title: Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone.
abstract: 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.
authors.affiliation: The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Lin
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Zhen-Jia
authors.initials: ZJ
authors.lastname: Li
authors.forename: Shi-Qi
authors.initials: SQ
authors.lastname: Ren
authors.forename: Guo-Nan
authors.initials: GN
authors.lastname: Chen
authors.forename: Xi-Tang
authors.initials: XT
authors.lastname: Ying
authors.forename: Jin-Ming
authors.initials: JM
authors.lastname: Lin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D002138:Calibration; D002417:Cattle; D005260:Female; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007124:Immunoenzyme Techniques / Q000379:methods*; D007700:Kinetics; D049449:Luminescence; D008165:Luminol / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D011247:Pregnancy; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000378:metabolism*; D013972:Thyrotropin / Q000032:analysis* / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D008165:Luminol; D001710:Biotin; D013972:Thyrotropin; D019809:Streptavidin; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase