Antibodies Of Predefined Specificity Research Articles

Sequential affinity purification of peroxidase tagged bispecific anti-SARS-CoV antibodies on phenylboronic acid agarose

Hybrid hybridomas (quadromas) are derived by fusing at least two hybridomas, each producing a different antibody of predefined specificity. The resulting cell secretes not only the immunoglobulins of both parents but also hybrid molecules manifesting the binding characteristics of the individual fusion partners. Purification of the desired bispecific immunoprobe with high specific activity from a mixture of bispecific and monospecific monoclonal antibodies requires special strategies. Using a dual, sequential affinity chromatography (Protein-G chromatography followed by m-aminophenyleboronic acid agarose column), we have purified bispecific monoclonal antibodies (BsMAb) as a preformed HRPO (Horseradish Peroxidase) complex (BsMAb–HRPO). The quadroma culture supernatant was initially processed on a Protein-G column to isolate all the species of immunoglobulins. This pre-enriched fraction was subsequently passed through the aminophenyleboronic acid column super saturated with HRPO. The column matrix has the ability to bind to proteins such as HRPO with vicinal diols. The enzyme loaded column captures the desired bispecific anti-SARS-CoV × anti-HRPO species with the elimination of the monospecific anti-SARS-CoV MAb to result in a high specific activity diagnostic probe. The presence of anti-HRPO MAb is an acceptable impurity as it will not bind to the target SARS-CoV NP antigen and will get washed out during the ELISA procedure.

Enzyme Immunoassay for 2,4,6-Trichloroanisole Based on Monoclonal Antibodies

Monoclonal antibodies (mabs) are of great interest according to their homologous abilities, high specificity, and unlimited possibility of production. Mabs against different substances have been developed. They are used in many fields of application (e.g., medicine and pesticide analysis). Immunoassays performed with mabs have the advantages of easy handling and high sensitivity. The goal of this study was to develop mabs against 2,4,6-trichloroanisole (TCA). TCA has been identified as the most potent contaminant in wine causing a mouldy taste. Several mice have been immunized with two different TCA haptens. Spleen cells have been gained and prepared following the technique developed by Köhler and Milstein (Köhler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256, 495–497). After a screening period with a competitive immunoassay two cells lines have been detected that produce TCA-specific antibodies. Both different antibodies have been used to develop ELISAs. One ELISA with the hapten B mab (Hbab) and one ELISAs with the hapten C mab (Rami). The ELISA with the hapten C mab was afterwards optimized with signal amplification. Each ELISA shows a remarkable detection limit for TCA. The ELISA with signal amplification performed with the mab Rami detects 20 ng/L and the ELISA with the mab Hbab 10 ng/L TCA. Both mabs have been used to measure spiked wine samples. Only with the mab Hbab were spiked cork samples also determined.

The study of the toxic site in ammodytoxin A with antibodies of pre-defined specificity: V. Curin-Serbec and F. Gubensek (J. Stefan Institute, Dept of Biochemistry, Jamova 39, Ljubljana, Slovenia)

The study of the toxic site in ammodytoxin A with antibodies of pre-defined specificity: V. Curin-Serbec and F. Gubensek (J. Stefan Institute, Dept of Biochemistry, Jamova 39, Ljubljana, Slovenia)

Reproducible production of protective human monoclonal antibodies by fusion of peripheral blood lymphocytes with a mouse myeloma cell line.

The production of monoclonal antibodies of human origin may represent a significant advance in immunotherapy for disease in humans. Although human monoclonal antibody has been produced from human lymphocytes by fusion with human myeloma cell lines or by Epstein-Barr viral transformation, fusion of postimmunization human lymphocytes with a mouse myeloma cell line is a relatively simple and reproducible alternative. Mouse-human hybrid cell lines were obtained in 205 (53%) of the microtiter wells initially seeded. Thirty-one (15%) of these hybrid cell lines secreted antibody of predefined specificity. Cloning was attempted with eight of the hybrid cell lines, and long-term antibody production was established in four of the lines: two hybridomas secreted antibody to the capsule of Haemophilus influenzae type b, one secreted antibody to tetanus toxoid, and one secreted antibody to diphtheria toxin. The production of mouse-human hybridomas appears to be a reliable method for obtaining human monoclonal antibody of predefined specificity.

Production of hybrid lines secreting monoclonal anti-idiotypic antibodies by cell fusion on membrane filters.

Following the demonstration by Kohler and Milstein (1) that permanent cell lines secreting antibodies of predefined specificity can be obtained by cell fusion of myeloma cells with spleen cells of immunized mice, much effort has been devoted to improve the efficiency of hybrid formation between cells of the immune system and to enlarge the spectrum of monoclonal antibodies which such hybrid lines (“hybridomas”) can supply. The high efficiency of polyethylene-glycol (PEG) as a fusing agent for fibroblasts and its ability to yield hybrids in cell combinations recalcitrant to Sendai virus (2,3,4) has led most groups to substitute this simple chemical for the agglutinating virus. But PEG concentration is critical for the yield of viable hybrids ; this variable is difficult to control locally in most reported fusion procedures which rely on “gentle pipetting” for the removal of the culture medium and time-controlled resuspension of the cell pellets in a PEG solution. This prompted us to develop a simpler method which insures the formation of an intimate layer of the two parental cell types essentially free of culture medium, closely mimicking the conditions used for the efficient fusion of cells growing attached to a support. We describe this method, analyze some parameters which control its efficiency for the production of intraspecific and interspecific hybrids and demonstrate that it can be successfully utilized for the isolation of hybridomas secreting anti-idiotypic antibodies.