Potential factors influencing antigen detection in immunoassays for measuring rat or mouse aeroallergen (i.e., assay setup, antigen specificities, standard extracts used, and antigen decay) were investigated in a three-country study (the UK, The Netherlands, Sweden). An inhibition enzyme immunoassay (EIA) setup gave nominal rat urinary allergen (RUA) sample values seven times higher than a sandwich EIA setup utilizing identical antibodies and standards. In immunoblotting experiments, pooled patient serum and polyclonal rabbit antibodies partly detected different rat antigens; monoclonal antibody specificity could not be determined. Immunoblot detection of mouse urinary antigens (MUA) by the polyclonal rabbit antibodies from all laboratories was similar. In both the RUA and the MUA assays, urinary antigen standards were detected with similar potency, except purified Rat n 1, which was an inefficient inhibitor in the RUA RAST inhibition. In the sandwich EIA RUA assays, a rat room-dust extract was detected with 700–800-fold less sensitivity than rat urine, whereas in the RAST RUA assay, dust inhibited equally with rat urine. Simulated decay did not decrease the potency of urinary antigen in any assay. Thus, assay setup and choice of detection antibodies strongly influence the nominal allergen levels. We recommend the use of standardized and characterized antibodies and standard extracts in sandwich EIAs to measure airborne rodent urinary allergens.
Abbreviations: NHLI: National Heart and Lung Institute, London, UK; WAU: Wageningen Agricultural University, Wageningen, The Netherlands; NIWL: National Institute for Working Life, Solna, Sweden; Ab: antibody; mAb: monoclonal antibody; EIA: enzyme immunoassay; RAST: radioallergosorbent test; RIA: radioimmunoassay; MUA: mouse urinary allergen; RUA: rat urinary allergen; BSA: bovine serum albumin; HSA: human serum albumin; PBS: phosphate-buffered saline; PTFE: polytetrafluoroethylene (Teflon); TBS: Tris-buffered saline.