CN103558372B - The method of preparing an antigen standards and the sample dilutions used in a elisa - Google Patents

The method of preparing an antigen standards and the sample dilutions used in a elisa Download PDF

Info

Publication number
CN103558372B
CN103558372B CN201310555174.XA CN201310555174A CN103558372B CN 103558372 B CN103558372 B CN 103558372B CN 201310555174 A CN201310555174 A CN 201310555174A CN 103558372 B CN103558372 B CN 103558372B
Authority
CN
China
Prior art keywords
added
further
solution
plate
step
Prior art date
Application number
CN201310555174.XA
Other languages
Chinese (zh)
Other versions
CN103558372A (en
Inventor
章登吉
程禹
段新伟
张波
Original Assignee
博弈诺生物科技(无锡)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 博弈诺生物科技(无锡)有限公司 filed Critical 博弈诺生物科技(无锡)有限公司
Priority to CN201310555174.XA priority Critical patent/CN103558372B/en
Publication of CN103558372A publication Critical patent/CN103558372A/en
Application granted granted Critical
Publication of CN103558372B publication Critical patent/CN103558372B/en

Links

Abstract

本发明涉及免疫检测技术领域,提供了一种ELISA用的抗原标准品和样本稀释液的制备方法,包括制备抗体标被;对BDNF板和Prolaction板分别进行浇注、封闭、洗涤、晾干后,再保存起来待用;配制标准稀释液,作为空白液;配制样本稀释液;将BDNF板和Prolaction板放入孵育振荡器中孵育抗体;加入检测抗体后加入试剂盒SA‑HRP中;放在酶标仪下读取光密度OD值;绘制BDNF和prolactin的回收率标准曲线并测算稀释样品的回收率。 The present invention relates to the field of immunoassay techniques, provides an antigen standards and the sample dilutions were prepared with an ELISA comprising antibodies prepared by standard; BDNF plate to plate and pouring Prolaction respectively, closed, washed, dried, then saved stand; dilutions standard formulation, as the blank solution; prepared sample diluent; and BDNF into the plate and the plate was incubated shaker Prolaction antibody incubation; detection antibody was added after addition of SA-HRP in the kit; on enzyme reading the optical density OD value meter index; drawn curve BDNF and recovery standard prolactin and measure the recovery of the diluted sample. 本发明有效解决了背景值偏高,信噪比低,灵敏度低,并且具有降低基质效应所产生的干扰,使检测结果更加稳定可靠的特点。 BACKGROUND The present invention effectively solves the high value, low SNR, low sensitivity, and has reduced interference matrix effects generated, the detection results are more stable and reliable characteristics.

Description

一种EL ISA用的抗原标准品和样本稀释液的制备方法 Antigen dilutions standard and sample preparation method with EL ISA

技术领域 FIELD

[0001]本发明涉及免疫检测技术领域,特别涉及一种ELISA用的抗原标准品和样本稀释液的制备方法。 [0001] The present invention relates to immunoassay techniques, and more particularly relates to a method for preparing an antigen standards and the sample dilutions using an ELISA.

背景技术 Background technique

[0002] ELISA是酶联免疫吸附剂测定(Enzyme-Linked Immunosorbnent Assay)的简称, 是继免疫荧光和放射免疫技术之后发展起来的一种免疫酶技术。 [0002] ELISA enzyme linked immunosorbent assay (Enzyme-Linked Immunosorbnent Assay) short, following an immune immunofluorescence and radioimmunoassay after enzyme technology developed. 由于ELISA的实验方法具有敏感、特异、经济、简便、安全等特点,因此是目前免疫学实验室中最常用的一种检测方法。 Since the ELISA test method has a sensitive, specific, economical, simple, and security features, so is the immunology laboratory of the most commonly used detection method.

[0003] ELISA操作过程中选用优质的试剂、良好的仪器以及正确的操作是保证ELISA检测结果准确可靠的必要条件,ELISA的操作因固相载体的形成不同而有所差异,国内医学检验一般均采用板式点。 [0003] ELISA process operation of high quality reagents, good equipment and proper operation is essential to ensure accurate and reliable ELISA test results, operating ELISA varies form a solid phase support vary, generally domestic medical tests using plate points.

[0004] 无论ELISA技术如何发展,更高的灵敏度和准确性,更高的数据可重复性始终是广大科研工作者追求的目标。 [0004] No matter how ELISA technology development, higher sensitivity and accuracy, higher repeatability data is always scientific researchers to pursue. 建立一个好的ELISA方法的基础是首先要找到好的抗原与抗体, 然而目前该技术仍然面临许多问题和挑战:(1)灵敏度有待提高,ELISA方法在医院,药企和科研院所被应用于与疾病相关的各种因子的检测,这些因子往往都是一些内源性的细胞因子、趋化分子、粘附分子等等,然而有些内源性分子表达风度低,难于被检测,高灵敏度的试剂盒有利于解决这个问题。 Establish a good foundation ELISA method is to first find a good antigen and antibody, but now the technology still faces many problems and challenges: (1) the sensitivity needs to be improved, ELISA method is applied in hospitals, pharmaceutical companies and research institutes detection of various factors associated with the disease, these factors are often some of the endogenous cytokine, chemokine, adhesion molecules, etc., but some low-style endogenous molecule expression, difficult to be detected with high sensitivity the kit help solve this problem. (2)基质效应问题,ELISA所检测的样本的基质种类多种多样,不同基质成分、物理性质不同,会对样本检测产生干扰,造成结果的不准确或不稳定;并且在内源性因子的定量检测中,配制标准曲线的缓冲液和待测样本真实基质间的客观差异也会影响到最后的检测结果。 (2) a matrix effect problem, the matrix type detected by ELISA samples varied, different matrix component, different physical properties, sample testing will interfere, resulting in inaccurate or inconsistent results; and endogenous Factor quantitative detection, the objective difference between the real buffer and a test sample matrix formulation of the standard curve will affect the final detection result. (3)非特异性吸附,非特异性吸附的原因复杂,其中一个结果就是导致空白的背景值偏高,信噪比偏低,从而影响了整个试剂盒的定量灵敏度。 (3) non-specific adsorption due to nonspecific adsorption complex, wherein the result is a white background results in high value, low signal to noise ratio, thus affecting the sensitivity of the overall quantitation kit.

[0005] 酶联免疫反应是一种敏感性很高的反应,如果血清、血浆或其它基质样本不稀释, 不可避免会产生很强的非特异性反应,出现假阳性,因此,由于待测样本机制的复杂性也可能造成基质效应,干扰检测结果。 [0005] The enzyme immunoassay is a highly sensitive reaction, if the serum, plasma samples or other substrate without dilution, will inevitably produce a very strong non-specific reaction, false positive, and therefore, since the sample to be tested Mechanism the complexity of the matrix effects may also cause interference detection result.

[0006] 因此,免疫检测技术领域急需一种降低背景值偏高,提高信噪比,增加灵敏度,消除基质效应所产生的干扰,使检测结果更加稳定可靠的ELISA用的抗原标准品和样本稀释液的制备方法。 [0006] Accordingly, an urgent need FIELD standard immunoassay techniques for reducing background and diluted value is high, to improve signal to noise ratio, increase the sensitivity, to eliminate the interference matrix effects generated, the detection results are more reliable sample for ELISA antigen liquid preparation.

发明内容 SUMMARY

[0007] 本发明提供了一种ELISA用的抗原标准品和样本稀释液的制备方法,技术方案如下: [0007] The present invention provides a method for preparing an antigen standards and the sample dilutions used in an ELISA, the following technical solutions:

[0008] -种ELISA用的抗原标准品和样本稀释液的制备方法,其特征在于,包含如下步骤: [0008] - antigen standards and the sample dilutions were prepared with the types of ELISA, wherein, comprising the steps of:

[0009] 第一步,制备抗体包被; [0009] The first step, preparation of antibody-coated;

[0010]将捕获的脑源性神经营养因子BDNF抗体加入碳酸盐缓冲液中,并且将其加入ELI SA板的孔中,标记为BNDF板,置于2-8 °C的温度下包被16-24小时; [0010] brain-derived neurotrophic factor BDNF antibody captured in the carbonate buffer was added, which was added to the wells and the ELI SA plate, labeled BNDF plate, is placed at a temperature of 2-8 ° C packets are 16-24 hours;

[0011]进一步地,将催乳素Prolactin抗体加入另一组碳酸盐缓冲液中,并且将其加入ELISA板的孔中,标记为Prolaction板,置于2-8°C的温度下包被16-24个小时; [0011] Further, the prolactin Prolactin antibody was added to another set of carbonate buffer, and added ELISA plate wells, labeled Prolaction plate, is placed at a temperature of 2-8 ° C is 16 packets -24 hours;

[0012] 第二步,对第一步中BDNF板和Pro lact i on板分别进行浇注、封闭、洗涤、晾干后,再保存起来待用; [0012] The second step of the first step and BDNF Pro lact i on the plate were cast plate, blocking, washed, dried, and then save them stand;

[0013]将PH值为7.4 ± 0.2的含有5 %牛血清白蛋白BSA的磷酸盐缓冲液roS向第一步中的BNDF板和Prolaction板分别浇注;或者将PH值为7.4 ±0.2的含有5-10 %脱脂奶粉向第一步中的BNDF板和Prolaction板分别饶注; [0013] The PH value of 7.4 ± 0.2 roS phosphate buffer containing 5% bovine serum albumin BSA respectively casting plate and a first step BNDF Prolaction plate; or PH value of 7.4 ± 0.2 containing 5 Rao, respectively -10% skim milk powder injection into the plate in the first step and BNDF Prolaction plate;

[0014] 进一步地,将该浇注好的BDNF板和Prolaction板分别放在2-8°C的温度下封闭放置16-24小时; [0014] Further, the plate and pouring good BDNF Prolaction plates were placed at a temperature of 2-8 ° C for 16-24 hours in a closed place;

[0015] 进一步地,用含有非离子型表面活性剂TWeen20的roS溶液对封闭好的BDNF板和Prolaction板进行洗涤、晾干后,置于2-8°C的温度下保存待用; [0015] Further, BDNF was washed well plate and the closing plate roS Prolaction solution containing a nonionic surfactant TWeen20 after drying, storage stand is placed at a temperature of 2-8 ° C;

[0016] 第三步,配制标准稀释液,作为空白液; [0016] The third step is to prepare a standard dilution of a blank solution;

[0017] 首先将0.05%聚氧乙烯山梨醇单月桂酸单脂Tween 20加入含有5%BSA的ros中, 并且搅拌均匀; [0017] First, 0.05% polyoxyethylene sorbitan monolaurate Tween 20 aliphatic mono ros containing 5% BSA was added in and stirred uniformly;

[0018] 进一步地,向该溶液中加入百万分之十五的液体防腐剂Proclin,搅拌均匀后作为标准稀释液,即空白液; [0018] Further, this solution was added a liquid preservative 15 ppm Proclin, stir dilution as a standard, i.e. the blank solution;

[0019] 第四步,配制样本稀释液; [0019] The fourth step to prepare a sample diluent;

[0020] 样本稀释液1:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2%表面活性剂3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐CHAPS 搅拌均勾;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0020] Sample Diluent 1: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% surfactant 3- [3- (prop-amido bile yl) dimethylammonio] propanesulfonate CHAPS were stirred hook; further, this solution was added 15 ppm Proclin were stirred hook;

[0021] 样本稀释液2:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步的,向该溶液中加入5mmo 1 /1的螯合剂乙二胺四乙酸EDTA搅拌均勾;进一步地,向该溶液中加入百万分之十五的Procl in搅拌均匀; [0021] Sample Diluent 2: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS Stir; further, this solution was added 5mmo chelating 1/1 EDTA ethylenediaminetetraacetic acid were stirred hook; further, this solution was added 15 ppm in Procl stir;

[0022] 样本稀释液3:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 05 %牛血清γ -球蛋白BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0022] Sample dilution 3: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added 05% bovine serum square γ - globulin BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0023] 样本稀释液4:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 10 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0023] 4 Sample Diluent: firstly addition of 0.05% Tween 20 in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 10% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0024] 样本稀释液5:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 15 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0024] sample 5 Diluent: firstly addition of 0.05% Tween 20 in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 15% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0025] 样本稀释液6:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入ο. 20 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0025] Sample Diluent 6: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added ο 20% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0026] 样本稀释液7:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 25 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0026] 7 Sample Diluent: firstly addition of 0.05% Tween 20 in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 25% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0027] 第五步,将BDNF板和Prolaction板放入孵育振荡器中孵育抗体; [0027] The fifth step, the BDNF plate into plate and incubated shaker Prolaction antibody incubation;

[0028]分别将第三步中的标准稀释液、第四步中的样本稀释液1-7、一份正常人血清样本,重复的分别加入第二步中晾干后待用的BDNF板和Prolaction板的孔中,再向各个反应孔中加入处理酶; [0028] The samples were dilutions of the standard dilution in the third step, the fourth step 1-7, a normal human serum sample, repeating the second step was added to dry after the stand plate and BDNF, respectively Prolaction aperture plate, each reaction well was added again processing enzyme;

[0029] 进一步地,将加好溶液的BDNF板和Prolaction板放入孵育振荡器中,在18-25°C的室温条件下反应; [0029] Further, the plate BDNF plus a good solution and incubated into Prolaction plate shaker at room temperature conditions of 18-25 ° C;

[0030] 第六步,加入检测抗体; [0030] The sixth step, detection antibody;

[0031] 用含有Tween20的PBS作为洗液,对第五步中反应后的BDNF板和Prolaction板分别洗涤,再相应的向BDNF板的每个反应孔中加入BDNF检测抗体,并且相应的向Prolaction板的每个反应孔中加入Prolaction检测抗体; [0031] with PBS containing Tween20 as a lotion, after washing the fifth step of the reaction plate and the Prolaction plates are respectively BDNF, BDNF and then the detection antibody was added to each respective reaction well plate BDNF, and the corresponding Prolaction each reaction well of the plate was added Prolaction detection antibody;

[0032]第七步,将稀释液加入试剂盒中; [0032] The seventh step, the dilution was added to the kit;

[0033] 用含有Tween20的PBS作为洗液,对第六步中反应后的BDNF板和Prolaction板分别洗涤后对其进行稀释,再分别向试剂盒SA-HRP中的每个孔中加入稀释后的溶液; [0033] washed with PBS containing Tween20 as a lotion, after washing the sixth step of the reaction plate and BDNF Prolaction plates are subjected to dilution, were then added to each well and diluted with kits of the SA-HRP The solution;

[0034]第八步,放在酶标仪下读取光密度0D值; [0034] The eighth step, the optical density read on a microplate reader at 0D value;

[0035]用含有TWeen20的PBS作为洗液,对第七步中反应后的试剂盒SA-HRP分别洗涤,再向该洗涤好的试剂盒SA-HRP中加入四甲基联苯胺TMB底物; [0035] As with PBS containing TWeen20 washings after washing of the reaction kit seventh step SA-HRP, respectively, which washed again good kit SA-HRP was added tetramethyl benzidine TMB substrate;

[0036] 进一步地,将加好TMB的试剂盒SA-HRP放在酶标仪下读取0D值; [0036] Further, the good plus TMB kit SA-HRP 0D value read on microplate reader;

[0037] 第九步,根据0D值计算出非特异性吸附数据,通过比较非特异性吸附数据,进一步确定出牛血清γ球蛋白BGG作为抗原对降低背景噪音具有直接并且积极的作用。 [0037] The ninth step, the calculated value 0D nonspecific adsorption data, by comparing the non-specific adsorption data, is further determined as bovine serum γ-globulin BGG antigen has a direct and positive effect on reducing background noise.

[0038]首先,第三步中的标准稀释液、第四步中的样本稀释液1-7和一份正常人血清样本,在BDNF板上所测得的非特异性吸附NSB的信号结果; [0038] First, in the third step dilutions standard, sample diluent fourth step 1-7 and a normal serum samples, the resulting signal measured in BDNF nonspecific adsorption of NSB board;

[0039]进一步地,第三步中的标准稀释液、第四步中的样本稀释液1-7和一份正常人血清样本,在Prolactin板上所测得的非特异性吸附NSB的信号结果; [0039] Further, in the third step dilutions standard, sample diluent fourth step 1-7 and a normal human serum samples, the resulting signal measured in Prolactin board non-specific adsorption of NSB;

[0040] 进一步地,发现在稀释液中加入牛血清γ球蛋白BGG的样本稀释液3-7,在两种包被不同抗体的EL ISA板,即BNDF板、Pro 1 ac ti η板中的非特异性吸附NSB都很小,从而说明所产生的背景值都非常低; [0040] Further, it was found, the sample diluent BGG bovine serum γ-globulin solution is diluted 3-7, in both plates coated EL ISA different antibodies, i.e. BNDF plate, Pro 1 ac ti η plate NSB non-specific adsorption is very small, so that the generated description background values ​​were very low;

[0041] 进一步地,由于样本稀释液3-7中的BGG浓度不同,但是都降低了ELISA反应中的背景ί目号; [0041] Further, due to the different BGG concentration in sample diluent 3-7, but were reduced background ELISA reactivity ί number of mesh;

[0042]进一步地,说明任意浓度的牛血清γ球蛋白BGG抗原对降低背景噪音具有直接且积极的作用。 [0042] Further, any explanation of bovine serum γ-globulin concentration of BGG antigen has a direct and positive effect on reducing background noise.

