Olaf Behrsing

Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter

Hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and human alpha-fetoprotein (AFP) were obtained by fusing two hybridoma lines and selecting the fused cells by a fluorescence activated cell sorter (FACS III). The hybridoma cells were labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) respectively and heterofluorescent cells were sorted out after fusion. Several clones were found to produce bispecific antibodies, and one clone, designated T1, was subjected to growth in ascitic fluid in mice in order to obtain large quantities of hybrid antibodies. Bispecific antibodies could be separated from the monospecific antibody populations by one-step hydroxylapatite chromatography. SDS-polyacrylamide gel electrophoresis demonstrated that the hybrid antibody molecules contained the heavy chains of both anti-HRP and anti-AFP origin. The bispecific antibodies were used to build up a sensitive two-site binding enzyme immunoassay.

Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP)

Hybrid hybridomas producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) were obtained by fusing two hybridoma lines and selecting the fused cells using a fluorescence activated cell sorter (FACS). FITC was used to label different monoclonal antibodies and the bispecific antibodies acted as a linking agent between FITC-labelled antibody and the marker enzyme HRP. This system was used in enzyme immunoassays for the detection of different antigens. The results suggest a wide application of bispecific anti-FITC/anti-HRP antibodies as a detection system in EIA.

Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages

Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.

A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer.

Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope.

Production and Characterization of Monoclonal Antibodies Against Urea Derivatives

A panel of monoclonal antibodies was generated against the ureabased haptenN-(2-N-chloroacetylaminobenzyl)-N′-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Crossreactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.

Monoclonal anti-diuron antibodies prevent inhibition of photosynthesis by diuron

Two monoclonal anti‐diuron antibodies were generated that bind to diuron with an extremely low equilibrium dissociation constant. The antibodies prevented and restored in vitro and in vivo the diuron‐dependent inhibition of photosynthesis. In isolated thylakoids prepared from spinach leaves (Spinacia oleracea L.) the diuron‐inhibited Hill reaction was reconstituted immediately after the addition of the monoclonal antibodies. The antibodies also restored the diuron‐dependent inhibition of the photosynthetic oxygen evolution of the cell wall‐deficient mutant cw15 of the green alga Chlamydomonas reinhardtii Dangeard.

Bispecific IgA/IgM antibodies and their use in enzyme immunoassay

Two hybrid hybridomas secreting polymeric bispecific antibodies to human chorionic gonadotropin and calf intestinal alkaline phosphatase were produced by fusion of IgA- and IgM-secreting mouse hybridomas. Both hybrid antibodies were purified from ascitic fluid by size exclusion chromatography. An IgM-like fraction was shown to exhibit bispecific activity. Bispecificity was completely lost following mild reduction and alkylation. Both bispecific antibodies were used to develop a sensitive enzyme immunoassay for hCG.

A simple and sensitive method to study effects mediated by soluble lymphokines as demonstrated by the interaction of CD4+ and CD8+ cell subsets during T cell activation

A method is described for the study of lymphokine-mediated cellular interactions using triple wells, which permits co-culture of cell subpopulations without direct physical contact. The triple wells are constructed by slitting the walls to half height between three adjacent wells of a 96-well microtiter plate. The cells under study are positioned in the outer two wells, whereas the middle well serves to separate the cells. The half slits permit the wells to be treated independently before filling the triple well with the culture medium and prevents cell leakage thereafter. The feasibility of the method was established by studying the interaction of isolated CD4+ and CD8+ T cell subsets during T cell proliferation induced by immobilized anti-CD3 and anti-CD28 monoclonal antibodies.

Monoklonale Antikörper: Grundlagen und ihre Bedeutung in Diagnostik und Therapie

In den letzten 30 Jahren wurden auf den verschiedenen Gebieten der Biowissenschaften rasante Fortschritte erzielt, ermoglicht durch eine Reihe wichtiger methodischer Entwicklungen, von denen an prominenter Stelle sicher die Gentechnik zu nennen ist.