Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone.

A highly sensitive "two-site" chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin-streptavidin system (BSAS). The HRP-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP-streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1-40 mU L(-1). Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L(-1) which was 10-fold higher than the HRP-MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.

Development of high-performance magnetic chemiluminescence enzyme immunoassay for alpha-fetoprotein (AFP) in human serum.

BACKGROUND: A high-performance chemiluminescence enzyme immunoassay (CLEIA) for alpha-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma (HCC), was constructed by using magnetic particles (MPs) as both the immobilization matrix and separation tools. METHODS: A double sandwiched immunocomplex was formed through the reaction among anti-fluorescein isothiocyanate (FITC) antibody coated MPs, FITC-labeled anti-AFP antibody, AFP antigen, and alkaline phosphatase (ALP)-labeled anti-AFP antibody. The subsequent chemiluminescence reaction of ALP with 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD) gave light intensity that was directly proportional to the amount of analyte present in the samples. The effects of several physicochemical parameters, including the concentration of FITC-labeled anti-AFP antibody, the dilution ratio of ALP-labeled anti-AFP antibody, the volume of MPs and substrate, the immunoreaction time and other relevant variables upon the immunoassay were studied and optimized. RIA and microplate CLEIA were used as comparison methods. RESULTS: The proposed method had a sensitivity of 3.0 ng/ml, low cross reactivities, and an assay time of 1 h. The linear range was 0-1200 ng/ml through using MPs and is useful for samples with extremely high AFP concentrations without dilution while avoiding the hook effect. The intra- and inter-assay precision was <3% and <5%. The present method has been successfully applied to the detection of AFP human serum with recoveries from 90 to 108%, and showed a good correlation with the commercially available AFP RIA kit. CONCLUSIONS: This proposed assay provided apparent advantages over microplate CLEIA and RIA, and facilitated the development of high-throughput screening and automated operation systems in the clinical practice.