Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody rea ctions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fuse d with spleen cells from mice previously immunized with TNP-ovalbumin (TNP- OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used t o detect anti-TNP antibodies in the supernatants, and five cultures were fo und to be strictly positive for TNP. Three of these were subsequently clone d by limiting dilution, and 15 clones were chosen for expansion based on th e criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG(1), and purified by Sepharose-protein G affini ty cromatography from ascites developed in BALB/c mice. Two purified MAbs ( 1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The re sulting conjugates were evaluated in ELISA tests for interferon-gamma and i nterleukin-4 detection, in which the secondary anti-cytokine antibodies wer e coupled either to TNP or biotin. The performance of anti-TNP conjugates i n these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r(2) (linear correlation coefficient) close to unity value. Growth studies of on e of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(- 1) and a doubling time of 16.5 h. Data discussed here show that at least tw o MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.