Immunoresonance Scautering Spectral Assay for the Determination of Antithrombin-Ⅲ
JIANG Bo1,2,JIANG Zhi-liang1*
1. School of Environment and Resource, Guangxi Normal University, Guilin 541004, China 2. Pharmaceutical Science Department, Zunyi Medical College, Zunyi 563003, China
Abstract：A new sensitive, selective and simple immune resonance scattering spectral assay was proposed for the determination of trace amounts of Antithrombin-Ⅲ（AT-Ⅲ）. It was based on the immune reaction of AT-Ⅲ with the goat-anti-human AT-Ⅲ antibody and the resonance scattering effect of the immunocomplex particles in pH 7.2 Tris-HCl buffer solution and in the presence of polyethelene glycol （PEG）6000. The results showed that the resonance scattering signal of AT-Ⅲ and goat-anti-human AT-Ⅲ antibody was very weak. However, AT-Ⅲ was combined with goat-anti-human AT-Ⅲ antibody specifically, and aggregated to form immunocomplex particles, which enhanced the resonance scattering intensity greatly and produced three resonance scattering peaks at 368 nm, 491 nm and 536 nm respectively. The strongest resonance scattering peak was at 491 nm. In the present paper, the influences of pH, goat-anti-human AT-Ⅲ antibody and PEG-6000 concentration, incubation temperature, reactive time, and foreign substances were investigated. The result showed that the resonance scattering intensity at 491 nm (IRS) is linear to the AT-Ⅲ concentration in the range of 62.5 to 850 ng · mL-1, under the optimum conditions of 0.30 mL goat-anti-human AT-Ⅲ antibody, 30 mg·mL-1 polyethelene glycol-6000, being incubated at 37 ℃ for 15 min, the voltage at 400 V, and the excitation and emission slit width both at 5.0 nm. Its regress equation is ΔIRS=0.062 5c+1.36，and a relative coefficient of 0.996, with a detection limit of 29.4 ng·mL-1. The results of co-existing substance tolerance test showed that 1.40×105 ng·mL-1 glycine, 9.0×103 ng·mL-1 L-glutamic acid, 5.0×105 ng·mL-1 glucose, 5.0×104 ng · mL-1 urea, 1.5×104 ng·mL-1 IgG, 3.0×104 ng·mL-1 human serum albumin, and 1.5×104 ng·mL-1 bovine serum albumin did not interfere with the resonance scattering determination of 2.50×102 ng·mL-1 AT-Ⅲ, when the relative error was within ±10%. Four polyethelene glycols, i.e. polyethelene glycol-4000, polyethelene glycol-6000, polyethelene glycol-10000 and polyethelene glycol-20000, on the immune resonance scattering spectral system were examined in details. The results show that polyethelene glycol-6000 has low blank and high ΔIRS value, and was chosen for use. The new resonance scattering spectral method features high sensitivity, good selectivity, simplicity and rapidity, and was applied to the quantitative analysis of AT-Ⅲ in plasma and serum samples with satisfactory results. Its recovery is in the range of 90.2%-108.9%.
