抗苗勒氏管激素的生物素-链霉亲和素-辣根过氧化物酶酶促化学发光免疫分析方法的建立
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摘要
目的:抗苗勒氏管激素(AMH)在评价卵巢储备功能方面具有更高的准确性,将其用于青春期、生育期女性的生育指导时具有很好的临床价值。目前对于AMH的检测,临床上常采用的是酶联免疫分析法,但存在灵敏度低、检测范围窄等缺点,而影响AMH实际水平的测定,使得对病情的预估不准确对疾病的治疗产生影响。因此,建立一种低成本、高敏感度的检测AMH水平的方法尤为重要。方法:利用生物素-亲和素放大系统,将一组抗AMH的第一抗体包被微孔板,同时用生物素标记第二抗体,与第一抗体-抗原复合物结合之后,再与生物素-链霉亲和素-辣根过氧化物酶结合物结合,加入发光底物,建立AMH酶促化学发光免疫分析方法,同时对这一体系的线性、灵敏度、特异度、稳定性、回收率等参数进行检测,并与常规ELISA法进行相比检测AMH浓度。结果:线性检测范围为(0.42～50.00)U/ml,该体系稳定性好,灵敏度0.42U/ml,批内差异小于10.0%,批间差异小于10.0%,与常规ELISA对比差异有统计学意义。结论:AMH的生物素-链霉亲和素-辣根过氧化物酶酶促化学发光免疫分析易于操作、灵敏度高、价格低廉,适用于临床检测。
Objective:Anti-Mullerian hormone(AMH)has a higher accuracy in evaluating ovarian reserve function,and it is of great clinical value in the reproductive guidance of female during puberty and growing period.At present,the enzyme-linked immunoassay is commonly used in the detection of AMH,but there are some disadvantages,such as low sensitivity and narrow detection range,which affect the actual level of AMH,which makes the prediction of the condition inaccurate to the treatment of the disease.Therefore,it is very important to establish a low cost and high sensitivity method to detect AMH level.Method:Using the Biotin-affinity amplification System,agroup of anti-AMH first antibody packages were coated with microporous plates,a method of AMH enzyme-induced chemiluminescence immunoassay was established by combining the biotin-streptavidin-horseradish peroxidase binding with biotin-labeled second antibody and the first antibody-antigen complex,and adding the luminescent substrate,the parameters such as linearity,sensitivity,specificity,stability and recovery rate of the system were detected,and compared with the conventional Elisa method to detect AMH concentration.Result:The linear detection range is(0.42-50.00)U/ml,the system has good stability,sensitivity 0.42 U/ml,the difference between batches is less than 10%,the difference between batches is less than 10%,and the difference is statistically significant compared with the conventional ELISA.Conclusion:AMH biotin-streptavidin-horseradish peroxidase enzymatic chemiluminescence immunoassay is easy to operate,high sensitivity and low price,and is suitable for clinical detection.
Objective:Anti-Mullerian hormone(AMH)has a higher accuracy in evaluating ovarian reserve function,and it is of great clinical value in the reproductive guidance of female during puberty and growing period.At present,the enzyme-linked immunoassay is commonly used in the detection of AMH,but there are some disadvantages,such as low sensitivity and narrow detection range,which affect the actual level of AMH,which makes the prediction of the condition inaccurate to the treatment of the disease.Therefore,it is very important to establish a low cost and high sensitivity method to detect AMH level.Method:Using the Biotin-affinity amplification System,agroup of anti-AMH first antibody packages were coated with microporous plates,a method of AMH enzyme-induced chemiluminescence immunoassay was established by combining the biotin-streptavidin-horseradish peroxidase binding with biotin-labeled second antibody and the first antibody-antigen complex,and adding the luminescent substrate,the parameters such as linearity,sensitivity,specificity,stability and recovery rate of the system were detected,and compared with the conventional Elisa method to detect AMH concentration.Result:The linear detection range is(0.42-50.00)U/ml,the system has good stability,sensitivity 0.42 U/ml,the difference between batches is less than 10%,the difference between batches is less than 10%,and the difference is statistically significant compared with the conventional ELISA.Conclusion:AMH biotin-streptavidin-horseradish peroxidase enzymatic chemiluminescence immunoassay is easy to operate,high sensitivity and low price,and is suitable for clinical detection.
引文
[1] Chinya A,Ratan SK,Aggarwal SK,et al.Association of Levels of Serum Inhibin B and Follicle-stimulating Hormone with Testicular Vascularity,Volume,and Echotexture in Children with Undescended Testes[J].J Indian Assoc Pediatr Surg,2017,22:3-8.
