RATIONALE: Mus m 1 is a major allergen found in mouse urine and has
been shown to contribute to asthma prevalence in inner city homes.
Commercially available mouse epithelium extracts are non-standardized.
Our goal is to develop monoclonal-antibody-based sandwich ELISA
assays for measurement of Mus m 1.
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 we... More
RATIONALE: Mus m 1 is a major allergen found in mouse urine and has
been shown to contribute to asthma prevalence in inner city homes.
Commercially available mouse epithelium extracts are non-standardized.
Our goal is to develop monoclonal-antibody-based sandwich ELISA
assays for measurement of Mus m 1.
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.