α-Gal抗原定量检测试剂盒的研发
|
摘要
目的:通过优化α-Gal抗原检测的方法及改善相关试剂的稳定性,研发α-Gal抗原定量检测试剂盒,并评价该试剂盒的稳定性、可靠性和适用性。方法:以医疗器械行业标准(YY/T 1561—2017组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测)中的α-Gal抗原检测酶联免疫抑制方法为基础,通过评价回收率、相关系数R2、最大吸收值和一抗的最高结合抑制率,对一抗、酶标二抗和固相抗原包被反应条件进行优化。预先制备好抗原包被板以及即用型试剂,制备成套试剂盒。通过考察Gal-BSA标准品的Gal抗原含量,评价试剂盒的检测回收率、标准曲线的R2值、最高反应的吸收度(A)和最高结合抑制率,考察其稳定性。使用优化好的试剂盒检测几种样品的Gal抗原,考察试剂盒的适用性。结果:经过优化后的试剂盒Gal抗原最低检测限为5.64×1012个/每反应,检测回收率80%~120%;相关系数R2>0.98;6批次的板内、板间检测的相对标准偏差RSD<5%;阴性参考品无Gal抗原检出。使用该试剂盒能够准确检测小鼠脏器Gal抗原含量,其结果显示小鼠各脏器Gal抗原表达趋势与Gal抗原调控基因的m RNA表达趋势完全一致,间接地佐证了该试剂盒的准确性和可信性。检测生物型硬脑膜补片原材料Gal抗原含量为(8.34±1.17)×1014个·mg-1,产品Gal抗原含量为(4.16±2.56)×1012个·mg-1,计算得抗原清除率为99.5%。结论:该试剂盒具有较好的灵敏度,特异性,适用于依照行业标准YY/T 1561—2017进行动物源医疗器械/生物材料α-Gal抗原残留量的定量和Gal抗原清除率检测,为行业标准的实施提供技术保障。
objective:To optimize the α-Gal antigen detection method and to improve the stability of related reagents,to develop α-Gal antigen quantitative detection Kit,and evaluate the stability,reliability and applicability of the Kit.Methods:Based on the α-Gal antigen detection method,inhibitive enzyme linked immunosorbent assay(ELISA)in the medical device industry standard(YY/T 1561-2017,Tissue engineering medical device products-remnant α-Gal antigen determination in scaffold materials utilizing animal tissues and their derivatives),the following were further optimized:the reaction conditions of primary antibody,enzyme-labeled second antibody and solid-phase antigen-coating by evaluation of the recovery ratio,correlation coefficient R2,maximum absorbance value and maximum binding inhibition rate of primary antibody.And by combining with pre-prepared antigen-coated plate and ready-to-use reagents a Kit was developed.The stability of the Kit was evaluated by investigating the Gal antigen content of Gal-BSA reference material,the detection recovery rate of the Kit,the R2 value of the standard curve,the maximum reaction absorbance(A)and the maximum binding inhibition rate.The Gal antigen of several samples was detected by optimized Kit,and the applicability of the Kit was investigated.Results:The minimum detection limit of Gal antigen of the optimizedk it was 5.64×1012 per reaction,the recovery rate of detection was 80%-120%;the correlation coefficient R2 was more than 0.98;the RSD value of internal and among plate of 6 batch plates was less than 5%.There was no Gal antigen was detected in the negative reference material.The use of the Kit can accurately detect Gal antigen in organs of mice, and the results showed that the trend of Gal antigen expression to be detected in the main organs of mice was very consistent with the trend of m RNA expression which regulated the Gal antigen expression,indirectly evidenced the accuracy and reliability of the Kit.The results of Gal antigen content detected by the Kit in the raw material and the product of biological Dural mater patch were(8.34±1.17)× 1014·mg-1 wet tissues and(4.16±2.56)×1012·mg-1 wet tissues,and the antigen clearance rate was 99.5%.Conclusion:This Kit has good sensitivity and specificity,and it is suitable for detecting the quantity of α-Gal antigen residue and the clearance of Gal antigen of animal source medical device/biological material according to the industry standard YY/T 1561-2017,and provides technical support for the implementation of the industry standard.
