技术领域technical field
本发明涉及多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，属于酶联免疫技术领域。The invention relates to a method for preparing a bisphenol A-coated antigen with polylysine as a carrier and an application thereof, and belongs to the technical field of enzyme-linked immunity.
背景技术Background technique
双酚A（Bisphenol A,BPA）为2, 2-双（4-羟基苯基）丙烷，在体内通过与雌激素受体结合或影响细胞信号传导途径等方式模仿或干扰内源雌激素作用，对人的皮肤、呼吸道、消化道和角膜有刺激性。当体内的双酚A达到一定量时就会破坏肝细胞和肾细胞，造成慢性中毒，使人出现不同程度的头昏、头痛、皮疹、精神不安和腹泻等症状。近年来，世界各地桶装饮用水、婴儿奶瓶和各种水体中都发现有雌激素类似作用的双酚A存在（Science. 2007,317(5840):884-885），由于仪器分析方法不适应现场和快速对桶装饮用水及婴儿奶瓶中双酚A进行检测，而免疫分析基于抗原-抗体间的特异性结合反应，选择性好，通过选用合适的标记体系，可实现对复杂体系中痕量物质的快速灵敏检测。酶联免疫分析方法灵敏度高、特异性强、快速、经济，非常适合BPA残留的筛选检测。Bisphenol A (BPA) is 2,2-bis(4-hydroxyphenyl)propane, which imitates or interferes with endogenous estrogen in the body by binding to estrogen receptors or affecting cell signal transduction pathways, etc. Irritant to human skin, respiratory tract, digestive tract and cornea. When the bisphenol A in the body reaches a certain amount, it will destroy the liver cells and kidney cells, causing chronic poisoning, causing people to experience symptoms such as dizziness, headache, rash, nervousness and diarrhea in varying degrees. In recent years, bisphenol A, which has estrogen-like effects, has been found in bottled drinking water, baby bottles and various water bodies around the world (Science. 2007, 317(5840): 884-885), due to inappropriate instrumental analysis methods On-site and rapid detection of bisphenol A in bottled drinking water and baby bottles, while immunoassay is based on the specific binding reaction between antigen and antibody, with good selectivity, and by selecting a suitable labeling system, it can realize the detection of traces in complex systems Rapid and sensitive detection of large quantities of substances. The enzyme-linked immunoassay method has high sensitivity, strong specificity, rapidity and economy, and is very suitable for the screening and detection of BPA residues.
研究双酚A的酶联免疫分析方法，首先必须制备得到双酚A的抗体。双酚A为小分子化合物，结构简单，不能直接免疫动物产生抗体，必须先将其与载体蛋白质偶联制备出完全抗原，利用免疫抗原注射动物诱发产生抗体。目前常用的方法是利用经典的碳二亚胺法或戊二醛法，选择牛血清白蛋白或卵清蛋白为载体来合成双酚A人工抗原。申请号为200810234850.2的专利中，胥传来等介绍了以双酚酸为半抗原与OVA偶联制备包被抗原的方法。多聚左旋赖氨酸（PLL）是一种人工合成的多聚肽，自身免疫原性很差但可以增加半抗原的免疫性，且具有更多的自由氨基，可以大大提高载体蛋白质与半抗原的偶联率，所以近来年被越来越多的用以人工免疫抗原的制备中，如中国专利号为200810051488.5中公开的以多聚赖氨酸为载体制备三聚氰胺完全抗原的方法；中国专利专利号200610041918.6提供了伊维菌素人工抗原的合成新方法。但是均未见以多聚赖氨酸为载体制备双酚A人工包被抗原的专利。To study the enzyme-linked immunoassay method of bisphenol A, the antibody to bisphenol A must be prepared first. Bisphenol A is a small molecular compound with a simple structure and cannot directly immunize animals to produce antibodies. It must first be coupled with a carrier protein to prepare a complete antigen, and the immunized antigen is used to inject animals to induce antibody production. The current commonly used method is to use the classic carbodiimide method or glutaraldehyde method to select bovine serum albumin or ovalbumin as the carrier to synthesize the bisphenol A artificial antigen. In the patent application No. 200810234850.2, Xu Chuanlai et al. introduced the method of preparing coated antigen by coupling bisphenolic acid as hapten with OVA. Poly-L-lysine (PLL) is an artificially synthesized polypeptide, which has poor autoimmunity but can increase the immunity of haptens, and has more free amino groups, which can greatly improve the interaction between carrier protein and hapten. The coupling rate is high, so it has been used more and more in the preparation of artificial immune antigens in recent years, such as the method for preparing melamine complete antigens with polylysine as a carrier disclosed in Chinese Patent No. 200810051488.5; Chinese Patent No. No. 200610041918.6 provides a new synthesis method of ivermectin artificial antigen. However, there are no patents on preparing bisphenol A artificially coated antigens with polylysine as a carrier.
发明内容Contents of the invention
针对上述不足，本发明的目的在于提供一种合成方法简单的以多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，并最终建立了双酚A检测的酶联免疫试剂盒。In view of the above-mentioned deficiencies, the object of the present invention is to provide a method for the preparation and application of a bisphenol A-coated antigen with a simple synthesis method using polylysine as a carrier, and finally establish an enzyme-linked immunosorbent reagent for bisphenol A detection box.
