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
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摘要
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 precipitation of CIC in serum(preparation of standard HBsAg-anti-HBs CIC),which was dissociated by immune complex dissociation 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 335 samples of five HBV serological markers(HBV-M)were detected by HBsAg-CIC,respectively.Results The best precipitation condition 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℃,centrifugal time was 10 min,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 complexes 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 pressure 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.
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 precipitation of CIC in serum(preparation of standard HBsAg-anti-HBs CIC),which was dissociated by immune complex dissociation 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 335 samples of five HBV serological markers(HBV-M)were detected by HBsAg-CIC,respectively.Results The best precipitation condition 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℃,centrifugal time was 10 min,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 complexes 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 pressure 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.
引文
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[10]管晓龙,王海永,周莹,等.类风湿关节炎血清免疫复合物的质谱鉴定与差异蛋白分析[J].医学研究生学报,2017,30(5):495-501.Guan XL,Wang HY,Zhou Y,et al.Differentially expressed proteins in serum immune complexes of rheumatoid arthritis:Analysis by mass spectrometry[J].Journal of Medical Postgraduates,2017,30(5):495-501.
[11]Sobenin IA,Salonen JT,Zhelankin AV,et al.Low density lipoprotein-containing circulating immune complexes:Role in atherosclerosis and diagmostic value[J].Bio Med Res Int,2014(2014):205697.
[12]Yao X,Wang X,Zhao C,et al.Transcriptional analysis of immune-related genes in dendritic cells from hepatitis B surface antigen(HBsAg)-positive transgenic mice and regulation of Fc gamma receptor IIB by HBsAg-anti-HBs complex[J].J Med Virol,2011,83(1):78-87.
[13]Takeda K,Maruki M,Yamagaito T,et al.Highly sensitive detection of hepatitis B virus surface antigen by use of a semiautomated immune complextransfer chemiluminescence enzyme immunoassay[J].J Clin Microbiol,2013,51(7):2238-2244.
[2]Iwasaki A,Medzhitov R.Control of adaptive immunity by the innate immune system[J].Nat Immunol,2015,16(4):343-353.
[3]Rojko JL,Evans MG,Price SA,et al.Formation,clearance,deposition pathogenicity and identification of biopharmaceutical-related immune complexes:review and case studies[J].Toxicol Pathol,2014,42(4):725-764.
[4]Takeda K,Maruki M,Yamagaito T,et al.Highly sensitive detection of hepatitis B virus surface antigen by use of a semiautomated immune complex transfer chemiluminescence enzyme immunoassay[J].J Clin Microbiol,2013,51(7):2238-2244.
[5]Dai Y,Hu Z,Chen Y,et al.A novel general and efficient technique for dissociating antigen in circulating immune complexes[J].Electrophoresis,2018,39(2):406-416.
[6]Moore TL.Immune complexes in juvenile idiopathic arthritis[J].Front Immunol,2016(7):177.
[7]Reshetnyak T,Alexandrova EN,Seredavkina NV,et al.AB0434antibody to dsdna(anti-dsdna),circulating immune complex(cic)in systemic lupus erithematosus(sle)and antiphospholipid syndrome(aps)[J].Ann Rheum Dis,2013,72(suppl 3):A921.
[8]成军,戴玉柱,闫利,等.一种免疫复合物缓冲解离剂及其应用,中国专利:ZL 2014 10033277.4[P].2016-4-6.Cheng J,Dai YZ,Yan L,et al.An immune complex dissociation buffer agent and its application,CN:ZL2014 10033277.4[P].2016-4-6.
[9]成军,孙长贵,戴玉柱,等.一种用于免疫复合物缓冲解离剂的CIC复溶剂,中国专利:ZL201410034039.5[P].2016-4-6.Cheng J,Sun CG,Dai YZ,et al.An immune complex dissociation buffer agent CIC complex solvent,CN:ZL201410034039.5[P].2016-4-6.
[10]管晓龙,王海永,周莹,等.类风湿关节炎血清免疫复合物的质谱鉴定与差异蛋白分析[J].医学研究生学报,2017,30(5):495-501.Guan XL,Wang HY,Zhou Y,et al.Differentially expressed proteins in serum immune complexes of rheumatoid arthritis:Analysis by mass spectrometry[J].Journal of Medical Postgraduates,2017,30(5):495-501.
[11]Sobenin IA,Salonen JT,Zhelankin AV,et al.Low density lipoprotein-containing circulating immune complexes:Role in atherosclerosis and diagmostic value[J].Bio Med Res Int,2014(2014):205697.
[12]Yao X,Wang X,Zhao C,et al.Transcriptional analysis of immune-related genes in dendritic cells from hepatitis B surface antigen(HBsAg)-positive transgenic mice and regulation of Fc gamma receptor IIB by HBsAg-anti-HBs complex[J].J Med Virol,2011,83(1):78-87.
[13]Takeda K,Maruki M,Yamagaito T,et al.Highly sensitive detection of hepatitis B virus surface antigen by use of a semiautomated immune complextransfer chemiluminescence enzyme immunoassay[J].J Clin Microbiol,2013,51(7):2238-2244.