[0043]如上所述的一种ELISA用的抗原标准品和样本稀释液的制备方法,还包含:第十步,将第四步中的样本稀释液6作为新的标准稀释液,仅将CHAPS的含量分别调整为0.1 %、 0.15%、0.25%、0.3%、0.45%、0.5%、0.6%,从而形成7种新的样本稀释液8-14,其它步骤完全按照第一步至八步进行处理,读取0D值,进一步计算出非特异性吸附系数;并对测得的非特异性吸附系数进行分析,得出在牛血清γ球蛋白BGG含量一定的情况下,CHAPS浓度位于0.2%-0.45 %之间时,BDNF板和Prolactin板的非特异性吸附NSB都会降低,从而使背景信号降低。 [0043] The standard antigen preparation and the sample dilutions using an ELISA as described above, further comprising: a tenth step, the sample diluent in the fourth step 6 as a new standard dilutions, only the CHAPS the contents were adjusted to 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, to form seven new sample diluent 8-14, further steps follow step full eight steps process, reads the value 0D, further calculates the nonspecific adsorption coefficient; and analyzes non-specific adsorption coefficient measured, the results of bovine serum γ-globulin content BGG certain circumstances, CHAPS concentration from 0.2% -0.45% It is between, BDNF plate and the plate Prolactin NSB will reduce nonspecific adsorption, thereby reducing background signals.

[0044] 如上所述的一种ELISA用的抗原标准品和样本稀释液的制备方法,还包含:第十一步,从第四步和第十步中选取出三种信噪比高,非特异性信号低的三种样本稀释液来绘制BDNF和prolactin的回收率标准曲线并测算稀释样品的回收率;根据测试结果证明了向含有5%BSA的PBS中分别加入0.05%Tween 20、0.2-0.45%〇^卩5、5111111〇1/^细0了厶、任意浓度的牛血清γ球蛋白BGG、百万分之十五的Proclin作为稀释液,是一种效果非常好的适用于ELISA的样本稀释液,具体步骤为: [0044] The standard antigen preparation and the sample dilutions using an ELISA as described above, further comprising: an eleventh step, extraction or high signal to noise ratio from the fourth and the tenth step selected, nonspecific heterosexual low signal to draw three kinds of sample diluent recovery BDNF and prolactin standard curve and measures the recovery of the diluted sample; the test results demonstrate the PBS containing 5% BSA was added, respectively, 0.05% Tween 20,0.2-0.45 % ^ square Jie 5,5111111〇1 / bovine serum γ ^ 0 the Si thin, globulin BGG any concentration, 15 ppm Proclin as a diluent, the effect is a very good sample suitable for ELISA diluent, the specific steps:

[0045] 首先,分别将BDNF和Prolactin标准抗原加入BDNF和prolactin阴性血清中; [0045] First, respectively BDNF and BDNF added Prolactin standard antigen negative sera and prolactin;

[0046] 进一步地,向该阴性血清样本中分别加入样本稀释液3、样本稀释液10、样本稀释液12中其他步骤与第五步至第八步完全一致,读出光密度0D值和回收率; [0046] Further, to the negative serum samples were added to the sample dilution 3, 10 sample dilution, sample dilutions additional step 12 fifth step to fully consistent with the eighth step, the readout value and the optical density 0D recovery rate;

[0047] 进一步地,两种样本稀释液3和样本稀释液12分别是CHAPS为0.2%和0.45%的2个临界点,经实验表明三种样本稀释液3、10、12配制的BDNF和Prolactin的标准曲线分别互相平行,相关性很好,并且与阴性血清相比回收率提高; [0047] Further, two kinds of sample dilutions and sample dilutions 3 are 12 and 0.2% CHAPS to 0.45% of two critical point, the experiment showed that BDNF and Prolactin three kinds of sample diluent prepared 3,10,12 the standard curves were parallel to each other, a good correlation, and the recovery rate as compared with negative serum;

[0048] 进一步地,证明向PH值为7 · 4 ± 0 · 2的含有5 % BSA的PBS中分别加入0 · 05 % Tween 20、0.2-0.45 % CHAPS、5mmo 1 /1的EDTA、任意浓度的牛血清γ球蛋白BGG、百万分之十五的Proclin作为稀释液,是一种效果非常好的适用于ELISA的样本稀释液。 [0048] Further, to prove to the PH value of PBS containing 5% BSA 7 · 4 ± 0 · 2 were added in 0 · 05% Tween 20,0.2-0.45% CHAPS, EDTA 5mmo 1/1, and any concentration bovine serum γ-globulin BGG, 15 ppm Proclin as a diluent, an effect that is very well suited for the ELISA sample dilution.

[0049] 本发明的有益效果是: [0049] Advantageous effects of the present invention are:

[0050] 1.本发明用牛血清γ球蛋白BGG作为抗原,从而起到降低背景噪音的作用。 [0050] 1. The present invention BGG immunoglobulin as an antigen, and thus play a role in reducing the background noise bovine serum γ.

[0051 ] 2.本发明确定了在牛血清γ球蛋白BGG含量一定的情况下,CHAPS浓度位于0.2 % -0.45%之间时,BDNF板和Prolactin板的非特异性吸附NSB都会降低,降低了背景信号。 When [0051] 2. The present invention is determined in bovine serum γ-globulin content BGG certain circumstances, CHAPS concentration of 0.2% -0.45% is located, nonspecific adsorption of BDNF NSB plate are reduced and Prolactin plate, reducing background signal. [0052] 3.本发明提供了一种非常好的适用于ELISA的抗原标准品和样本稀释液,弥补了行业的空白,促进科学进步。 [0052] 3. The present invention provides a very well suited for ELISA antigen standard and sample dilution, to make up for the gaps in the industry, promote scientific progress.

附图说明 BRIEF DESCRIPTION

[0053]下面结合附图和具体实施方式来详细说明本发明: [0053] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0054]图1是本发明BDNF在三种稀释液中的回收率标准曲线。 [0054] FIG. 1 is a standard curve of BDNF of the present invention is the recovery of the three dilutions.

[0055]图2是本发明Prolactin在三种稀释液中的回收率标准曲线。 [0055] FIG. 2 is a standard curve Prolactin present invention recovery of the three dilutions.

具体实施方式 Detailed ways

[0056] 为了使本发明的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体图示,进一步阐述本发明。 [0056] In order to make the technical means of the present invention, the creation of features, to achieve the purpose and effect readily apparent appreciated that specifically illustrated below with reference to further illustrate the invention.

[0057] 本发明提供了一种ELISA用的抗原标准品和样本稀释液的制备方法,具体步骤如下: [0057] The present invention provides a method for preparing an antigen standards and the sample dilutions used in an ELISA, the following steps:

[0058]第一步,制备抗体包被; [0058] The first step, preparation of antibody-coated;

[0059]将捕获的脑源性神经营养因子BDNF抗体加入PH值为9.6的0.05mol/l碳酸盐缓冲液中,配制成浓度为2μg/ml的溶液,并且将其加入ELI SA板的孔中,标记为BNDF板,置于2-8 °C的温度下包被16-24小时; [0059] brain-derived neurotrophic factor BDNF antibody captured 0.05mol / l carbonate buffer PH value of 9.6, was added to prepare a concentration of 2μg / ml solution, which was added to the wells and the plate ELI SA , the labeled BNDF plate coated is placed for 16-24 hours at a temperature of 2-8 ° C;

[0060] 进一步地,将催乳素Prolactin抗体加入另一组PH值为9.6的0.05mol/l碳酸盐缓冲液中,配制成浓度为2.5μg/ml的溶液,并且将其加入ELISA板的孔中,标记为Prolaction 板,置于2-8 °C的温度下包被16-24个小时; [0060] Further, the antibodies were added to another set of prolactin Prolactin PH 0.05mol / l carbonate buffer, pH value of 9.6, formulated at a concentration of 2.5μg / ml solution, which was added to the wells and the ELISA plate , the labeled Prolaction plate, placed in a temperature of 2-8 ° C for 16-24 hours is next packet;

[0061 ] 第二步,对第一步中BDNF板和Pro lact i on板分别进行浇注、封闭、洗涤、晾干后,再保存起来待用; [0061] The second step of the first step and BDNF Pro lact i on the plate were cast plate, blocking, washed, dried, and then save them stand;