为解决上述技术问题，本发明的前一技术方案是这样的：一种以多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，以多聚赖氨酸为载体蛋白，与双酚酸偶联制备双酚A包被抗原。In order to solve the above-mentioned technical problems, the previous technical scheme of the present invention is as follows: a method for preparing a bisphenol A-coated antigen using polylysine as a carrier and its application, using polylysine as a carrier protein, Coupling antigen with bisphenol A was prepared by coupling with bisphenol A.
上述的以多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，依次包括下述步骤：1）将双酚酸完全溶解在有机溶剂中；2）将NHS(N-羟基琥珀酰亚胺)和EDC·HCL分别完全溶解在机溶剂中；3）将步骤2）制得的溶液滴加至步骤1）制得的溶液中，室温避光搅拌1-24h，离心分离取上清液得A液；4）将多聚赖氨酸溶于磷酸缓冲溶液中成B液；5）将步骤3）制备的 A液滴加B液中，室温避光搅拌1-24h，离心分离，上清液转移到透析袋，用磷酸缓冲溶液透析24-96h，每12 h换一次透析液，最后将透析液经冷冻干燥得到双酚A人工包被抗原即BVA-PLL；The preparation method and application of the above-mentioned bisphenol A-coated antigen with polylysine as a carrier include the following steps in sequence: 1) completely dissolving bisphenol A in an organic solvent; 2) dissolving NHS (N-hydroxyl succinimide) and EDC·HCL were completely dissolved in the organic solvent; 3) the solution prepared in step 2) was added dropwise to the solution prepared in step 1), stirred at room temperature for 1-24h in the dark, and centrifuged to obtain The supernatant was obtained as liquid A; 4) Polylysine was dissolved in phosphate buffer solution to form liquid B; 5) Liquid A prepared in step 3) was added dropwise to liquid B, stirred at room temperature for 1-24 hours in the dark, and centrifuged Separate, transfer the supernatant to a dialysis bag, dialyze with phosphate buffer solution for 24-96 hours, change the dialysate every 12 hours, and finally freeze-dry the dialysate to obtain bisphenol A artificially coated antigen, namely BVA-PLL;
其中：双酚酸与NHS、EDC·HCL、多聚赖氨酸的摩尔比为：30～200﹕30～400﹕30～400﹕1。Among them: the molar ratio of bisphenolic acid to NHS, EDC·HCL, and polylysine is: 30-200:30-400:30-400:1.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，所述的多聚赖氨酸为多聚左旋赖氨酸或者多聚右旋赖氨酸或者多聚左旋赖氨酸和多聚右旋赖氨酸混合使用。Further, the above-mentioned method for preparing bisphenol A-coated antigen with polylysine as a carrier and its application, the polylysine is poly-L-lysine or poly-D-lysine or Poly-L-lysine and poly-D-lysine are used in combination.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，其特征在于，步骤1）和步骤2）所述的有机溶剂为四氢呋喃或者N,N-二甲基甲酰胺或者二甲基亚砜的其中之一。Further, the above-mentioned method for preparing bisphenol A-coated antigen with polylysine as a carrier and its application is characterized in that the organic solvent in step 1) and step 2) is tetrahydrofuran or N,N-di One of methylformamide or dimethyl sulfoxide.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原制备方法及其应用，步骤4）和步骤5）所述的磷酸缓冲溶液的 pH值为6-8。Further, the pH value of the phosphate buffer solution described in step 4) and step 5) of the above-mentioned preparation method and application of bisphenol A coated antigen with polylysine as a carrier is 6-8.
