The Development of a Highly Sensitive Electrochemiluminescence Method for the Accurate Quantification of Thyroid Stimulating Hormone in Human Serum
Objective To develop a high-sensitivity chemiluminescence immunoassay method for the determination of thyroid stimulating hormone (TSH)in human serum based on the principle of double-site sandwich chemiluminescence immunoassay.Methods The study samples were co-incubated with superparamagnetic beads pre-coated with anti-thyrotropin antibody (TSH)and anti-TSH-alkaline phosphate conjugate in the reaction tube.TSH in the sample was combined with magnetic beads and anti-TSH-alkaline phosphate conjugate by sandwich method to form a two-site complex.The complex bound to the magnetic bead was adsorbed to the magnetic field and the unbound material was washed away with a washing buffer.The luminescent substrate, 3-(2-spiroadamantane )-4-methoxy-4-(3-phosphoryloxy )-phenyl-1 ,2-dioxetane (AMPPD)was added,and the relative photon intensity (RLU)was measured.The concentration of TSH in the sample was calculated using a standard curve.Results TSH detection method established in this study showed that the minimum detection limit was 0.003μIU/mL,in-batch CV was less than 1％,and total CV was less than 3％.The relative deviation between the measured value and the target value of the sample was within the range of ±12.5％,while the linear range was from 0.005 to 100μIU/mL, and the correlation coefficient (r)was higher than 0.9900.The relative deviation of the stability was less than 10％.Conclusion The proposed method in this study demonstrates a high sensitivity,high accuracy,wide linear range and good stability for quantifying serum TSH,and all indicators meet the requirements of clinical testing and can be popularized for future applications.
万方数据
维普
