Preparation and Characterization of Monoclonal Antibodies against Neisseria meningitidis

Abstract

This thesis was intended for the production of monoclonal antibodies against Neisseria meningitidis antigens type A and C and their characterization in favor of their utilization within diagnostic applications.

We were actively pursuing too approaches of producing immune mouse spleen cells capable of producing the highest antibody levels against these two antigens. The Neisseria meningitidis antigens were emulsified with Freund’s adjuvant to produce a stable emulsion and were inoculated intraperitoneally in BALB/c mice. The subsequent doses were emulsified with incomplete Freund’s adjuvant and taken within time intervals sufficient for the mice immune system to produce antibodies. The serum samples were experienced for the existence of antibodies against Neisseria meningitidis antigens using ELISA technique. Excellent responding mice were chosen for additional immunizations.

Another schedule of immunization was performed using the chemically prepared Aluminum phosphate adjuvant (Alum). ELISA was performed to choose the mainly responder mice. The data recorded by ELISA have revealed antibody levels with the utilization of Alum which were more elevated than those obtained with the utilization of complete and incomplete Freund's oil adjuvants. The results comparison confirmed the utilization of Alum as an adjuvant.

The spleen of the excellent responder mouse was taken out to be fused with Myeloma cell line (P3NS1) using polyethylene glycol of Mwt 1500 (40% concentration) as a fusogenic agent. Hybridomas (fused spleen – Myeloma cells) were selected from nonfused cells using HAT medium as a selective media. Twelve days post fusion, the produced hybridomas were screened for production of antibodies specific to Neisseria meningitidis antigens type A and C using ELISA. At the fourteenth day after fusion, the manufactured hybridomas assumed extra expansions to be preserved by freezing and highly positive hybridomas were planned for cloning. At this stage, HT medium substitute the HAT one.

Three highly positive hybridomas producing monoclonal antibodies against Neisseria meningitidis antigens type A and C namely (I1A3), (I2B6) and (II III D2) were successfully selected and cloned using the limiting dilution method. The well established clones generating monoclonal antibodies against Neisseria meningitidis antigens type A and C were selected by ELISA technique, expanded in special media in tissue culture flasks and then submitted to the characterization procedure.

Further defining and characterization procedures as supernatants screenings, specificity and immunoglobulin isotyping tests were performed to provide us with a product that can be utilized as a reagent capable of detecting these two antigens in recently infected patients' specimens. ELISA test was performed to differentiate between type A and C prepared monoclonal antibodies. Results have revealed that the majority of monoclonal antibodies produced belong to type A meningococcal meningitis antigen. Furthermore, using agar gel procedure and mouse immunoglobulin isotyping kits, the obtained clones have been identified and all were found to belong to IgM isotype.

In this attending work, we had begun the first step towards the preparation of a serodiagnostic kit against the meningococcal meningitis antigens type A and C. The resulting supernatants provide a semi- product of monoclonal antibodies developed against Neisseria meningitidis antigens type A and type C which needed up-streaming processes for a large scale production in VACSERA in favor of their uses as diagnostic kits serving for the diagnosis of Neisseria meningitidis antigens in recent infections in Egypt.