A rapid and sensitive chemiluminescence dot-immunobinding assay for screening hybridoma supernatants

Abstract

The present report describes a simple and rapid dot-immunobinding assay combined with a chemiluminescence detection system for screening hybridoma supernatants for specific monoclonal antibodies (MAbs). Small rectangular nitrocellulose filters dotted with either crude mixtures of antigens, or with control samples, were placed in six well plates, incubated with hybridoma supernatants, then stained with peroxidase-conjugated anti-mouse IgG. The reaction was performed with a chemiluminescence detection system. We used this method to screen hybridoma supernatants for MAbs against a 354 amino acid polypeptide of hog cholera virus (HCV) gp33-gp55 protein expressed as a fusion protein. We also extended it for the screening of MAbs against foot-and-mouth disease virus (FMDV). The chemiluminescence dot-immunobinding assay (CDIA) was compared with neutralization (N) and immunofluorescence (IF) screening tests and some FMDV seroneutralizing MAbs were found to either poorly reactive or undetected by the IF test. The advantage of the present method is that it detects in only one step all MAbs detected in the IF and/or N tests together with some MAbs not detected by either of these methods. The present method is at least 356 times more sensitive than the IF test.