不同基质溶液与渗透压对PEG沉淀血清循环免疫复合物的影响

Effects of Different Matrix Solutions and Osmotic Pressure on Serum Circulating Immune Complex in PEG Precipitation

目的探讨聚乙二醇(PEG)沉淀预处理的实验影响因素,建立一种高效的PEG沉淀体系用于血清循环免疫复合物(CIC)前处理。方法对PEG沉淀预处理的实验条件(溶液基质、离子强度、pH值和渗透压)优化,沉淀血清(制备标准HBsAg-anti-HBs CIC)中的CIC后,采用自主研制的免疫复合物解离剂进行解离,然后再用化学发光法检测游离HBsAg。通过计算解离量来比较最适沉淀体系,最后分别采用传统沉淀和优化后的沉淀条件结合自主研发的解离技术,对335份五种乙肝血清标志物模式(HBV-M)标本进行HBsAg-CIC检测。结果基质溶液为0.15mol/L,pH8.2,500mOsm/kg的硼酸盐缓冲液的70g/L PEG 6000沉淀剂,4℃过夜沉淀后选择离心力为18 009×g,离心温度4℃,离心时间为10min,其沉淀分离效果最佳;五种乙肝血清标志物模式(HBV-M)标本分别采用传统和改良法检测免疫复合物,其中改良法的阳性率高于传统法分别为:0,95.2%,87.2%,80%和8.7%,另外在HBV-M 2,3,4组中改良法的沉淀CIC含量高于传统法且差异存在统计学意义(P<0.05)。结论 PEG沉淀被广泛用于血清CIC检测及前处理,受到多重因素的影响,其中基质溶液及渗透压的选择也十分重要。该文为提高血清中循环免疫复合物(CIC)的检出率及缩短沉淀时间提供了理论基础。

Objective To explore the influence factors of PEG precipitation pretreatment and establish a highly efficient PEG precipitation system for serum circulating immune complex （CIC） preprocessing. Methods The experimental conditions of pretreatment （solution matrix,ionic strength,pH value and osmotic pressure） PEG precipitation was optimized. After pre- cipitation of CIC in serum （preparation of standard HBsAg-anti-HBs CIC）, which was dissociated by immune complex disso- ciation agent developed independently,and then free HBsAg was detected by using the chemiluminescence, comparison of the optimum precipitation system by calculating the amount of dissociation. Optimized techniques of precipitation dissociation, and traditional technology were combined with immune complex dissociation technique independently in the 335samples of five HBV serological markers （HBV-M） were detected by HBsAg-CIC,respectively. Results The best precipitation condi- tion as follow：0.15 mol/L,pH：8.2,500 mOsm/kg borate buffer solution including 70 g/L PEG precipitation 6000 mixed with serum overnight in 4℃. After precipitation centrifugal speed was 18 009 × g, centrifugal temperature was 4 ℃, centrifu- gal time was 10min,the precipitation separation effect was best. HBsAg-CIC of the five kinds of HBV serological markers （HBV-M） were detected by the traditional method and the improved method, respectively. The positive rate of immune com- plexes by the improved method was higher than the traditional method, and the positive rates were 0,95.2 %, 87.2 %, 80 % and 8.7 % respectively. Additional, the amount of HBsAg-CIC in the HBV-M 2,3 and 4 were higher by the improved method than by the traditional method,and the difference had statistical significance. Conclusion PEG precipitation is widely used for the detection of serum CIC and pretreatment,influenced by multiple factors,including matrix solution and osmotic pres- sure on the detection of CIC also is very important,in order to improve the circulating immune complex （PEG precipitation） provides a theoretical basis for the detection rate and shortened the settling time.