[0062]将PH值为7.4 ± 0.2的含有5 %牛血清白蛋白BSA的磷酸盐缓冲液roS向第一步中的BNDF板和Prolaction板分别浇注,每个孔中浇注300μ1;或者将PH值为7.4±0.2的含有5-10%脱脂奶粉向第一步中的BNDF板和Prolaction板分别浇注,每个孔中浇注300μ1; Phosphate buffer roS [0062] to the PH value of 7.4 ± 0.2 containing 5% bovine serum albumin BSA, respectively, in the first step to the casting plate and BNDF Prolaction plate, each well pouring 300μ1; or PH value containing 5-10% of skim milk powder were poured in a first step to the plate and BNDF Prolaction plate of 7.4 ± 0.2, each well pouring 300μ1;

[0063] 进一步地,将该浇注好的BDNF板和Prolaction板分别放在2-8 °C的温度下封闭放置16-24小时; [0063] Further, the plate and pouring good BDNF Prolaction plates were placed at a temperature of 2-8 ° C for 16-24 hours in a closed place;

[0064] 进一步地,用含有0.05%非离子型表面活性剂Tween的PBS溶液对封闭好的BDNF板和Prolaction板进行洗涤、晾干后,置于2-8°C的温度下保存待用; [0064] Further, containing 0.05% of Tween in PBS nonionic good for closing plate and BDNF Prolaction plate washed, dried, placed in storage stand at a temperature of 2-8 ° C;

[0065]第三步,配制标准稀释液,作为空白液; [0065] The third step is to prepare a standard dilution of a blank solution;

[0066] 首先将0.05%聚氧乙烯山梨醇单月桂酸单脂Tween 20加入含有5%BSA的roS中, 并且搅拌均匀; [0066] First, 0.05% polyoxyethylene sorbitan monolaurate Tween 20 aliphatic mono roS containing 5% BSA was added in and stirred uniformly;

[0067]进一步地,向该溶液中加入百万分之十五的液体防腐剂Proclin,搅拌均匀后作为标准稀释液,即空白液; [0067] Further, this solution was added a liquid preservative 15 ppm Proclin, stir dilution as a standard, i.e. the blank solution;

[0068]第四步,配制样本稀释液; [0068] The fourth step to prepare a sample diluent;

[0069] 样本稀释液1:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2%表面活性剂3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐CHAPS 搅拌均勾;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0069] Sample Diluent 1: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% surfactant 3- [3- (prop-amido bile yl) dimethylammonio] propanesulfonate CHAPS were stirred hook; further, this solution was added 15 ppm Proclin were stirred hook;

[0070] 样本稀释液2:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步的,向该溶液中加入5mmo 1 /1的螯合剂乙二胺四乙酸EDTA搅拌均勾;进一步地,向该溶液中加入百万分之十五的Procl in搅拌均匀; [0070] Sample Diluent 2: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS Stir; further, this solution was added 5mmo chelating 1/1 EDTA ethylenediaminetetraacetic acid were stirred hook; further, this solution was added 15 ppm in Procl stir;

[0071] 样本稀释液3:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 05 %牛血清γ -球蛋白BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0071] Sample dilution 3: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added 05% bovine serum square γ - globulin BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0072] 样本稀释液4:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 10 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0072] 4 Sample Diluent: firstly addition of 0.05% Tween 20 in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 10% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0073] 样本稀释液5:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 15 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0073] sample 5 Diluent: firstly addition of 0.05% Tween 20 in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 15% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0074] 样本稀释液6:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 20 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0074] Sample Diluent 6: First, 0.05% Tween 20 was added in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 20% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0075] 样本稀释液7:首先将0.05 % Tween 20加入含有5 % BSA的PBS中,并且搅拌均匀;进一步地,向该溶液中加入0.2 % CHAPS搅拌均匀;进一步地,向该溶液中加入5mmol/l的EDTA 搅拌均匀;进一步地,向该溶液中加入〇. 25 % BGG;进一步地,向该溶液中加入百万分之十五的Proclin搅拌均勾; [0075] 7 Sample Diluent: firstly addition of 0.05% Tween 20 in PBS containing 5% BSA, and stir; Further, this solution was added 0.2% CHAPS stir; Further, this solution was added 5mmol / l of EDTA stir; further, this solution was added square 25% BGG;. further, this solution was added 15 ppm Proclin were stirred hook;

[0076] 第五步,将BDNF板和Prolaction板放入孵育振荡器中孵育抗体; [0076] The fifth step of BDNF into the plate and incubated shaker plate Prolaction antibody incubation;

[0077]分别将第三步中的标准稀释液、第四步中的样本稀释液1-7、一份正常人血清样本,以10个重复分别加入第二步中晾干后待用的BDNF板和Prolaction板的孔中,再向各个反应孔中加入1〇〇μ1处理酶, [0077] The samples were dilutions of the standard dilution in the third step, the fourth step 1-7, a normal human serum sample were added to 10 repeating the second step after the drying stand BDNF Prolaction aperture plate and the plate, each reaction well was added again 1〇〇μ1 enzyme treatment,

[0078] 进一步地,将加好溶液的BDNF板和Prolaction板放入孵育振荡器中,以500转/分的速度在18_25°C的室温条件下反应2小时。 [0078] Further, the BDNF plus a good solution and the plate incubated plate into Prolaction shaker at a speed of 500 rev / min at room temperature the reaction 18_25 ° C for 2 hours.

[0079] 第六步,加入检测抗体; [0079] The sixth step, detection antibody;

[0080] 用含有0.05%Tween20的PBS作为洗液,对第五步中反应后的BDNF板和Prolaction 板分别洗涤4次,再相应的向BDNF板的每个反应孔中加入100μ1的BDNF检测抗体,并且相应的向Prolaction板的每个反应孔中加入100μ1的Prolaction检测抗体; [0080] with PBS containing 0.05% Tween20 as a lotion, after the fifth step of the reaction plate and BDNF Prolaction plates were washed four times, then the corresponding BDNF detection antibody was added to each well 100μ1 BDNF plate , and the corresponding detection antibody to each well of the plate was added 100μ1 Prolaction of Prolaction;

[0081 ]第七步,将稀释液加入试剂盒中; [0081] The seventh step, the dilution was added to the kit;

[0082] 用含有0.05%Tween20的PBS作为洗液,对第六步中反应后的BDNF板和Prolaction 板分别洗涤4次,再以1:10000对其进行稀释后,分别向试剂盒SA-HRP中的每个孔中加入100 μ 1的稀释后的洛液; [0082] with PBS containing 0.05% Tween20 as a lotion, after the sixth step of the reaction plate and BDNF Prolaction plates were washed four times, and then to 1: 10,000 dilution thereof, respectively, to the SA-HRP kit in Los solution was added to each well diluted to 100 μ 1;

[0083] 第八步,放在酶标仪下读取光密度0D值; [0083] The eighth step, the optical density read on a microplate reader at 0D value;

[0084]用含有0.05%TWeen20的roS作为洗液,对第七步中反应后的试剂盒SA-HRP分别洗涤4次,再向该洗涤好的试剂盒SA-HRP中加入四甲基联苯胺TMB底物; [0084] with roS containing 0.05% TWeen20 as a lotion, a kit for the SA-HRP after the seventh step the reaction washed 4 times, respectively, which again the washed SA-HRP kit was added tetramethyl benzidine TMB substrate;

[0085]进一步地,将加好TMB的试剂盒SA-HRP放在波长为450nm的酶标仪下读取0D值; [0086]第九步,根据0D值计算出非特异性吸附数据,通过比较非特异性吸附数据,进一步确定出牛血清γ球蛋白BGG作为抗原对降低背景噪音具有直接并且积极的作用; [0085] Further, the good plus TMB kit SA-HRP on the read wavelength values ​​0D microplate reader at 450nm; [0086] The ninth step is calculated according to the nonspecific adsorption 0D value data, by comparing nonspecific adsorption data, is further determined as bovine serum γ-globulin BGG antigen has a direct and positive role in reducing background noise;

[0087]首先,第三步中的标准稀释液、第四步中的样本稀释液1-7、一份正常人血清样本, 在BDNF板上所测得的非特异性吸附NSB的信号结果,如表一: [0087] First, the standard dilution in the third step, the fourth step in sample diluent 1-7, a normal human serum samples, the resulting signal measured in BDNF nonspecific adsorption of NSB board, such as Table I:

[0088]表一 [0088] Table I

[0089] [0089]

Figure CN103558372BD00111

[0090] 进一步地,第三步中的标准稀释液、第四步中的样本稀释液1-7、一份正常人血清样本,在Prolactin板上所测得的非特异性吸附NSB的信号结果,如表二: [0090] Further, the standard dilution in the third step, the fourth step in sample diluent 1-7, a normal human serum samples, the resulting signal measured Prolactin in nonspecific adsorption of NSB board, as shown in table II:

