抗凝血酶Ⅲ的免疫共振散射光谱分析

Immunoresonance Scautering Spectral Assay for the Determination of Antithrombin-Ⅲ

在pH7．2的Tris-HCI缓冲溶液中和聚乙二醇6000（PEG-6000）存在下，羊抗人抗凝血酶Ⅲ与抗凝血酶Ⅲ（AT-Ⅲ）发生免疫反应形成疏水性的免疫复合物微粒，导致体系的共振散射强度增强，在波长为368，491和538nm处出现3个共振散射峰，其中491nm处的峰最强。分别考察了pH、AT-Ⅲ和PEG-6000浓度、温育时间和温度、共存物质的影响。在选定条件下，AT-Ⅲ浓度在62．5-875lag·mL^-1范围内与491nm处体系的散射强度呈良好的线性关系，其回归方程为△IRS=62．5c＋1．36，相关系数为0．996，检出限为29．4ng·mL^-1。该方法简便、灵敏和选择性好，用于人血中AT-Ⅲ含量的测定，结果满意，回收率在90．2％～108．9％之间。

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-HC1 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-11 concentration in the range of 62. 5 to 850 ng · mL^-1 , under the optimum conditions of 0. 30 mL goat-anti-hurnan 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 nrn. Its regress equation is AIRs=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 1.40X 10s ng · mL^-1 glycine, 9.0X103 ng · mL^-1 L-glutamic acid, 5.0X10s ng · mL^-1 glucose, 5.0X104 ng · mL^-1 urea, 1. SX 104 ng · mL^-1 IgG, 3.0X104 ng · mL^-1 human serum albumin, and 1.5X104 ng · mL^-1 bovine serum albumin did not interfere with the resonance scattering determination of 2. 50X 10z ng ~ mL-~ AT-Ill, when the relative error was within ＋10~. Four polyethelene glycols, i. ~ 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 AIRs 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%.