objective:To optimize the α-Gal antigen detection method and to improve the stability of related reagents,to develop α-Gal antigen quantitative detection Kit,and evaluate the stability,reliability and applicability of the Kit.Methods:Based on the α-Gal antigen detection method,inhibitive enzyme linked immunosorbent assay(ELISA)in the medical device industry standard(YY/T 1561-2017,Tissue engineering medical device products-remnant α-Gal antigen determination in scaffold materials utilizing animal tissues and their derivatives),the following were further optimized:the reaction conditions of primary antibody,enzyme-labeled second antibody and solid-phase antigen-coating by evaluation of the recovery ratio,correlation coefficient R2,maximum absorbance value and maximum binding inhibition rate of primary antibody.And by combining with pre-prepared antigen-coated plate and ready-to-use reagents a Kit was developed.The stability of the Kit was evaluated by investigating the Gal antigen content of Gal-BSA reference material,the detection recovery rate of the Kit,the R2 value of the standard curve,the maximum reaction absorbance(A)and the maximum binding inhibition rate.The Gal antigen of several samples was detected by optimized Kit,and the applicability of the Kit was investigated.Results:The minimum detection limit of Gal antigen of the optimizedk it was 5.64×1012 per reaction,the recovery rate of detection was 80%-120%;the correlation coefficient R2 was more than 0.98;the RSD value of internal and among plate of 6 batch plates was less than 5%.There was no Gal antigen was detected in the negative reference material.The use of the Kit can accurately detect Gal antigen in organs of mice, and the results showed that the trend of Gal antigen expression to be detected in the main organs of mice was very consistent with the trend of m RNA expression which regulated the Gal antigen expression,indirectly evidenced the accuracy and reliability of the Kit.The results of Gal antigen content detected by the Kit in the raw material and the product of biological Dural mater patch were(8.34±1.17)× 1014·mg-1 wet tissues and(4.16±2.56)×1012·mg-1 wet tissues,and the antigen clearance rate was 99.5%.Conclusion:This Kit has good sensitivity and specificity,and it is suitable for detecting the quantity of α-Gal antigen residue and the clearance of Gal antigen of animal source medical device/biological material according to the industry standard YY/T 1561-2017,and provides technical support for the implementation of the industry standard.
引文
[1]HENION TR,MACHER BA,ANARAKI F,et al.Defining the minimal size of catalytically active primateα1,3galactosyltransferase:structure function studies on the recombinant truncated enzyme[J].Glycobiology,1994,4(2):193
[2]GALILI U,SHOHET SB,KOBRIN E,et al.Man,apes,and old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells[J].J Biochem,1988,263(33):17755
[3]GALILI U,MANDRELL RE,HAMADEH RM,et al.Interaction between human natural anti-α-galactosyl immunoglobulin G and bacteria of the human flora[J].Infect Immun,1988,56(7):1730
[4]CHOI HJ,KIM MK,LEE HJ,et al.Effect of alpha Gal on corneal xenotransplantation in a mouse model[J].Xenotransplantation,2011,18(3):176
[5]NASO F,GANDAGLIA A,IOP L,et al.First quantitative assay of alpha-Gal in soft tissues:presence and distribution of the epitope before and after cell removal from xenogeneic heart valves[J].Acta Biomater,2011,7(4):1728
[6]SHADDY RE,HAWKINS JA.Immunology and failure of valvedallografts in children[J].Ann Thorac Surg.2002,74(4):1271
[7]PETER G,GOLDSTEIN IJ.The use of fluorescein-conjugated Banderiaeasimplicifolia B4-lectin as a histochemical reagent for the detection of alpha-D-galactopyranosyl groups.Their occurrence in basement membranes[J].Exp Cell Res,1979,120:321
[8]GALILI U,LATEMPLE DC,RADIC MZ.A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody[J].Transplantation,1998,65(8):1129
[9]孙晓霞,刘佳,刘成虎,等.应用抑制性ELISA法测定动物源性生物材料中α-Gal抗原[J].药物生物技术,2015,22(1):33SUN XX,LIU J,LIU CH,et al.Determination ofα-Gal Epitope in animal-derived biomaterials Using ELISA Inhibition Assay[J].Pharm Biotechnol,2015,22(1):33
