Comparison of polyclonal and monoclonal antibody methods for detecting cockroach antigen in dust samples

Abstract

BACKgrOUND: Previously we have demonstrated excellent correlation between polyclonal (PC) and monoclonal (MC) antibody methods for detecting dust mite, cat and dog antigen levels in dust (correlation coefficients > 0.6) but not for cockroach (CR). The purpose of this study was to determine whether detection of different CR antigens using these two assays accounted for this discrepancy.METHODS: Dust samples with high PC/MC, high PC/low MC and low PC/high MC levels for CR antigen were selected for analysis. SDS-PAGE and western blotting were performed using a polyclonal rabbit IgG antibody generated against German CR antigen (Greer labs). Separate experiments were performed using human serum from CR sensitized subjects as the detection antibody.RESULTS: Western blotting revealed 8 protein bands against the CR antigen standard ranging from 26 to 120 kd. High MC/high PC dust samples yielded several bands spanning this molecular weight (MW) range. The low MC/high PC samples yielded only high MW bands (> 75 kd) whereas the high MC/low PC samples yielded no visible bands. When human serum from CR sensitized subjects was used as the detection antibody, multiple bands between 50 - 120 kd were observed for all dust samples analyzed.CONCLUSION: PC antibody CR assays detected several high MW proteins not identified by MC assays. Although MC antibody CR assays are more sensitive and specific for detecting Bla g 1 (20-25 kd) and Bla g 2 (36 kd) in dust, they may underestimate CR exposure as CR-sensitized individuals reacted to several CR antigens above 36 kd. BACKgrOUND: Previously we have demonstrated excellent correlation between polyclonal (PC) and monoclonal (MC) antibody methods for detecting dust mite, cat and dog antigen levels in dust (correlation coefficients > 0.6) but not for cockroach (CR). The purpose of this study was to determine whether detection of different CR antigens using these two assays accounted for this discrepancy. METHODS: Dust samples with high PC/MC, high PC/low MC and low PC/high MC levels for CR antigen were selected for analysis. SDS-PAGE and western blotting were performed using a polyclonal rabbit IgG antibody generated against German CR antigen (Greer labs). Separate experiments were performed using human serum from CR sensitized subjects as the detection antibody. RESULTS: Western blotting revealed 8 protein bands against the CR antigen standard ranging from 26 to 120 kd. High MC/high PC dust samples yielded several bands spanning this molecular weight (MW) range. The low MC/high PC samples yielded only high MW bands (> 75 kd) whereas the high MC/low PC samples yielded no visible bands. When human serum from CR sensitized subjects was used as the detection antibody, multiple bands between 50 - 120 kd were observed for all dust samples analyzed. CONCLUSION: PC antibody CR assays detected several high MW proteins not identified by MC assays. Although MC antibody CR assays are more sensitive and specific for detecting Bla g 1 (20-25 kd) and Bla g 2 (36 kd) in dust, they may underestimate CR exposure as CR-sensitized individuals reacted to several CR antigens above 36 kd.