奶牛孕酮ELISA检测试剂盒的研制及初步应用
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摘要
监测奶牛孕酮的含量变化是进行奶牛发情识别、妊娠诊断、人工授精效果监测、繁殖障碍诊断的技术手段。本研究旨在通过制备孕酮单克隆抗体,建立奶牛孕酮检测方法,研制奶牛孕酮的快速检测试剂盒,为科学开展奶牛繁殖状态的监控及提高奶牛繁殖效率提供简便快速的技术手段。
本研究采用实验室保存的杂交瘤细胞,经过扩大培养、接种小鼠、收集腹水、腹水的纯化等程序得到了孕酮单克隆抗体,并对孕酮单克隆抗体的效价进行了测定。通过对ELISA检测条件的优化,建立了孕酮间接竞争ELISA检测方法;根据ELISA的检测原理,成功的组装了试剂盒,完成了试剂盒各种试剂的稳定剂筛选,实验结果表明试剂盒可以在4℃保存一年;对组装的试剂盒进行了各种指标的测定,试剂盒的灵敏度为0.26ng/mL,试剂盒的批内差异为2.22%,试剂盒的批间误差为2.37%,试剂盒与雌二醇、雌三醇无交叉反应,与雌酮的交叉反应为1.2%,试剂盒的准确度控制在90%~105%之间;优化了试剂盒的标准曲线,筛选出了与样品测定时反应体系相同的空白奶样的制备方法,提高了实验结果的准确性。本实验建立的ELISA检测方法、试剂稳定性数据、试剂盒的相关参数等对孕酮ELISA检测试剂盒的商品化生产提供了重要的理论依据。
Progesterone is a kind of Steroid parahormone,we may monitoring dairy cow ovaries’s function,gravidity and breed condition by determining the content of Progesterone.This research on the basis of fundamental principle about ELISA,established the method of detecting the Progesterone,and explored a kind of kit that can detect the content of progesterone quickly,sensitively,accurately and is inexpensive.On the basis of that assemble and install to a kit,we grading-up the reaction of kit.
To utilize antigen and Progesterone McAb that we prepared, on the basis of fundamental principle about ELISA,we established indirect competition ELISA method for detecting Progesterone, moreover,we optimized reaction condition of ELISA detection method: the enzyme-label plate homogenicity intra-hole variation coefficient is 2.513%; The best coating concentration of detected antigen is 3.125μg/ml; The best antigen coating condition is 37℃2 hours; The best confining liquid is 2% goat blood serum;The best block time is 37℃2 hours; The best working concentration of Mab is 0.1μg/ml; Indirect competition ELISA standard curve is y = -1.8444x - 0.1401 R2= 0.9817;Enzyme labelled antibody the best reaction time is 40 minutes; The best reaction concentration of substrate is 0.1mg/mL; The best reaction concentration of Hydrogen dioxide is 0.1μL/mL; The best recaction time of substrate is 25 minutes.
According to reagent’s nature of indirect competition ELISA, we did the experiment about stability of all reagent: detection antigen, Progesterone McAb, enzyme labelled antibody, standard Progesterone solution,substrate, hydrogen dioxide, eluant and so on.all the reagent homoenergetic to achieve detecting requirement, stabilizing agent to the ELISA reagent may prolong their conservation time, According to the difference of nature of reagent,we may addition different concentration and variety classes of stabilizing agent and conservative.It play considerable role in detecting consequence of the kit.
According to ELISA method, we optimize the standard curve in experiment. In the course of preparing Standard Progesterone, we use polyglucosan G-25 coating activated carbon,then we admixed fresh milk and activated carbon,we can obtain des-hormone milk,The method we prepared standard solution may to approach the reaction condition maximum extent. It guarantees the identical ELISA reaction system,so that the detected consequence is rationality.
We assemble and install all reagent to kit: Enzyme labeled borad(one piece);Standard preparation(seven bottles);Progesterone Mab(one bottle);Enzyme labelled antibody(one bottle);Substrate solution(solution A one bottle, solution B one bottle);Stop buffer(one bottle);Dilution(one bottle);Eluant(one bottle).
We test the correlated parameter about kit. Response curve-dose Parameter’s statistical method that we uses mathematic model [log(dose)-logit(B/B0)];The response curve-dose fitting equations is y =- 2.0126 x + 1.6396 R2=0.9981;The sensitivity of kit is 0.26ng/mL;The intra-batch difference of the kit is 2.22%;The inter- batch difference of the kit is 2.37%;Estradiol and Estriol are no consensual reaction with kit;Estrone consensual reaction rate is 1.2% with kit;The degree of accuracy of kit between 90%~105%.
本研究采用实验室保存的杂交瘤细胞,经过扩大培养、接种小鼠、收集腹水、腹水的纯化等程序得到了孕酮单克隆抗体,并对孕酮单克隆抗体的效价进行了测定。通过对ELISA检测条件的优化,建立了孕酮间接竞争ELISA检测方法;根据ELISA的检测原理,成功的组装了试剂盒,完成了试剂盒各种试剂的稳定剂筛选,实验结果表明试剂盒可以在4℃保存一年;对组装的试剂盒进行了各种指标的测定,试剂盒的灵敏度为0.26ng/mL,试剂盒的批内差异为2.22%,试剂盒的批间误差为2.37%,试剂盒与雌二醇、雌三醇无交叉反应,与雌酮的交叉反应为1.2%,试剂盒的准确度控制在90%~105%之间;优化了试剂盒的标准曲线,筛选出了与样品测定时反应体系相同的空白奶样的制备方法,提高了实验结果的准确性。本实验建立的ELISA检测方法、试剂稳定性数据、试剂盒的相关参数等对孕酮ELISA检测试剂盒的商品化生产提供了重要的理论依据。
Progesterone is a kind of Steroid parahormone,we may monitoring dairy cow ovaries’s function,gravidity and breed condition by determining the content of Progesterone.This research on the basis of fundamental principle about ELISA,established the method of detecting the Progesterone,and explored a kind of kit that can detect the content of progesterone quickly,sensitively,accurately and is inexpensive.On the basis of that assemble and install to a kit,we grading-up the reaction of kit.