[2]郭丽,银铎.子宫切除术中预防性切除输卵管对卵巢储备功能的影响[J].中华实用诊断与治疗杂志,2016,30(2):127-129.
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[4] Shen Z,Bao J,Zeng Y,et al.Influence of Cyclophosphamidum Used in Different Time during Menstrual Cycle on Ovary Anti-mullerian Hormone[J].China Pharmacist,2018,1:.
[5] Anna J,Malgorzata S,Anna G,et al.The Relationship between AMH and AMHR2Polymorphisms and the Follicular Phase in Late Reproductive Stage Women[J].Int J Environ Res Public Health,2016,13:185-185.
[6] Mumford SL,Legro RS,Diamond MP,et al.Baseline AMH Level Associated With Ovulation Following Ovulation Induction in Women With Polycystic Ovary Syndrome[J].J Clin Endocrinol Metab,2016,101:3288-3296.
[7]陈华干,杨婷,伍萍芝,等.抗苗勒管激素在高龄妇女卵巢功能评估中的价值探讨[J].中国妇幼保健,2016:31(19),3895-3898.
[8]路锦,李杭生,韦多,等.抗苗勒管激素及体质量指数与多囊卵巢综合征患者控制性促排卵结局的相关性分析[J].中华实用诊断与治疗杂志,2018,32(6):529-532.
[9] FeiDing XU.The Value of Age,Basal Follicle Stimulating Hormone and the Number of Sinus Follicle in Predicting Ovarian Response[J].Medical Innovation of China,2016.
[10]Wang H,Chen L,Jiang Y,et al.Association of gene polymorphisms of estrogen receptor,follicle-stimulating hormoneβand leptin with follicular cysts in Large White sows[J].Theriogenology,2017,103:143-148.
[11]Zhang H,Luo Q,Lu X,et al.Effects of hPMSCs on granulosa cell apoptosis and AMH expression and their role in the restoration of ovary function in premature ovarian failure mice[J].Stem Cell Res Ther,2018,9:20.
[12] Kereilwe O,Pandey K,Borromeo V,et al.AntiMüllerian hormone receptor type 2is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion[J].Reprod Fertil Dev,2018,14.
[13]Pankhurst MW,Leathart BL,Batchelor NJ,et al.The Anti-Müllerian Hormone Precursor(proAMH)Is Not Converted to the Receptor-Competent Form(AMHN,C)in the Circulating Blood of Mice[J].Endocrinology,2016,157:1622.
[14]Demirdjian G,Bord S,Lejeune C,et al.Performance characteristics of the Access AMH assay for the quantitative determination of anti-Müllerian hormone(AMH)levels on the Access family of automated immunoassay systems[J].Clinical Biochemistry,2016,49:1267-1273.
[15]Pearson K,Long M,Prasad J,et al.Assessment of the Access AMH assay as an automated,high-performance replacement for the AMH Generation II manual ELISA[J].Reproductive Biology&Endocrinology,2016,14:1-9.
[16]Zhu W,Zeng J,Tong LI,et al.Evaluation of the application of Chemiluminescence immunoassay(CLIA)in blood screening[J].Chinese Journal of Blood Transfusion,2015,29:493-497.
[17]Mei S,Wang J,Zhu L,et al.A UPLC-MS/MS method for analysis of vancomycin in human cerebrospinal fluid and comparison with the chemiluminescence immunoassay[J].Biomed Chromatogr,2017,31.
[18]Salehi N,Peng CA.Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography[J].Biotechnology Progress,2016,32:949-958.
[19]Wetzel D,Müller JM,Flaschel E,et al.Fed-batch production and secretion of streptavidin by Hansenula polymorpha:Evaluation of genetic factors and bioprocess development[J].Journal of Biotechnology,2016,225:3-9.
[20]Yoon JG,Hwang HJ,Jin A C.Application of the biotin-labeled toxin mutant for affinity isolation of associated proteins in the mammalian cells[J].Journal of Bioscience&Bioengineering,2018,125:497-504.
[21]Freitag S,Le IT,Chilkoti A,et al.Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex[J].Journal of Molecular Biology,2016,279:211-221.
[22]Wang Q,Huang X,Fu X,et al.A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles[J].Spectrochim Acta A Mol Biomol Spectrosc,2016,162:75-80.
