Monoclonal antibodies against alpha toxin of Clostridium perfringens

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.     

A sensitive and rapid luminescence sandwich assay for antibodies to hepatitis-B surface antigen (Anti-HBsAg)

A chemiluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5.26-8.11% and has a lower detection limit for hepatitis-B surface antigen of 1.3U/I.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb Monoclonal antibody Nomenclature C _AConcentration of any (mol m^-3) component A D Dilution rate (s^-1) K _dDeath-rate constant (mol m^-3) K _lLysis-rate constant (mol m^-3) K _sMonod constant (mol m^-3) m Maintenance coefficient (mol cell^-1 s^-1) q Specific consumption (mol cell^-1 s^-1) or production rate t Time (s) X Cell concentration (cell m^-3) Y Yield coefficient (cell mol^-1) Greek symbols _d Specific death rate (s^-1) _l Specific lysis rate (s^-1) of viable cells _net Net specific growth (s^-1) rate _true True specific growth (s^-1) rate     

Factors inhibiting IFN activity

Several factors may inhibit the activity of IFNs. Some of these occur naturally, others are therapy-induced or artificial. Naturally occurring antibodies appear to have a much broader reactivity than therapy-induced antibodies. Naturally induced antibodies are reported in patients suffering from chronic graft-versus-host disease after bone marrow transplantation. Differences in the reported immunogenicity between interferons may not be due to the minor variation in amino acid sequence. The clinical significance of therapy-induced antibodies has been unclear. In patients treated for chronic hepatitis C, antibody formation is closely related to relapse. In animal studies the efficacy of treatments targeting the IFN receptor interaction has been shown. Soluble IFN- receptor inhibits the development of autoimmune diseases in mice. Monoclonal antibodies to the IFN- receptor protects against allograft rejections in monkeys. Two naturally occurring inhibitors of IFN action were reported. The clinical significance and structure of these inhibitors remain elusive.     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Detection of microbial surface antigens that bind Lewisa antigen

Abstract There is evidence that the Lewis^a blood group antigen is one of the receptors for a number of potentially pathogenic microorganisms. To determine how widely distributed the microbial adhesins are that bind this antigen, anti-idiotypic antibodies produced against monoclonal anti-Lewis^a were used in coagglutination assays to screen a variety of species. The following were agglutinated: 7/7 strains of Staphylococcus aureus ; 10/19 (53%) strains of Neisseria meningitidis ; 8/13 (62%) strains of Haemophilus influenzae ; 1/3 strains of Helicobacter pylori ; 1/2 strains of Neisseria gonorrhoeae ; 1/2 strains of Candida albicans . The application of the anti-idiotypic antibodies to studies of host cell receptors, isolation of adhesins and development of new epidemiological typing reagents is discussed.     

Human T Cell Leukemia Virus Type 1 (HTLV-1) Antibodies in Healthy Populations and Renal Transplanted Patients in the North-East of Brazil

The seroprevalence of human T cell leukemia virus type 1 (HTLV-1) infection was investigated in Brazilians (570): native inhabitants (298) and descendants from Japanese (272) living in Recife and its neighborhoods—North-east of Brazil. Furthermore, polytransfused renal transplanted patients (54) were also examined for the serological status to this virus. The seropositivity to HTLV-1, screened by enzyme-linked immunosorbent assay (ELISA), was low: 1.34% for the local population and 0.73% for the descendants from Japanese. However, the seropositivity for the renal transplanted patients was found to be 11.1%. This higher value suggests that this retrovirus infection seems to be of importance in this clinical condition.     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline