一种通用循环免疫复合物抗体解离技术的建立及应用评价
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摘要
目的:建立一种通用循环免疫复合物(CIC)抗体解离技术,并评价其应用价值。方法:采用已建立的聚乙二醇(PEG)二次沉淀分离技术对标本中的CIC进行高效分离,然后运用Gly-HCl缓冲系统和正交设计方法对HBs Ag-IC中的anti-HBs解离实验条件进行优化选择,建立通用的CIC抗体解离技术,并对其方法学性能及应用进行初步评价。结果:HBs Ag-IC中anti-HBs的最佳解离条件为:CIC抗体解离剂p H1. 80,15℃解离5～10 min,振荡频率>60次/min,加入CIC抗体中和剂后10min内测定;该技术对HBs Ag-IC的anti-HBs平均解离率为64. 3%,重现性<5. 97%,线性良好(r=0. 993 2),平均回收率95. 4%,特异性100%,试剂稳定性均>12个月; HCV-IC、HIV-IC、Ins-IC、TG-IC的解离条件与HBs Ag-IC基本相同,但CIC中抗体解离率有差异;该技术对不同HBV-M模式HBs Ag-IC中的anti-HBs含量和检出率具有统计学差异(P<0. 05),对HCV-IC、HIV-IC、INS-IC、TG-IC中的相应抗体检出率分别为34. 8%、66. 7%、20. 0%、14. 3%。结论:CIC抗体解离技术是一种通用前处理技术,它能够对多种CICs进行沉淀、分离、解离后直接测定抗体,具有一定的应用价值。
Objective: To establish a general dissociation technique for antibodies in circulating immune complexes( CICs) and evaluate its application. Methods: CICs in specimens were efficiently separated by an established polyethylene glycol( PEG) doubleprecipitation separation technique. Then the experimental conditions of anti-HBs dissociation from HBs Ag-IC were tested using a GlyHCl buffer system and the orthogonal design method. A general CIC antibody dissociation technique was established,and its methodological performance and application was preliminarily evaluated. Results: The optimal conditions for anti-HBs dissociation from HBs Ag-IC were as follows: CIC antibody dissociation agent at p H1. 80( Glycine-HCl buffer system) was preferred for the dissociation of anti-HBs from HBs Ag-IC,with incubation for 5-10 min at 15℃,an oscillation frequency of >60/min and detection within 10 min after the addition of CIC antibody neutralizing agent. Additionally,the mean dissociation rate,reproducibility,the correlation coefficient,the mean recovery rate and specificity were 64. 3%,<5. 97%,0. 993 2,95. 4%,100%,respectively. The reagents remained stable for longer than12 months. The conditions of antibody dissociation in HCV-IC,HIV-IC,INS-IC,TG-IC and HBs Ag-IC were basically the same by the technique,however,the antibody dissociation rate for CICs was different. This technique resulted in significant differences in the level and detection rate of anti-HBs in HBSAg-IC among different HBV-M patterns( P<0. 05),and the detection rates of corresponding anti-bodies in HCV-IC,HIV-IC,INS-IC and TG-IC were 34. 8%,66. 7%,20. 0% and 14. 3%,respectively. Conclusion: CIC antibody dissociation technique is a general pre-treatment technique for the detection of antibodies after precipitation,separation and dissociation of multiple CICs,so that it has some application value.
Objective: To establish a general dissociation technique for antibodies in circulating immune complexes( CICs) and evaluate its application. Methods: CICs in specimens were efficiently separated by an established polyethylene glycol( PEG) doubleprecipitation separation technique. Then the experimental conditions of anti-HBs dissociation from HBs Ag-IC were tested using a GlyHCl buffer system and the orthogonal design method. A general CIC antibody dissociation technique was established,and its methodological performance and application was preliminarily evaluated. Results: The optimal conditions for anti-HBs dissociation from HBs Ag-IC were as follows: CIC antibody dissociation agent at p H1. 80( Glycine-HCl buffer system) was preferred for the dissociation of anti-HBs from HBs Ag-IC,with incubation for 5-10 min at 15℃,an oscillation frequency of >60/min and detection within 10 min after the addition of CIC antibody neutralizing agent. Additionally,the mean dissociation rate,reproducibility,the correlation coefficient,the mean recovery rate and specificity were 64. 3%,<5. 97%,0. 993 2,95. 4%,100%,respectively. The reagents remained stable for longer than12 months. The conditions of antibody dissociation in HCV-IC,HIV-IC,INS-IC,TG-IC and HBs Ag-IC were basically the same by the technique,however,the antibody dissociation rate for CICs was different. This technique resulted in significant differences in the level and detection rate of anti-HBs in HBSAg-IC among different HBV-M patterns( P<0. 05),and the detection rates of corresponding anti-bodies in HCV-IC,HIV-IC,INS-IC and TG-IC were 34. 8%,66. 7%,20. 0% and 14. 3%,respectively. Conclusion: CIC antibody dissociation technique is a general pre-treatment technique for the detection of antibodies after precipitation,separation and dissociation of multiple CICs,so that it has some application value.
引文
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[10]Zhang ZH,Li L,Zhao XP,et al.Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance[J].J Viral Hepat,2011,18(6):424-433.
[11]Takeda K,Maruki M,Yamagaito T,et al.Highly sensitive detection of hepatitis B virus surface antigen by use of asemiautomated immune complex transfer chemiluminescence enzyme immunoassay[J].J Clin Microbiol,2013,51(7):2238-2244.
[12]Ohyama K,Ueki Y,Kawakami A,et al.Immune complexome analysis of serum and its application in screening for immune complex antigens in rheumatoid arthritis[J].Clin Chem,2011,57(6):905-909.
