Comparison of Three Immunoassays for Screening Anti-Hepatitis B Hybridomas

Three micro solid phase immunoassays were developed for screening secreting anti-hepatitis B hybridoma clones: a micro-solid phase radioimmunoassay (micro-SPRIA)1 and two enzyme-linked immunosorbent assays (ELISA). In all three procedures, aliquots of hybridoma culture fluid were first added to antigen-coated wells. Goat anti-mouse IgG (GtaM) was used as second antibody. For micro-SPRIA, Gt α m was iodinated by the chloramine T method. For the ELISAs, we compared two methods of coupling the antibody to alkaline phosphatase (AP): with glutaraldehyde (ELISA-glutaraldehyde) and with N-succinimidyl 3-(2-pyridyldi-thio) propinate (SPDP) (ELISA-SPDP). All three tests were more sensitive than the commercially available AUSAB (Abbott), which could not detect low enough levels of antibody to be used for hybridoma screening. ELISA-glutaraldehyde was the least sensitive of the three and produced a number of false positive results. Micro-SPRIA was 5 times more sensitive than ELISA-glutaraldehyde, and ELISA-SPDP was 2.5 times more sensitive than micro-SPRIA. This higher sensitivity of ELISA-SPDP allowed us to detect a greater number of secreting clones at an earlier time after fusion.