[0091] 表二 [0091] Table II

[0092] [0092]

Figure CN103558372BD00112

[0094]根据上述表一和表二中的结果进行分析,发现在稀释液中加入牛血清γ球蛋白BGG的样本稀释液3-7,在两种包被不同抗体的ELISA板,即BNDF板、Prolactin板中的非特异性吸附NSB都很小,从而说明所产生的背景值都非常低,为0.02-0.05之间; [0094] The analysis in Table I and Table II the results, found that the addition of bovine serum in the dilution liquid sample diluent γ-globulin BGG 3-7, two kinds of plates coated with different antibodies in ELISA, i.e., BNDF plate , nonspecific adsorption NSB Prolactin plate are small, so that the generated instructions are very low background value, is between 0.02 to 0.05;

[0095] 进一步地,由于样本稀释液3-7中的BGG浓度不同,但是都降低了ELISA反应中的背景ί目号; [0095] Further, due to the different BGG concentration in sample diluent 3-7, but were reduced background ELISA reactivity ί number of mesh;

[0096] 进一步地,说明牛血清γ球蛋白BGG抗原对降低背景噪音具有直接且积极的作用; [0096] Furthermore, bovine serum γ-globulin described BGG antigen has a direct and positive role in reducing background noise;

[0097] 第十步,将第四步中的样本稀释液6作为新的标准稀释液,仅将CHAPS的含量分别调整为0.1%、0.15%、0.25%、0.3%、0.45%、0.5%、0.6%,从而形成7种新的样本稀释液8-14。 [0097] The tenth step, the sample diluent in the fourth step 6 as a new standard dilutions, only the CHAPS contents were adjusted to 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, to form seven new sample dilutions 8-14. 其它步骤完全按照第一步至八步进行处理,读取0D值; Other steps in full accordance with the first step to eight steps for processing the read values ​​0D;

[0098]第十一步,根据0D值计算出非特异性吸附NSB的值,从而得到在牛血清γ球蛋白BGG含量一定的情况下,CHAPS浓度位于0.2%-0.45%之间时,BDNF板和Prolactin板的非特异性吸附NSB都会降低,从而使背景信号的降低; [0098] eleventh step, the calculated value 0D NSB nonspecific adsorption value, resulting in the content of bovine serum γ-globulin BGG certain circumstances, when positioned between the concentration of CHAPS 0.2% -0.45%, BDNF and plate Prolactin plate NSB will reduce nonspecific adsorption, thereby reducing the background signal;

[0099] 第十步中的样本稀释液6、样本稀释液8-14和一份正常人血清样本在BDNF板上所测得的非特异性吸附NSB的信号结果,如表三: [0099] Sample Diluent tenth step 6, the resulting signal sample NSB nonspecific adsorption dilutions 8-14 and a normal human serum sample plate BDNF measured, as shown in Table III:

[0100] 表三 [0100] Table III

Figure CN103558372BD00121

[0101] [0101]

[0102] [0102]

[0103]进一步地,第十步中的样本稀释液6、样本稀释液8-14、一份正常人血清样本,在Prolactin板上所测得的非特异性吸附NSB的信号结果,如表四: [0103] Further, the tenth sample diluent in step 6, 8-14 sample diluent, a normal human serum samples, the resulting signal measured Prolactin in nonspecific adsorption of NSB board, as shown in Table IV:

[0104] 表四 [0104] Table IV

[0105] [0105]

Figure CN103558372BD00131

[0106] EDTA,Tween20皆为本领域常用添加剂,但CHAPS并不是大家普遍使用的添加剂。 [0106] EDTA, Tween20 are all common additives in the art, but not CHAPS all commonly used additives. 因此,根据上述表三和表四中的结果进行分析,发现在牛血清γ球蛋白BGG含量一定的情况下,CHAPS浓度位于0.2%-0.45%之间时,80即板和?仰1&(^11板的非特异性吸附吧8都会降低,从而使背景信号的降低; Therefore, according to the analysis in Table III and Table IV the results found in the bovine serum γ-globulin content BGG certain circumstances, CHAPS concentration of 0.2% -0.45% is located between, i.e. plate 80 and? 1 & Yang (^ nonspecific adsorption plate 11 of the bar 8 will be reduced, thereby reducing the background signal;

[0107] 第十二步,从第四步和第十步中选取出三种信噪比高,非特异性信号低的三种样本稀释液来绘制BDNF和prolactin的回收率标准曲线,并测算稀释样品的回收率;图1是本发明BDNF在三种稀释液中的回收率标准曲线,图2是本发明Pro lact in在三种稀释液中的回收率标准曲线;根据测得的回收率证明了向含有5 % BSA的PBS中分别加入0.05 % Tween 20、 0.2-0.45%CHAPS、5mmol/l的EDTA、任意浓度的牛血清γ-球蛋白BGG、百万分之十五的Proclin作为稀释液,是一种效果非常好的适用于ELISA的样本稀释液。 [0107] The twelfth step, removed or high signal to noise ratio and low nonspecific signal Three dilutions of the sample from the fourth and the tenth step and prolactin BDNF selected to draw a standard curve of recovery, and calculation was diluted the recovery was; FIG. 1 is a standard curve of BDNF of the present invention is the recovery of the three dilutions, Pro lact FIG. 2 is a standard curve of the present invention in recovery of the three dilutions; demonstrated according to the measured recovery to the PBS containing 5% BSA was added, respectively, 0.05% Tween 20, 0.2-0.45% CHAPS, 5mmol / l of EDTA, any concentration of bovine serum γ- globulin BGG, 15 ppm Proclin as diluent , it is a very good effect ELISA suitable sample dilutions.

[0108] 首先,分别将4000pg/ml的BDNF和? [0108] First of all, respectively BDNF 4000pg / ml and? 1'〇1&(:1:;[11标准抗原加入130册和口1'013(31:;[11阴性血清中; 1'〇1 & (: 1:; [11 130 standard antigen was added and 1'013 port (31:; [11 negative sera;

[0109] 进一步地,分别以1:2,1:4,1:8对配制好的阴性血清进行稀释,稀释后分别加入样本稀释液3、样本稀释液10、样本稀释液12中,其他步骤与第五步至第八步完全一致,读出光密度0D值,进一步的计算出回收率,并且根据回收率绘制标准曲线,图1是本发明BDNF在三种稀释液中的回收率标准曲线,图2是本发明Prolactin在三种稀释液中的回收率标准曲线; [0109] Further, each of 1: 2,1: 4,1: eight pairs of the prepared negative sera were diluted, added to each diluted sample dilution 3, 10 sample dilution, sample diluent 12, the additional step of and a fifth step to an eighth step exactly the same, the optical density read 0D value, the recovery of further calculations, according to the standard curve and recovery of the present invention, FIG. 1 is a BDNF recovery of the three dilutions standard curve FIG 2 is a standard curve Prolactin present invention recovery of the three dilutions;

[0110]三种样本稀释液所配制BDNF标准曲线的光密度0D值与回收率,如表五: [0110] The optical density 0D value of three kinds of recovery BDNF standard curve sample dilutions formulated as shown in Table V:

[0111] 表五 [0111] Table V

[0112] [0112]

Figure CN103558372BD00141

[0113] 三种样本稀释液所配制Prolactin标准曲线的光密度0D值与回收率,如表六: [0113] Prolactin three kinds of sample dilutions standard curve formulated optical density 0D recovery value, as shown in Table VI:

[0114] 表六 [0114] Table VI

[0115] [0115]

Figure CN103558372BD00142

[0116] 进一步地,以阴性血清样本作为对照,比较样本稀释液的平均回收率; [0116] Further, as a control in the negative serum samples, comparison of the average recovery of sample diluent;

[0117] BDNF在三种样本稀释液中的平均回收率,如表七: [0117] The average recovery of the three BDNF in the sample diluent, as shown in Table VII:

[0118] 表七 [01] Table VII

[0119] [0119]

Figure CN103558372BD00151

[0120] prolactin在三种样本稀释液中的平均回收率,如表八: [0120] prolactin average recovery of the three sample dilutions, as shown in Table VIII:

[0121] 表八 [0121] Table VIII

[0122] [0122]

