Labeled antigen capture assay: A method for detecting monoclonal antibodies to cloned gene products

Abstract

We report here a relatively easy and highly sensitive assay for detecting monoclonal antibodies to the product of virtually and cloned gene. The protocol, termed labeled antigen capture assay (LACA), is a solid-phase type radioimmunoassay which uses a bacteriophage T7 expression system to generate exclusively radiolabeled antigen. Thus, to generate radiolabeled antigen for screening, the gene encoding the protein of interest need only be subcloned downstream of a T7 promoter, and the new construct transformed into an Escherichia coli strain harboring a compatible plasmid which encodes a thermal inducible copy of the T7 RNA polymerase. Expression of the T7-promoted gene in the presence of rifampicin and [ 35S]cysteine (or methionine) yields labeled antigen, which is then “captured” by specific monoclonal antibody and detected by autoradiography. Our results indicate that as little as 30 ng of specific monoclonal antibody can be detected using the LACA protocol. The protocol is applicable to the product of any cloned gene but is particularly useful in the case where the biochemical properties of the gene product of interest are unavailable. In this report we use the LACA protocol to screen a hybridoma library for monoclonal antibodies to the STb heat-stable enterotoxin (STb) of E. coli. Mice were immunized with a genetically constructed, affinity-purified Protein A-STb hybrid protein, and following spleen cell fusion and HAT selection, hybridomas were screened by the described LACA protocol for production of STb-specific monoclonal antibody. Of over 1500 hybridomas tested 138 were positive, by primary LACA screening, for STb-specific IgG monoclonal antibodies.