Christian Stähli

High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis.

Hybridomas producing antibodies against soluble antigens have in most cases been difficult to establish. After fusion of myeloma cells with spleen cells obtained from mice immunized with a soluble protein, hybridomas secreting specific antibodies have been observed to occur very rarely among non-specific hybridomas. We found that the frequency of specific hybridomas correlates directly with the increase over background of the frequency of blast and/or plasma cells in the spleen (measured by cell size analysis) after antigenic stimulation. High yields of specific hybridomas were obtained simply by following a novel immunization technique consisting of several conventional preimmunization courses followed by 4 very high doses of antigen in saline on each of the last 4 days before fusion.

A Gaba/Benzodiazepine Receptor Complex from Bovine Brain: Purification, Reconstitution and Immunological Characterization

A GABA/benzodiazepine receptor complex was purified from bovine cerebral cortex. The receptor fraction displayed binding sites for benzodiazepines as well as high and low affinity binding sites for GABA which are characteristics of the membrane-bound receptor. Two monoclonal antibodies of which one was directed against the 50 kd and the other against the 55 kd subunit were used for immunoprecipitation studies. Both of them were shown to quantitatively precipitate the entire receptor population. These results indicate that the binding sites for benzodiazepines and GABA (high and low affinity sites) reside on the same receptor complex containing a mixture of 50 kd and 55 kd subunits. Reconstitution of the receptor in phospholipid vesicles was achieved.

Monoclonal antibodies against antigens on breast cancer cells

Of 360 mAb obtained in a cell fusion experiment with the spleen cells of a mouse immunized with a mixture of different human breast carcinoma cells lines, 30 mAb were selected which reacted more strongly with tumor cells than with (noncancerous) fibroblasts. Theses mAb were tested for reactivity with additional types of cancerous and noncancerous tissues. Two mAb showed high tumor selectivity, but the corresponding epitopes on individual tumor cells were heterogeneously expressed. The mAb will be evaluated for in vivo applications.

[76] A rapid quantitative assay of high sensitivity for human leukocyte interferon with monoclonal antibodies

Publisher Summary This chapter describes the use of two monoclonal antibodies in a simple and rapid solid phase sandwich immunoassay for the detection and quantitative determination of human leukocyte interferon. Monoclonal antibodies provide a novel opportunity to select two or more antibodies directed against different epitopes of an antigen. For a sandwich immunoassay, these antibodies should have the following properties: (1) lack of mutual interference with antigen binding, (2) optimal characteristics for solid phase function, that is, antigen collection, (3) suitability for revealing antigen, that is, stability to labeling with radioisotope, enzyme, fluorescent dye, and such, and (4) high antigen affinity and specificity of both antibodies. The use and proper selection of two suitable monoclonal antibodies for measuring an antigen in a sandwich immunoassay provides a novel and universal strategy for designing and developing this type of assay for any antigen that has at least two independent epitopes. For some antigens, this approach may be the only possible solution; for other large substances such as carcinoembryonic antigen it should improve the specificity, sensitivity, and long-term reproducibility of already existing sandwich immunoassays. This interferon radioimmunoassay has already proven extremely useful for monitoring interferon during purification.

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.

Monoclonal antibodies to thymosin α1

Abstract Sixteen monoclonal antibodies specific for thymosin α 1 [Low et al. , J. biol. Chem. 254 , 981–986 (1979)] obtained from two fusions with the spleens of three mice [Stahli et al. , Meth. Enzym. 92 , 26–36 (1982 b )] all react with an epitope in the C-terminal half of thymosin α 1. Human and foetal calf serum contain substances which cross-react with this epitope. A simple procedure to selectively remove the cross-reactive material and a sensitive RIA are described.

Monoclonal Antibodies Against two Antigens of Clinical Interest

Abstract With six monoclonal antibodies (mAb) against β-thromboglobulin (βTG) and with over 20 mAb against prostatic acid phosphatase (PAP) sandwich assays for these clinically important proteins were developed. The analysis of the epitope specificities, development of the assays as well as some preliminary clinical results (with the PAP-assay) are described.

Monoclonal Antibodies to the Human Leukocyte Interferons and their Use for Interferon Purification and a Quantitative Interferon Assay

Abstract Thirteen monoclonal antibodies to human leukocyte interferon have been obtained. They exhibit different patterns of binding to purified leukocyte interferon species that are consistent with the structural multiplicity of the human leukocyte interferons. These antibodies are useful as probes into the structure of the human leukocyte interferons and for their purification by affinity chromatography. A simple and highly sensitive assay for leukocyte interferon was developed with the use of two monoclonal antibodies.