Figure CN103558372BD00152

[0123] 根据对表五、六、七、八的分析,两种样本稀释液3和样本稀释液12分别是CHAPS为0.2 %和0.45 %的2个临界点;经实验表明三种样本稀释液3、10、12配制的BDNF和Prolactin 的标准曲线分别互相平行,相关性很好;用这三种样本稀释液来分别稀释含有BDNF和Prolactin的血清样本,与阴性血清样本相比,其回收率从65-77%提高到85-115%,回收率有了大大的提高,结果准确度提高,从而满足检测的要求; [0123] The analysis of Table V, six, seven, eight, two kinds of sample dilutions and sample dilutions 3 are 12 and 0.2% CHAPS to 0.45% of two critical points; Experiments show that three kinds of Sample Diluent 3,10,12 formulated BDNF and Prolactin standard curves are parallel to each other, it correlates well; dilutions with three samples were diluted to contain BDNF and Prolactin serum samples compared to the negative serum samples, the recovery rate increased from 65-77% to 85-115% and the recovery has been greatly improved, to improve the accuracy of the result, to meet the requirements of detection;

[0124] 进一步地,证明向PH值为7 · 4 ± 0 · 2的含有5 % BSA的PBS中分别加入0 · 05 % Tween 20、0.2-0.45 % CHAPS、5mmo 1 /1的EDTA、任意浓度的牛血清γ球蛋白BGG、百万分之十五的Proclin作为稀释液,是一种效果非常好的适用于ELISA的样本稀释液。 [0124] Further, to prove to the PH value of PBS containing 5% BSA 7 · 4 ± 0 · 2 were added in 0 · 05% Tween 20,0.2-0.45% CHAPS, EDTA 5mmo 1/1, and any concentration bovine serum γ-globulin BGG, 15 ppm Proclin as a diluent, an effect that is very well suited for the ELISA sample dilution.

[0125] 本发明用牛血清γ球蛋白BGG作为抗原,从而起到降低背景噪音的作用。 [0125] The present invention is bovine γ globulin serum BGG as the antigen, and thus play a role to reduce background noise.

[0126] 本发明确定了在牛血清γ球蛋白BGG含量一定的情况下,CHAPS浓度位于0.2 % -0.45%之间时,BDNF板和Prolactin板的非特异性吸附NSB都会降低,降低了背景信号。 [0126] The present invention is determined in the case of bovine serum γ-globulin BGG certain content, when positioned between the concentration of CHAPS 0.2% -0.45%, BDNF plate and the plate Prolactin NSB will reduce nonspecific adsorption, reducing the background signal.

[0127] 本发明提供了一种非常好的适用于ELISA的抗原标准品和样本稀释液,弥补了行业的空白,促进科学进步。 [0127] The present invention provides a very well suited for ELISA antigen standard and sample dilution, to make up for the gaps in the industry, promote scientific progress.

[0128] 以上显示和描述了本发明的基本原理、主要特征和本发明的优点。 [0128] The above and described the principles of the invention, the main features and advantages of the present invention. 本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。 The industry the art will appreciate, the present invention is not limited to the above embodiment, the above-described examples and embodiments described in the specification are only illustrative of the principles of the present invention, without departing from the spirit and scope of the present invention, the present invention also have the respective variations and modifications, changes and modifications which fall within the scope of the claimed invention. 本发明要求保护范围由所附的权利要求书及其等同物界定。 The scope of the invention as claimed by the appended claims and equivalents thereof defined.

Claims (3)

1. 一种ELISA用的抗原标准品和样本稀释液的制备方法,其特征在于,包含如下步骤: 第一步,制备抗体包被; 将捕获的脑源性神经营养因子抗体加入碳酸盐缓冲液中,并且将其加入ELISA板的孔中,标记为BNDF板,置于2-8 °C的温度下包被16-24小时; 进一步地,将催乳素抗体加入另一组碳酸盐缓冲液中,并且将其加入ELISA板的孔中, 标记为催乳素板,置于2-8 °C的温度下包被16-24个小时; 第二步,对第一步中的脑源性神经营养因子板和催乳素板分别进行浇注、封闭、洗涤、 晚干后,再保存起来待用; 将PH值为7.4 ± 0.2的含有5 %牛血清白蛋白的磷酸盐缓冲液向第一步中的脑源性神经营养因子板和催乳素板分别浇注;或者将PH值为7.4±0.2的含有5-10%脱脂奶粉向第一步中的脑源性神经营养因子板和催乳素板分别浇注; 进一步地,将该浇注好的脑源性神经营养因子板 Antigen standards and the sample preparation dilution ELISA A used, characterized in that it comprises the following steps: first, preparing antibody-coated; brain-derived neurotrophic factor antibody captured in carbonate buffer was added solution, and added ELISA plate wells, labeled BNDF plate coated is placed for 16-24 hours at a temperature of 2-8 ° C; further, the buffer is added another antibody prolactin carbonate solution, and added ELISA plate wells, labeled prolactin plate, placed coated 16-24 hours at a temperature of 2-8 ° C; the second step, the first step of brain-derived neurotrophic factors and the plates were poured prolactin plate, blocking, washing, dry night, and then saved stand; PH value of the phosphate buffer containing 5% bovine serum albumin 7.4 ± 0.2 in the first step the brain-derived neurotrophic factor prolactin plate and plates are poured; or PH value containing 5-10% skim milk powder in the first step to the brain-derived neurotrophic factor prolactin plate and the plates are respectively 7.4 ± 0.2 casting; further, the pouring good brain-derived neurotrophic factor plate 和催乳素板分别放在2-8°C的温度下封闭放置16-24小时; 进一步地,用含有非离子型表面活性剂的PBS溶液对封闭好的脑源性神经营养因子板和催乳素板进行洗涤、晾干后,置于2-8°C的温度下保存待用; 第三步,配制标准稀释液,作为空白液; 首先将0.05 %聚氧乙烯山梨醇单月桂酸单脂加入含有5 %牛血清白蛋白的磷酸盐缓冲液中,并且搅拌均匀; 进一步地,向该溶液中加入百万分之十五的液体防腐剂,搅拌均匀后作为标准稀释液, 即空白液; 第四步,配制样本稀释液; 样本稀释液1:首先将0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白的磷酸盐缓冲液中,并且搅拌均匀;进一步地,向该溶液中加入0.2%表面活性剂3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐搅拌均匀;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 样本稀释液2:首先 Prolactin and plates are placed at a temperature of 2-8 ° C for 16-24 hours in a closed place; further, with a PBS solution containing a nonionic surfactant for blocking good brain-derived neurotrophic factor and prolactin plate plates were washed, dried, placed in storage stand at a temperature of 2-8 ° C; the third step, preparation of standard dilutions, as the blank solution; first 0.05% polyoxyethylene sorbitan monolaurate was added monoester phosphate buffered saline containing 5% bovine serum albumin, and stirred uniformly; further, the liquid preservative added to the solution of 15 ppm, stir as the standard dilutions, i.e., blank solution; the first four steps to prepare the sample diluent; sample diluent 1: first, 0.05% polyoxyethylene sorbitan monolaurate was added monoester phosphate buffer containing 5% bovine serum albumin, and stirred uniformly; further, to to this solution was added 0.2% surfactant 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate stir; further, the liquid preservative added to the solution of 15 ppm stir agent; sample diluent 2: first 0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白的磷酸盐缓冲液中,并且搅拌均匀;进一步地,向该溶液中加入0.2 %的3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐搅拌均勾;进一步的,向该溶液中加入5mmo 1/1的螯合剂乙二胺四乙酸搅拌均匀;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 样本稀释液3:首先将0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白的磷酸盐缓冲液中,并且搅拌均匀;进一步地,向该溶液中加入0.2 %的3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐搅拌均勾;进一步地,向该溶液中加入5mmol/l的乙二胺四乙酸搅拌均匀;进一步地,向该溶液中加入0.05 %牛血清γ球蛋白;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 样本稀释液4:首先将0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白 0.05% polyoxyethylene sorbitan monolaurate was added monoester phosphate buffer containing 5% bovine serum albumin, and stirred uniformly; Further, this solution was added 0.2% of 3- [3- (bile acid aminopropyl) dimethylammonio] propanesulfonate was stirred hook; further, this solution was added 5mmo 1/1 chelating agent ethylenediaminetetraacetic acid stir; further, this solution was added one million liquid preservative 15 ppm stir; sample 3 diluent: first 0.05% polyoxyethylene sorbitan monolaurate was added monoester phosphate buffer containing 5% bovine serum albumin, and stirred uniformly; further to this solution was added 0.2% of 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate was stirred hook; further, this solution was added 5mmol / l of ethylenediamine stir acid; further, this solution was added 0.05% bovine serum γ-globulin; further, the liquid preservative added to the solution of 15 ppm stir; sample diluent 4: first 0.05 % polyoxyethylene sorbitan monolaurate was added monoester containing 5% bovine serum albumin 磷酸盐缓冲液中,并且搅拌均匀;进一步地,向该溶液中加入0.2 %的3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐搅拌均勾;进一步地,向该溶液中加入5mmol/l的乙二胺四乙酸搅拌均匀;进一步地,向该溶液中加入0.10 %牛血清γ球蛋白;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 样本稀释液5:首先将0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白的磷酸盐缓冲液中,并且搅拌均匀;进一步地,向该溶液中加入0.2 %的3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐搅拌均勾;进一步地,向该溶液中加入5mmol/l的乙二胺四乙酸搅拌均匀;进一步地,向该溶液中加入0.15 %牛血清γ球蛋白;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 样本稀释液6:首先将0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白的磷酸盐缓冲液中 Phosphate buffer, and stirred uniformly; Further, this solution was added 0.2% of 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate was stirred hook; further, to the solution was added to 5mmol / l EDTA stir; further, this solution was added 0.10% bovine serum γ-globulin; further, the liquid preservative added to the solution of 15 ppm stir; sample 5 diluent: first, 0.05% polyoxyethylene sorbitan monolaurate was added monoester phosphate buffer containing 5% bovine serum albumin, and stirred uniformly; further, this solution was added 0.2 % of 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate was stirred hook; further, this solution was added 5mmol / l EDTA stir; further, to this solution was added 0.15% bovine serum γ-globulin; further, the liquid preservative added to the solution of 15 ppm stir; 6 sample diluent: first, 0.05% polyoxyethylene sorbitan monolaurate acid monoester was added phosphate buffer containing 5% bovine serum albumin 并且搅拌均匀;进一步地,向该溶液中加入0.2 %的3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐搅拌均勾;进一步地,向该溶液中加入5mmol/l的乙二胺四乙酸搅拌均匀;进一步地,向该溶液中加入0.20 %牛血清γ球蛋白;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 样本稀释液7:首先将0.05%聚氧乙烯山梨醇单月桂酸单脂加入含有5%牛血清白蛋白的磷酸盐缓冲液中,并且搅拌均匀;进一步地,向该溶液中加入0.2 %的3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐搅拌均勾;进一步地,向该溶液中加入5mmol/l的乙二胺四乙酸搅拌均匀;进一步地,向该溶液中加入0.25 %牛血清γ球蛋白;进一步地,向该溶液中加入百万分之十五的液体防腐剂搅拌均匀; 第五步,将脑源性神经营养因子板和催乳素板放入孵育振荡器中孵育抗体; 分别将第三步中的标准稀释液、第四步 And stir; Further, this solution was added 0.2% of 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate was stirred hook; Further, this solution was added 5mmol / l EDTA stir; further, this solution was added 0.20% bovine serum γ-globulin; further, the liquid preservative added to the solution of 15 ppm stir; sample diluent 7: first, 0.05% polyoxyethylene sorbitan monolaurate was added monoester phosphate buffer containing 5% bovine serum albumin, and stirred uniformly; further, this solution was added 0.2% 3- [3 - (cholamidopropyl) dimethylammonio] propanesulfonate was stirred hook; further, this solution was added 5mmol / l EDTA stir; further, this solution was added 0.25 % bovine serum γ-globulin; further, the liquid preservative added to the solution of 15 ppm stir; a fifth step, the brain-derived neurotrophic factor into the plate and the plate was incubated shaker prolactin antibody incubation; each standard dilution in the third step, the fourth step 的样本稀释液1-7、一份正常人血清样本,重复的分别加入第二步中晾干后待用的脑源性神经营养因子板和催乳素板的孔中,再向各个反应孔中加入处理酶, 进一步地,将加好溶液的脑源性神经营养因子板和催乳素板放入孵育振荡器中,在18-25°C的室温条件下反应; 第六步,加入检测抗体; 用含有TWeen20的PBS作为洗液,对第五步中反应后的脑源性神经营养因子板和催乳素板分别洗涤,再相应的向BDNF板的每个反应孔中加入脑源性神经营养因子检测抗体,并且相应的向催乳素板的每个反应孔中加入催乳素检测抗体; 第七步,将稀释液加入试剂盒中; 用含有Tween20的F>BS作为洗液,对第六步中反应后的脑源性神经营养因子板和催乳素板分别洗涤后对其进行稀释,再分别向该试剂盒中的每个孔中加入稀释后的溶液; 第八步,放在酶标仪下读取光密度值; 用 The Sample Diluent 1-7, a normal human serum sample, repeat Step holes are added after the dry standby brain-derived neurotrophic factor prolactin plate and the plate, each reaction well again processing enzyme is added, and further, the solution was added a good brain-derived neurotrophic factor prolactin plate into plate and incubated shaker at room temperature conditions of 18-25 ° C; the sixth step, detection antibody; with PBS containing TWeen20 as lotion, washed with brain-derived neurotrophic factor on the plate and the plate prolactin fifth step after the reaction, respectively, and then added to the appropriate brain-derived neurotrophic factor BDNF to each well of the plate detection antibody, the detection antibody and prolactin corresponding prolactin was added to each well of the plate; a seventh step, the dilution was added to the kit; Tween20-containing F> BS as a lotion, a sixth step after the reaction brain-derived neurotrophic factor and the plates were washed prolactin plate subjected to dilution, to each well and then were in the kit was added after the dilution; eighth step, on the microplate reader reading the optical density value; with 有TWeen20的roS作为洗液,对第七步中反应后的试剂盒分别洗涤,再向该洗涤好的试剂盒中加入四甲基联苯胺底物; 进一步地,将加好TMB的试剂盒放在酶标仪下读取光密度值; 第九步,根据光密度值计算出非特异性吸附数据,通过比较非特异性吸附数据,进一步确定出牛血清γ球蛋白作为抗原对降低背景噪音具有直接并且积极的作用; 首先,第三步中的标准稀释液、第四步中的样本稀释液1-7、一份正常人血清样本,在脑源性神经营养因子板上所测得的非特异性吸附NSB的信号结果; 进一步地,第三步中的标准稀释液、第四步中的样本稀释液1-7、一份正常人血清样本, 在催乳素板上所测得的非特异性吸附NSB的信号结果; 进一步地,发现在稀释液中加入牛血清γ球蛋白的样本稀释液3-7,在两种包被不同抗体的ELISA板,即神经营养因子板和催乳素板中的非特 There TWeen20 of roS as washing liquid, the washing after the seventh step of the reaction kit were added, and the well was washed with the kit was added tetramethyl benzidine substrate; further, the TMB kit plus good discharge microplate reader reads the optical density value; ninth step, nonspecific adsorption is calculated according to optical density data by comparing the data non-specific adsorption, is further determined bovine serum γ-globulin as an antigen and decrease the background noise has a direct positive role; first, the standard dilution in the third step, the fourth step in sample diluent 1-7, a normal human serum sample, brain-derived neurotrophic factor in the board measured nonspecific adsorption NSB result signal; and further, a third step dilutions standard, sample diluent 1-7 in the fourth step, a normal human serum samples, the plate measured prolactin non-specific adsorption of NSB result signal; further, found that the addition of bovine serum sample diluent γ globulin 3-7 in the dilution liquid, in both non-specific ELISA plates coated with different antibodies, i.e., neurotrophin plate and the plate prolactin 异性吸附NSB都很小,从而说明所产生的背景值都非常低,为〇. 02-0.045之间; 进一步地,由于样本稀释液3-7中的牛血清γ球蛋白浓度不同,但是都降低了ELISA反应中的背景信号; 进一步地,说明牛血清γ球蛋白抗原对降低背景噪音具有直接且积极的作用。 NSB anisotropic adsorption is very small, so that the generated instructions are very low background value, is square between 02-0.045; Further, due to the different concentrations of bovine serum γ-globulin in the sample diluent 3-7, but were reduced the background signal of the ELISA reaction; further explained bovine serum γ-globulin and the antigen has a direct positive effect on reducing background noise.
2. 根据权利要求1所述的一种ELISA用的抗原标准品和样本稀释液的制备方法,还包含:第十步,将该第四步中的样本稀释液6作为新的标准稀释液,仅将该3-[3-(胆酰氨丙基) 二甲氨基]丙磺酸内盐的含量分别调整为0.1%、0.15%、0.25%、0.3%、0.45%、0.5%、 0.6%,从而形成7种新的样本稀释液8-14,其它步骤完全按照第一步至八步进行处理,读取光密度值,进一步计算出非特异性吸附系数;并对测得的非特异性吸附系数进行分析,得出在牛血清γ球蛋白含量一定的情况下,3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐浓度位于0.2%-0.45%之间时,脑源性神经因子板和催乳素板的非特异性吸附都会降低,从而使背景信号降低。 2. The ELISA according to claim 1 and a standard antigen diluent of sample preparation methods, further comprising: a tenth step, the sample diluent in the fourth step 6 as a new standard dilution the only 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate contents were adjusted to 0.1%, 0.15%, 0.25%, 0.3%, 0.45%, 0.5%, 0.6%, thereby forming seven new sample diluent 8-14, other steps are processed in a first step completely to eight steps, reading the optical density value, and further calculates the nonspecific adsorption coefficient; and non-specific adsorption coefficient is measured analysis, the results of bovine serum γ-globulin content in certain circumstances, 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate inner salt lies between 0.2% -0.45%, brain-derived nonspecific adsorption properties are neurotropic factor prolactin plate and the plate is reduced, thereby reducing background signals.
3. 根据权利要求2所述的一种ELISA用的抗原标准品和样本稀释液的制备方法,还包含:第十一步,从第四步和第十步中选取出三种信噪比高,非特异性信号低的三种样本稀释液来绘制BDNF和prolactin的回收率标准曲线并测算稀释样品的回收率;根据测试结果证明了向含有牛血清白蛋白的磷酸盐缓冲液中分别加入〇. 05 %聚氧乙烯山梨醇单月桂酸单月旨、0.2-0.45%的3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐、5111 111〇1/1的乙二胺四乙酸、任意浓度的牛血清γ球蛋白、百万分之十五的液体防腐剂作为稀释液,是一种效果非常好的适用于ELISA的样本稀释液,具体步骤为: 首先,分别将脑源性神经因子和催乳素标准抗原加入脑源性神经因子和催乳素阴性血清中; 进一步地,分别对配制好的阴性血清进行稀释,稀释后分别加入三种信噪比高,非特异性信号低的样本稀释液3、 The standard antigen preparation and sample dilution for ELISA according to one kind of claim 2, further comprising: an eleventh step, removed from the three kinds of high SNR fourth and tenth step selected nonspecific signal of the low three sample dilutions BDNF and draw standard curve and recovery prolactin measure the diluted sample recovery; square were added to a phosphate buffer containing bovine serum albumin based on the test results show. 05% polyoxyethylene sorbitan monolaurate monthly purpose, 0.2-0.45% ethylene 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate, 5111 111〇1 / 1 tetraacetic acid, any concentration of bovine serum γ-globulin, preservative liquid 15 ppm as a diluent, an effect is very well suited for sample dilution ELISA specific steps: first, respectively, brain-derived neurotrophic factor and prolactin standard antigen was added and brain-derived neurotrophic factor prolactin negative sera; further, each of the prepared negative sera diluted, were added after dilution or high signal to noise ratio, the non-specific signal low sample dilution 3, 本稀释液10、样本稀释液12中,其他步骤与第五步至第八步完全一致,读出光密度值和回收率; 进一步地,样本稀释液3和样本稀释液12分别是3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐为0.2%和0.45%的2个临界点,经实验表明三种样本稀释液3、10、12配制的脑源性神经因子和催乳素的标准曲线分别互相平行,相关性很好,并且与阴性血清相比回收率提高; 进一步地,证明向PH值为7.4 ± 0.2的含有牛血清白蛋白的磷酸盐缓冲液中分别加入0.05%聚氧乙烯山梨醇单月桂酸单脂、0.2-0.45%的3-[3-(胆酰氨丙基)二甲氨基]丙磺酸内盐、5mmol/l的乙二胺四乙酸、0.05%-0.2%的牛血清γ球蛋白、百万分之十五的液体防腐剂作为稀释液,是一种效果非常好的适用于ELISA的样本稀释液。 This solution was diluted 10, 12 in sample diluent, and the other steps are exactly the fifth step to an eighth step, the optical density value read out and recovery; Further, the sample 3 and the sample dilutions are dilutions 12 3- [ 3- (cholamidopropyl) dimethylammonio] propanesulfonate of 0.2% and 0.45% of two critical point, the experiment shows that three kinds of brain-derived neurotrophic factor prepared sample diluent 3,10,12 prolactin and standard curves were parallel to each other, a good correlation, and the recovery rate compared to the negative serum; further, the PH value of 7.4 ± proof phosphate buffer solution containing bovine serum albumin were added 0.2 0.05% polyoxyethylene sorbitan monolaurate monoester, 0.2-0.45% of 3- [3- (cholamidopropyl) dimethylammonio] propanesulfonate, 5mmol / l of ethylenediaminetetraacetic acid, 0.05% to 0.2% of bovine serum γ-globulin, preservative liquid 15 ppm as a diluent, an effect is very well suited for sample dilution for ELISA.
CN201310555174.XA 2013-11-08 2013-11-08 The method of preparing an antigen standards and the sample dilutions used in a elisa CN103558372B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310555174.XA CN103558372B (en) 2013-11-08 2013-11-08 The method of preparing an antigen standards and the sample dilutions used in a elisa