[2]郭丽,银铎.子宫切除术中预防性切除输卵管对卵巢储备功能的影响[J].中华实用诊断与治疗杂志,2016,30(2):127-129.
[3] Jamil Z,Fatima SS,Ahmed K,et al.Anti-Mullerian Hormone:Above and Beyond Conventional Ovarian Reserve Markers[J].Dis Markers,2016,5246217.
[4] Shen Z,Bao J,Zeng Y,et al.Influence of Cyclophosphamidum Used in Different Time during Menstrual Cycle on Ovary Anti-mullerian Hormone[J].China Pharmacist,2018,1:.
[5] Anna J,Malgorzata S,Anna G,et al.The Relationship between AMH and AMHR2Polymorphisms and the Follicular Phase in Late Reproductive Stage Women[J].Int J Environ Res Public Health,2016,13:185-185.
[6] Mumford SL,Legro RS,Diamond MP,et al.Baseline AMH Level Associated With Ovulation Following Ovulation Induction in Women With Polycystic Ovary Syndrome[J].J Clin Endocrinol Metab,2016,101:3288-3296.
[7]陈华干,杨婷,伍萍芝,等.抗苗勒管激素在高龄妇女卵巢功能评估中的价值探讨[J].中国妇幼保健,2016:31(19),3895-3898.
[8]路锦,李杭生,韦多,等.抗苗勒管激素及体质量指数与多囊卵巢综合征患者控制性促排卵结局的相关性分析[J].中华实用诊断与治疗杂志,2018,32(6):529-532.
[9] FeiDing XU.The Value of Age,Basal Follicle Stimulating Hormone and the Number of Sinus Follicle in Predicting Ovarian Response[J].Medical Innovation of China,2016.
[10]Wang H,Chen L,Jiang Y,et al.Association of gene polymorphisms of estrogen receptor,follicle-stimulating hormoneβand leptin with follicular cysts in Large White sows[J].Theriogenology,2017,103:143-148.
[11]Zhang H,Luo Q,Lu X,et al.Effects of hPMSCs on granulosa cell apoptosis and AMH expression and their role in the restoration of ovary function in premature ovarian failure mice[J].Stem Cell Res Ther,2018,9:20.
[12] Kereilwe O,Pandey K,Borromeo V,et al.AntiMüllerian hormone receptor type 2is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion[J].Reprod Fertil Dev,2018,14.
[13]Pankhurst MW,Leathart BL,Batchelor NJ,et al.The Anti-Müllerian Hormone Precursor(proAMH)Is Not Converted to the Receptor-Competent Form(AMHN,C)in the Circulating Blood of Mice[J].Endocrinology,2016,157:1622.
[14]Demirdjian G,Bord S,Lejeune C,et al.Performance characteristics of the Access AMH assay for the quantitative determination of anti-Müllerian hormone(AMH)levels on the Access family of automated immunoassay systems[J].Clinical Biochemistry,2016,49:1267-1273.
[15]Pearson K,Long M,Prasad J,et al.Assessment of the Access AMH assay as an automated,high-performance replacement for the AMH Generation II manual ELISA[J].Reproductive Biology&Endocrinology,2016,14:1-9.
[16]Zhu W,Zeng J,Tong LI,et al.Evaluation of the application of Chemiluminescence immunoassay(CLIA)in blood screening[J].Chinese Journal of Blood Transfusion,2015,29:493-497.
[17]Mei S,Wang J,Zhu L,et al.A UPLC-MS/MS method for analysis of vancomycin in human cerebrospinal fluid and comparison with the chemiluminescence immunoassay[J].Biomed Chromatogr,2017,31.
[18]Salehi N,Peng CA.Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography[J].Biotechnology Progress,2016,32:949-958.
[19]Wetzel D,Müller JM,Flaschel E,et al.Fed-batch production and secretion of streptavidin by Hansenula polymorpha:Evaluation of genetic factors and bioprocess development[J].Journal of Biotechnology,2016,225:3-9.
[20]Yoon JG,Hwang HJ,Jin A C.Application of the biotin-labeled toxin mutant for affinity isolation of associated proteins in the mammalian cells[J].Journal of Bioscience&Bioengineering,2018,125:497-504.
[21]Freitag S,Le IT,Chilkoti A,et al.Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex[J].Journal of Molecular Biology,2016,279:211-221.
[22]Wang Q,Huang X,Fu X,et al.A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles[J].Spectrochim Acta A Mol Biomol Spectrosc,2016,162:75-80.