IgA免疫复合物微粒的共振散射光谱研究及其分析应用

在pH 6.2的Na2HPO4-柠檬酸缓冲溶液中及聚乙二醇4 000存在下,免疫球蛋白A(IgA)与羊抗人免疫球蛋白A通过库力引力、范德华力、氢键结合力、疏水等作用力发生特异性结合形成抗原-抗体免疫复合物微粒.激光散射法测得该微粒的平均粒径约为1 100 nm;而且该微粒在340,390,420,450,470,520 nm有6个共振散射峰.考察了pH值、不同分子量聚乙二醇、羊抗人IgA血清用量、温度及反应时间对共振散射光谱测定IgA的影响.在最佳实验条件下,IgA浓度在0.133～4.67 μg·mL-1范围内与340和470 nm处的共振散射强度均呈线性关系,其回归方程分别为ΔI340 nm=18.61 cIgA 3.19,ΔI470 nm=18.57 cIgA 6.51,相关系数分别为0.998 5和0.998 7,检出限分别为0.068和0.072 μg·mL-1.该法用于人血清IgA的测定,相对标准偏差在2.2%～4.2%,并与免疫比浊法测定结果作线性回归分析,其斜率、截距和相关系数分别为1.064,-0.213和0.929 9,结果令人满意.

Resonance Scattering Spectral Study of IgA Immune Complex Particles and Its Analytical Application

Based on the resonance scattering (RS) effect of immune complex particles, a new resonance scattering spectral method for the determination of trace IgA in the human blood serum was developed. It was based on that goat anti-human IgA was combined with the antigen of IgA. It is known that antibody has C-terminal and N-terminal and the N-terminal is binding site of antigen, and it could combine antigen. Because the stereo structure anastomoses and the charge are opposite between goat anti-human IgA and IgA, they could attract and combine each other. The attraction and combination have high idiosyncrasy and they are done by Val der Waals force, hydrophobic force, Coulomb attracting force and hydrogen bond binding force, and aggregate to form immune complex particles that exhibit three resonance scattering peaks at 340, 390, 420, 450, 470 and 520 nm respectively, in the pH 6.2 Na2 HPO4-citrate buffer solutions and in the presence of polyethylene glycol (PEG) 4000. The laser scattering results indicate that the average diameter of the immune complex particles is about 1 100 nm. The influences of pH, PEG type and its concentration, goat anti-human immunoglobulin A concentration, incubation temperature and incubation time, foreign substance such as glucose, human serum albumin (HSA), urea and L-tyrosine were examined in details. Under the chosen conditions of pH 6.2 Na2 HPO4-citrate buffer solutions-60 mg x mL(-1) polyethylene glycol(PEG) 4000-0.35 mL anti-human immunoglobulin, the resonance scattering intensity at 340 nm deltaI340 nm and the resonance scattering intensity at 470 nm deltaI470 nm all are both proportional to the concentration of IgA in the range of 0.133-4.67 microg x mL(-1). Its regress equation was deltaI340 nm 18.61 C(IgA) 3.19, deltaI470 nm = 18.57C(IgA+) 6.51, with a detection limit of 0.068 and 0.072 microg x mL(-1), respectively. The assay has been applied to the analysis of IgA in human serum, which was also determined by the immunoturbidimetric method, with satisfactory results. The slope, intercept and correlation coefficient of the linear regress analysis were 1.064, -0.213 and 0.929 9, respectively.