Methods: Monoclonal antibodies were generated in rats by immunizing with natural Mus m 1 using a proprietary MonoExpress immunization protocol (GenScript USA). Anti-Mus m 1 polyclonal sera were generated in rabbits hyperimmunized with Mus m 1: rabbits were primed subcutaneously with 100 mcg of nMus m 1 in Freund’s Complete Adjuvant, followed by boosts of 100 mcg nMus m 1 in Freund’s Incomplete Adjuvant at weeks 3 and 7. Hybridoma supernatants and polyclonal sera were screened against nMus m 1 and mouse urine. Two sandwich ELISAs (sELISAs) were developed: one in which both the capture (12B10) and detection (biotinylated 11G6) antibodies were monoclonal, and one in which the detection antibody was rabbit polyclonal sera.
Results: The monoclonal assay was found to be highly specific and sensitive, and mouse urine contained 3.0 g/L of the antigen, but Mus m 1 in commercial extracts was below the limit of detection. Using polyclonal rabbit sera for detection, the assay was more sensitive. With this assay the Mus m 1 content of mouse urine was 6.7x10-1 g/L and the commercial extracts contained 1.5x10-4 to 2.5x10-3 g/L.
Conclusions: sELISA assays has been developed for determining Mus m 1 contents of non-standardized mouse allergen extracts.