补体4的免疫共振散射光谱分析

在pH7.3Na2HPO4-KH2PO4缓冲溶液中及聚乙二醇-6000存在下,补体4(C4)与羊抗人补体4(goat anti-human C4)通过库力引力、范德华力、氢键结合力、疏水等作用力发生免疫反应,可聚集形成疏水的免疫复合物微粒,该微粒在350,390,440nm有3个共振散射峰。激光散射法测得免疫复合物微粒的平均粒径为3440.0nm。分别研究了pH、羊抗人补体4和PEG浓度、温育时间和温度、共存物质的影响。在最佳条件下,补体4(C4)浓度在0.18~2.60μg.mL-1范围内与350,390nm处的散射强度均呈线性关系,其回归方程、相关系数、检出限分别为ΔI350nm=28.23c 9.17,ΔI390nm=31.36c 11.08,0.9939,0.9923,0.084μg.mL-1,0.11μg.mL-1。该法用于分析人血清中补体4(C4),结果与免疫透射比浊法结果一致,相对标准偏差在1.88%~4.36%,具有简便快速、灵敏度高、选择性好等特点,在临床检验上具有一定的应用价值。

Immunoresonance Scattering Spectral Assay of Complement Factor 4(C4)

Based on resonance scattering (RS) effect of immune complex particles, a new resonance scattering method for the determination of C4 in the human blood serum was developed. It was based on the fact that goat anti-human C4 was combined with complement factor 4 (C4) in the pH 7.3 Na2 HPO4-KH2PO4 buffer solution. It is known that antibody has C-terminal and N-terminal, and the N-terminal is the binding site of antigen and it could combine with antigen. Because the stereo structure anastomoses and the charge is opposite between goat ant-human C4 and C4, they could attract and combine with each other. The attraction and combination have high idiosyncrasy and they are done by Van der Waals force, hydrophobic force, Coulomb attracting force and hydrogen bond binding force, and form immune complex particles that exhibit three resonance scattering peaks at 350, 390 and 440 nm, respectively, in the presence of PEG-6000. The laser scattering results indicate that the average diameter of the immune complex particles is about 3 440.0 nm. The influence of pH, goat anti-human C4 and PEG-6000 concentration, incubation temperature and incubation time, foreign substance such as arginine, -phenyl alanine, L-glutamic acid, L-cystine, L-threonine, L-tryptophan, L-histidine, L-leucine, glucose, EDTA, human serum albumin (HSA), bovine serum albumin (BSA), L-proline, L-lysine on the determination of C4 was considered in details. Under the conditions chosen, C4 concentration in the range from 0.18 to 2.60 microg x mL(-1) is proportional to the resonance scattering intensity at 350 and 390 nm. Its regression equation is deltaI350 nm = 28.23c + 9.17 and deltaI390 nm = 31.36c + 11.08, the correlation coefficient is 0.993 9 and 0.992 3, and the detection limit is 0.084 microg x mL(-1) and 0.11 microg x mL(-1), respectively. The method has been applied to the determination of C4 in the human blood serum, and the results are in agreement with that of the immunoturbidity, with relative standard deviation of 1.88%-4.36%, and with some advantages including simplicity, rapidity, high sensitivity and selectivity.