Immunodot assay for determining the isotype and light chain type of murine monoclonal antibodies in unconcentrated hybridoma culture supernates

Abstract

We describe a procedure for determining immunoglobulin isotype and light chain class of murine monoclonal antibodies. Isotype and light chain-specific antibodies are immobilized as dots on a strip of nitrocellulose filter paper. The immobilized antibodies retain binding specificity, and the strips are used in a type of 'sandwich' immunoassay wherein murine monoclonal immunoglobulins bound to the appropriate anti-isotype dot are detected with a peroxidase-conjugated anti-mouse immunoglobulin reagent. The immunodot isotyping assay is specific and sensitive enough to detect mouse immunoglobulin present in ng/ml concentrations. It is as easy or easier to set up and perform than conventional isotyping techniques. It has the added advantages that it greatly conserves antisera reagents and can be performed on hybridoma culture supernates without the need to concentrate, expand, or purify them.