterating atherosclerotic lesions of ICAs, we found that: 1 the maximum improvement in HMFs appears by the 30th day of the postoperative period in comparison with preoperative parameters; 2 the most