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310555174.XA CN103558372B (en) 2013-11-08 2013-11-08 The method of preparing an antigen standards and the sample dilutions used in a elisa

Publications (2)

Publication Number Publication Date
CN103558372A CN103558372A (en) 2014-02-05
CN103558372B true CN103558372B (en) 2017-02-15

Family

ID=50012678

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310555174.XA CN103558372B (en) 2013-11-08 2013-11-08 The method of preparing an antigen standards and the sample dilutions used in a elisa

Country Status (1)

Country Link
CN (1) CN103558372B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106483282B (en) * 2016-09-29 2018-08-31 北京世纪沃德生物科技有限公司 A kind of antigen stabilizer and the preparation method and application thereof
CN106932563B (en) * 2017-01-19 2018-11-23 杭州联科生物技术股份有限公司 A kind of preparation method of the ELISA standard dilutions based on serum

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6984494B2 (en) * 2000-08-15 2006-01-10 Genentech, Inc. Analytical method
WO2006074076A1 (en) * 2004-12-31 2006-07-13 Genentech, Inc. Detecting human antibodies in non-human serum
CN101226199A (en) * 2007-01-15 2008-07-23 中国科学院上海生命科学研究院;中国科学院上海药物研究所 Effect of N-methyl-D-asparagic acid acceptor in epilepsy generation
WO2011069104A2 (en) * 2009-12-04 2011-06-09 Genentech, Inc. Multispecific antibodies, antibody analogs, compositions, and methods
WO2012138975A1 (en) * 2011-04-07 2012-10-11 Genentech, Inc. Anti-fgfr4 antibodies and methods of use

Also Published As

Publication number Publication date
CN103558372A (en) 2014-02-05

Similar Documents

Publication Publication Date Title
Hornbeck Enzyme‐linked immunosorbent assays
CN101713779B (en) Method for performing immunological test on biomolecules by avidin/streptavidin magnetic composite particles
CN102590524B (en) Neutrophil gelatinase-associated lipocalin protein assay kit
CN104897901B (en) A kind of method of the acridinium ester chemiluminescent immunology detection HE4 based on gold-magnetic particles
CN102628864A (en) Kit for determining heart-type fatty acid binding protein in serum or urine by latex enhanced turbidimetric immunoassay
CN101377492A (en) Bladder chalone C determining reagent kit
CN101813700B (en) Kit for detecting beta2-microglobulin by nanometer microsphere immunoturbidimetry
CN102192983B (en) Time-resolved immunoflourometric chromatographic test strip for quantitative detection as well as preparation method and application thereof
CA1146852A (en) Reagent for latex agglutination
CN101769932B (en) Full-range C-reactive protein detection kit
CN102628867B (en) Diabodies latex reinforcing retinol binding protein assay kit
CN101183105B (en) ELISA reagent kit for detecting melamine
CN104614535B (en) TMA detection kit and its preparation method and application
Birkmeyer et al. Application of novel chromium dioxide magnetic particles to immunoassay development.
CN104237522B (en) Kit and its preparation method of detecting the content of adiponectin
CN102621332B (en) Retinol binding protein assay kit based on latex particle coating
CN102944673A (en) Kit (Latex-enhanced immunoturbidimetry) for detecting content of glycocholic acid in blood serum
CN103760348A (en) Glycocholic acid immunodetection reagent and preparing method and detecting method thereof
CN101206223A (en) Direct racing method ELISA reagent box for detecting melamine
CN102998468B (en) 25-hydroxy-vitamin D3 detection kit, as well as preparation method and applications thereof
CN101699287B (en) Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof
CN105308458A (en) Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases
US20130011827A1 (en) Methods and kits for decreasing interferences in plasma or serum containing assay samples of specific binding assays
CN104407159B (en) An immunoglobulin IgM immunoturbidimetric assay kit
CN102590497A (en) Cysteine protease inhibitor C test kit

Legal Events

Date Code Title Description
C06 Publication
EXSB Decision made by sipo to initiate substantive examination
C41 Transfer of patent application or patent right or utility model
C14 Grant of patent or utility model