To utilize antigen and Progesterone McAb that we prepared, on the basis of fundamental principle about ELISA,we established indirect competition ELISA method for detecting Progesterone, moreover,we optimized reaction condition of ELISA detection method: the enzyme-label plate homogenicity intra-hole variation coefficient is 2.513%; The best coating concentration of detected antigen is 3.125μg/ml; The best antigen coating condition is 37℃2 hours; The best confining liquid is 2% goat blood serum;The best block time is 37℃2 hours; The best working concentration of Mab is 0.1μg/ml; Indirect competition ELISA standard curve is y = -1.8444x - 0.1401 R2= 0.9817;Enzyme labelled antibody the best reaction time is 40 minutes; The best reaction concentration of substrate is 0.1mg/mL; The best reaction concentration of Hydrogen dioxide is 0.1μL/mL; The best recaction time of substrate is 25 minutes.
According to reagent’s nature of indirect competition ELISA, we did the experiment about stability of all reagent: detection antigen, Progesterone McAb, enzyme labelled antibody, standard Progesterone solution,substrate, hydrogen dioxide, eluant and so on.all the reagent homoenergetic to achieve detecting requirement, stabilizing agent to the ELISA reagent may prolong their conservation time, According to the difference of nature of reagent,we may addition different concentration and variety classes of stabilizing agent and conservative.It play considerable role in detecting consequence of the kit.
According to ELISA method, we optimize the standard curve in experiment. In the course of preparing Standard Progesterone, we use polyglucosan G-25 coating activated carbon,then we admixed fresh milk and activated carbon,we can obtain des-hormone milk,The method we prepared standard solution may to approach the reaction condition maximum extent. It guarantees the identical ELISA reaction system,so that the detected consequence is rationality.
We assemble and install all reagent to kit: Enzyme labeled borad(one piece);Standard preparation(seven bottles);Progesterone Mab(one bottle);Enzyme labelled antibody(one bottle);Substrate solution(solution A one bottle, solution B one bottle);Stop buffer(one bottle);Dilution(one bottle);Eluant(one bottle).
We test the correlated parameter about kit. Response curve-dose Parameter’s statistical method that we uses mathematic model [log(dose)-logit(B/B0)];The response curve-dose fitting equations is y =- 2.0126 x + 1.6396 R2=0.9981;The sensitivity of kit is 0.26ng/mL;The intra-batch difference of the kit is 2.22%;The inter- batch difference of the kit is 2.37%;Estradiol and Estriol are no consensual reaction with kit;Estrone consensual reaction rate is 1.2% with kit;The degree of accuracy of kit between 90%~105%.
引文
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[2] 王建辰.动物生殖调控[M].安徽科学技术出版社,1998.
[3] Ruiz,F.J.,Oltenacu,P A.and Smith,R D.Evaluation of on-farm milk progesterone tests to determine non-pregnant cows and to prevent inseminationen-ors.J.Dairy Sci.1989,72:2718~2727.
[4] B.M.A.Oswin Perera,Reproduction in water buffalo:comparative aspectsand implications for management.J.Reproduction and Fertility Supplement,1999,54:157~168.
[5] Boni,Roviello,S.and Zicarelli,L.Repeated ovum pick-up in Italian Mediterranean buffalo cows,Theriogenology.1996,46:899~909.
[6] Lundstrom,K.,Abeygunawardena,H.,de Silva LNA and Perera, B.M.A.O. Environmental in influence on calving interval and estimates of its repeatability in the Mumah buffalo in Sri Lanka.Aminal Reproduction Science.1982,5:99~109.
[7] Laing,J.A.,and Heap,R.B.The concentration of progesterone in the mi1k of cows during the reproductive cycle. [J].Br Vet J.,1971,127:616~620.
[8] 蔡志强,徐宁迎,徐步进[J]. 中国畜牧杂志,2001,7(5).
[9] 刘智喜,武浩,王建辰等.固相 RIA 法测定血浆孕酮检测黄牛产后卵巢机能[J].中国兽医学报,1997, 3 (17).
[10] 吴美文,徐步进等.放射免疫分析法比较奶牛粪便和乳汁中的孕酮含量[J].浙江农业大学学报,1999,25(2):195~197.
[11] 蔡正华,卢雁平,罗应荣等.用奶孕酮放免测定技术监测奶牛人工授精效果的研究[J].中国农业科学,1997,30(5):88~90.
[12] Saucer,M.J.,Foulkes,J.A.and Cookson,A.D.Direct enzymeimmunoassay for progesterone in bovine milk,[J].Steroids,1981,38:45~53.
[13] Chang,C.F.,and Estergreen,V L.,Development of a direct enzyme immunoassay ofmilk progesterone and its application to pregnancy diagnosis in cows.[J].Steroids,1981,41:173~195.
[14] Sauer,M.J.,Foulkes,J.A.and ONeill,P M.Use of microtitre plate EIA for direct determination of progesterone in whole mills:application of heterologous systems for improved sensitivity[J].Br.Yet.J.1982,138:522~532.
[15] 朱立平,陈学清.免疫学常用实验方法[M].人民军医出版社,第二十章,342~349.
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