[13]成军,戴玉柱,闫利,等.一种免疫复合物缓冲解离剂及其应用[P].中国专利:ZL 2014 10033277.4,2016-4-6(2016).Cheng J,Dai YZ,Yan L,et al.An immune complex buffer dissociation agent and its application[P].Chinese patent:ZL 201410033277.4,2016-4-6(2016).
[14]de Carvalho CA,Partata AK,Hiramoto RM,et al.A simple immune complex dissociation ELISA for leishmaniasis:standardization of the assay in experimental models and preliminary results in canine and human samples[J].Acta Trop,2013,125(2):128-136.
[15]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.
[16]Oda M,Uchiyama S,Noda M,et al.Effects of antibody affinity and antigen valence on molecular forms of immune complexes[J].Mol Immunol,2009,47(2-3):357-364.
[17]Summa M,von Bonsdorff CH,Maunula L.Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce,sliced ham,and frozen raspberries[J].J Virol Methods,2012,183(2):154-160.
[18]Lee KB,Lee H,Ha SD,et al.Comparative analysis of viral concentration methods for detecting the HAV genome using real-time RT-PCR amplification[J].Food Environ Virol,2012,4(2):68-72.
[2]Stahl D,Sibrowski W.Warm autoimmune hemolytic anemia is an Ig M-IgG immune complex disease[J].J Autoimmun,2005,25(4):272-282.
[3]Glazer E,Ejaz A,Coley CJ 2nd,et al.Fibrin ring granuloma in chronic hepatitis C:virus-related vasculitis and/or immune complex disease[J].Semin Liver Dis,2007,27(2):227-230.
[4]杜君,田萍,陈陶阳,等.慢性乙肝病毒感染者进展为肝癌病程中血清循环免疫复合物的含量变化和意义[J].中华肿瘤杂志,2011,33(12):905-910.Du J,Tian P,Chen TY,et al.Progressive increase of serum circulating immune complexes and its significnace in patients during the progression from chronic hepatitis B to hepatocellular carcinoma[J].Chin J Oncol,2011,33(12):905-910.
[5]高德玉,陈芳芳,刘阳,等.抗瓜氨酸化肽特异性免疫复合物对类风湿关节滑膜细胞功能影响的研究[J].中国免疫学杂志,2013,29(2):115-120.Gao DY,Chen FF,Liu Y,et al.The effects of anti-citrullinated peptide antibody specific immune complexes on the functions of fibroblast-like synoviocyte in rheumatoid arthritis[J].Chin J Immunol,2013,29(2):115-120.
[6]Li X,Zhang W,Yu HJ,et al.Clinical and pathological study on patients with primary antineutrophil cytoplasmic autoantibody-associated vasculitis with renal immune complex deposition[J].J Clin Rheumatol,2015,21(1):3-9.
[7]Chia D,Barnett EV,Yamagata J,et al.Quantitation and characterization of soluble immune complexes precipitated from sera by polyethylene glycol(PEG)[J].Clin Exp Immunol,1979,37(3):399-407.
[8]周裕林,彭宣宪,柳珑,等.检测HBVDNA/Ig-双特异性循环免疫复合物的免疫捕捉法PCR[J].中国免疫学杂志,2000,16(2):71-72,75.Zhou YL,Peng XX,Liu L,et al.Immuno-capture PCR for HBVD-NA/Ig-two-component-determined circulating immune complexes[J].Chin J Immunol,2000,16(2):71-72,75.
[9]林晨,罗伟中,刘小澄,等.血清标本中HBsAg免疫复合物对检测HBsAg的影响[J].中国病理生理杂志,1996,12(2):172-174.Lin C,Luo WZ,Liu XC,et al.Acid dissociation of HBsAg-anti HBs antibody immune complexs improves diagnostic utility of HB-s Ag detection in HBV infection[J].Chin J Pathophysiol,1996,12(2):172-174.(上接第页)
[10]Zhang ZH,Li L,Zhao XP,et al.Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance[J].J Viral Hepat,2011,18(6):424-433.
[11]Takeda K,Maruki M,Yamagaito T,et al.Highly sensitive detection of hepatitis B virus surface antigen by use of asemiautomated immune complex transfer chemiluminescence enzyme immunoassay[J].J Clin Microbiol,2013,51(7):2238-2244.
[12]Ohyama K,Ueki Y,Kawakami A,et al.Immune complexome analysis of serum and its application in screening for immune complex antigens in rheumatoid arthritis[J].Clin Chem,2011,57(6):905-909.
[13]成军,戴玉柱,闫利,等.一种免疫复合物缓冲解离剂及其应用[P].中国专利:ZL 2014 10033277.4,2016-4-6(2016).Cheng J,Dai YZ,Yan L,et al.An immune complex buffer dissociation agent and its application[P].Chinese patent:ZL 201410033277.4,2016-4-6(2016).
[14]de Carvalho CA,Partata AK,Hiramoto RM,et al.A simple immune complex dissociation ELISA for leishmaniasis:standardization of the assay in experimental models and preliminary results in canine and human samples[J].Acta Trop,2013,125(2):128-136.
[15]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.
[16]Oda M,Uchiyama S,Noda M,et al.Effects of antibody affinity and antigen valence on molecular forms of immune complexes[J].Mol Immunol,2009,47(2-3):357-364.
[17]Summa M,von Bonsdorff CH,Maunula L.Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce,sliced ham,and frozen raspberries[J].J Virol Methods,2012,183(2):154-160.
[18]Lee KB,Lee H,Ha SD,et al.Comparative analysis of viral concentration methods for detecting the HAV genome using real-time RT-PCR amplification[J].Food Environ Virol,2012,4(2):68-72.