[10]冯卫,付丽,福兴,等.Alpha-Gal异种移植抗原在猪骨中的分布研究[J].中国实验诊断学,2009,13(3):343FENG W,FU L,FU X,et al.Distribution of the alpha-Gal epitope on porcine bone tissue[J].Chin J Lab Diagn,2009,13(3):343
[11]单永强,徐丽明,柯林楠,等.动物源性生物材料中残留α-Gal抗原检测方法[J].生物医学工程学杂志,2015,32(2):680SHAN YQ,XU LM,KE LN,et al.Assessment method of remnantα-13-Galactosyle epitopes in animal tissue-derived biomaterilas[J],J Biomed Eng,2015,32(2):680
[12]陆艳,单永强,邵安良,等.ELISA抑制法检测动物组织中α1,3-Gal抗原[J].药物分析杂志,2015,35(10):1729LU Y,SHAN YQ,SHAO AL,et al.Assessment ofα1,3-Gal antigen in animal tissues by ELISA inhibition method[J].Chin J Pharm Anal,2015,35(10):1729
[13]YY/T 1561—2017组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测.医疗器械行业标准[S].2017YY/T 1561-2017 Tissue engineering medical device productsremnantα-Gal antigen detection of animal tissue derived biomaterials and their derivatives.Industry Standard of Medical Device[S].2017
[14]MACHER BA,GALILI U.The Galα1,3Galβ1,4Glc NAc-R(α-Gal)epitope:a carbohydrate of unique evolution and clinical relevance[J].Biochim Biophys Acta,2008,1780(2):75
[15]LAVECCHIO JA,DUNNE AD,EDGE AS.Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies[J].Transplantation,1995,60(8):841
[16]GALILI U.Theα-Gal epitope(Galα1-3Galβ1-4Glc NAc-R)in xenotransplantation[J].Biochimie,2001,83(7):557
[2]GALILI U,SHOHET SB,KOBRIN E,et al.Man,apes,and old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells[J].J Biochem,1988,263(33):17755
[3]GALILI U,MANDRELL RE,HAMADEH RM,et al.Interaction between human natural anti-α-galactosyl immunoglobulin G and bacteria of the human flora[J].Infect Immun,1988,56(7):1730
[4]CHOI HJ,KIM MK,LEE HJ,et al.Effect of alpha Gal on corneal xenotransplantation in a mouse model[J].Xenotransplantation,2011,18(3):176
[5]NASO F,GANDAGLIA A,IOP L,et al.First quantitative assay of alpha-Gal in soft tissues:presence and distribution of the epitope before and after cell removal from xenogeneic heart valves[J].Acta Biomater,2011,7(4):1728
[6]SHADDY RE,HAWKINS JA.Immunology and failure of valvedallografts in children[J].Ann Thorac Surg.2002,74(4):1271
[7]PETER G,GOLDSTEIN IJ.The use of fluorescein-conjugated Banderiaeasimplicifolia B4-lectin as a histochemical reagent for the detection of alpha-D-galactopyranosyl groups.Their occurrence in basement membranes[J].Exp Cell Res,1979,120:321
[8]GALILI U,LATEMPLE DC,RADIC MZ.A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody[J].Transplantation,1998,65(8):1129
[9]孙晓霞,刘佳,刘成虎,等.应用抑制性ELISA法测定动物源性生物材料中α-Gal抗原[J].药物生物技术,2015,22(1):33SUN XX,LIU J,LIU CH,et al.Determination ofα-Gal Epitope in animal-derived biomaterials Using ELISA Inhibition Assay[J].Pharm Biotechnol,2015,22(1):33
[10]冯卫,付丽,福兴,等.Alpha-Gal异种移植抗原在猪骨中的分布研究[J].中国实验诊断学,2009,13(3):343FENG W,FU L,FU X,et al.Distribution of the alpha-Gal epitope on porcine bone tissue[J].Chin J Lab Diagn,2009,13(3):343
[11]单永强,徐丽明,柯林楠,等.动物源性生物材料中残留α-Gal抗原检测方法[J].生物医学工程学杂志,2015,32(2):680SHAN YQ,XU LM,KE LN,et al.Assessment method of remnantα-13-Galactosyle epitopes in animal tissue-derived biomaterilas[J],J Biomed Eng,2015,32(2):680
[12]陆艳,单永强,邵安良,等.ELISA抑制法检测动物组织中α1,3-Gal抗原[J].药物分析杂志,2015,35(10):1729LU Y,SHAN YQ,SHAO AL,et al.Assessment ofα1,3-Gal antigen in animal tissues by ELISA inhibition method[J].Chin J Pharm Anal,2015,35(10):1729
[13]YY/T 1561—2017组织工程医疗器械产品动物源性支架材料残留α-Gal抗原检测.医疗器械行业标准[S].2017YY/T 1561-2017 Tissue engineering medical device productsremnantα-Gal antigen detection of animal tissue derived biomaterials and their derivatives.Industry Standard of Medical Device[S].2017
[14]MACHER BA,GALILI U.The Galα1,3Galβ1,4Glc NAc-R(α-Gal)epitope:a carbohydrate of unique evolution and clinical relevance[J].Biochim Biophys Acta,2008,1780(2):75
[15]LAVECCHIO JA,DUNNE AD,EDGE AS.Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies[J].Transplantation,1995,60(8):841
[16]GALILI U.Theα-Gal epitope(Galα1-3Galβ1-4Glc NAc-R)in xenotransplantation[J].Biochimie,2001,83(7):557
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |