One- and two-step non-competitive avidin-biotin immunoassays for monomeric and heterodimeric antigen

In this study of one-step and two different two-step non-competitive avidin-biotin assays (NABAs) were developed for the measurement of a monomeric antigen (lactoferrin, LF) using polyclonal antibodies and the detection of a heterodimeric antigen (lutropin. LH) using monoclonal antibodies. The assays were based on the use of performed complexes of biotinylated antibody and avidin-peroxidase conjugate. The detection limits and intra-assay CVs of the one- and two-step NABAs were 0.1-0.5 mg/ml and 2.6-5.1% for LF, and 0.1-0.2 IU/l and 2.3-3.7% for LH, respectively. The working range was 1-100 ng/ml for the LF assay and 1-100 IU/l for the LH assay. A linear relationship with high correlation coefficients (0.979-0.992 for LF-NABAs: 0.949-0.990 for LH-NABAs) and good agreement was observed between the one- and two-step assays and the corresponding three-step NABAs used as reference methods. However, under stringent conditions the one-step assay for heterodimeric antigen was found to be sensitive to interference. The results indicate that it is possible to perform the multistep NABAs using convenient one- and two-step protocols. The one- and two-step assays also retained the advantages of the avidin-biotin system: rapidity and good sensitivity.