本发明的后一技术方案是这样的：应用双酚A包被抗原作为双酚A酶联免疫检测试剂盒，所述的双酚A酶联免疫检测试剂盒由包被抗原的固相载体、抗体工作液、双酚A标准溶液、酶标二抗溶液、抗体稀释液、洗涤浓缩液、底物溶液和终止液组成；The latter technical scheme of the present invention is as follows: the bisphenol A coated antigen is used as the bisphenol A ELISA kit, and the bisphenol A ELISA kit consists of a solid phase carrier coated with the antigen, Antibody working solution, bisphenol A standard solution, enzyme-labeled secondary antibody solution, antibody diluent, washing concentrate, substrate solution and stop solution;
其中：所述的固相载体为包被有双酚酸与多聚左旋赖氨酸偶联物的96孔聚苯乙烯酶标板，并封闭了微孔表面未吸附双酚A 抗原的位点；所述的双酚A 标准溶液的浓度分别为：0ng/mL， 3ng/mL， 8ng/mL，25ng/mL，75ng/mL，224ng/mL，673ng/mL，2018ng/mL。Wherein: the solid phase carrier is a 96-well polystyrene microtiter plate coated with bisphenolic acid and poly-L-lysine conjugates, and the sites on the surface of the micropores that do not adsorb bisphenol A antigens are blocked The concentration of described bisphenol A standard solution is respectively: 0ng/mL, 3ng/mL, 8ng/mL, 25ng/mL, 75ng/mL, 224ng/mL, 673ng/mL, 2018ng/mL.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原的应用，其特征在于，所述的抗体工作液为兔源多克隆抗双酚A的抗体。Furthermore, the application of the above-mentioned bisphenol A-coated antigen with polylysine as a carrier is characterized in that the antibody working solution is a rabbit-derived polyclonal anti-bisphenol A antibody.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原的应用，其特征在于，所述的底物液为含有四甲基联苯胺和过氧化氢的pH5.0磷酸柠檬酸缓冲溶液；所述的终止液为2mol/L硫酸溶液。Further, the application of the above-mentioned bisphenol A coated antigen with polylysine as a carrier is characterized in that the substrate liquid is pH5.0 lemon phosphate containing tetramethylbenzidine and hydrogen peroxide Acid buffer solution; the stop solution is 2mol/L sulfuric acid solution.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原的应用，其特征在于，所述的洗涤浓缩液为10X含5% 吐温-20，pH7.0，浓度为0.01mol/L的磷酸盐缓冲液。Further, the application of the above-mentioned bisphenol A-coated antigen with polylysine as the carrier is characterized in that the washing concentrate is 10X containing 5% Tween-20, pH 7.0, and the concentration is 0.01 mol/L phosphate buffer.
进一步的，上述的以多聚赖氨酸为载体的双酚A包被抗原的应用，其特征在于，所述的酶标二抗溶液为羊抗兔-辣根过氧化物酶。Furthermore, the application of the above-mentioned bisphenol A coated antigen with polylysine as the carrier is characterized in that the enzyme-labeled secondary antibody solution is goat anti-rabbit-horseradish peroxidase.
与现有技术相比，本发明具有如下优点：Compared with the prior art, the present invention has the following advantages:
1、抗原合成方法的优点：本发明的合成以多聚赖氨酸为载体，多聚赖氨酸来源广泛，价格便宜，易溶于水；在有机溶剂化学偶联过程中，多聚赖氨酸能保持其结构稳定性，不易变性，合成后的包被抗原具有良好地可溶性；多聚赖氨酸上含有的氨基数目远大于常用的白蛋白类物质（如 鸡卵清白蛋白， 牛血清白蛋白等），这些官能团可以和更多的半抗原分子偶联，进而增加了分子的偶联率，有利于偶联反应；多聚赖氨酸结构简单，可有效的减少非特异性反应，提高试剂盒的性能；常用的包被载体（鸡卵清白蛋白）属于生物大分子，拥有复杂的的三维结构，其合成的包被抗原与酶标板发生疏水性接触时，包被抗原易发生形变，小分子半抗原被大分子蛋白质屏蔽，进而不利于半抗原分子在酶标板上的呈现，然而合成的多聚赖氨酸属于网状结构，克服了这一缺点，可以更有利于半抗原在酶标板上呈现，提高了包被抗原与抗体之间的特异性识别；1. Advantages of the antigen synthesis method: the synthesis of the present invention uses polylysine as a carrier, and polylysine has a wide range of sources, is cheap, and is easily soluble in water; in the chemical coupling process of organic solvents, polylysine The acid can maintain its structural stability and is not easy to denature, and the coated antigen after synthesis has good solubility; the number of amino groups contained in polylysine is much larger than that of commonly used albumin substances (such as chicken ovalbumin, bovine serum albumin) Proteins, etc.), these functional groups can be coupled with more hapten molecules, thereby increasing the coupling rate of the molecule, which is conducive to the coupling reaction; poly-lysine has a simple structure, which can effectively reduce non-specific reactions and improve reagent efficiency. The performance of the box; the commonly used coating carrier (chicken ovalbumin) is a biological macromolecule with a complex three-dimensional structure. When the coated antigen synthesized by it is in hydrophobic contact with the microtiter plate, the coated antigen is prone to deformation. Small molecular haptens are shielded by large molecular proteins, which is not conducive to the presentation of hapten molecules on the microtiter plate. However, the synthetic poly-lysine has a network structure, which overcomes this shortcoming and can be more conducive to the hapten on the microtiter plate. Presented on the microtiter plate, which improves the specific recognition between the coated antigen and the antibody;
2、通过合成包被抗原，并将其按一定浓度包被在聚苯乙烯酶标板上，使用卵清蛋白封闭，制备了可以直接应用于酶联免疫分析的ELISA板条；通过制备好的双酚A抗体，建立了测定双酚A残留的试剂盒使用方法。2. By synthesizing the coated antigen, coating it on a polystyrene microplate at a certain concentration, and sealing it with ovalbumin, an ELISA strip that can be directly applied to enzyme-linked immunoassay was prepared; through the prepared Bisphenol A antibody, the kit method for the determination of bisphenol A residues was established.
3、本发明可用于地表水、地下水和饮用水中双酚A 的检测，具有前处理简单、快速准确、成本低廉，可用于现场大批量检测等优点，并且最低检测限为0.5ng/ml。3. The invention can be used for the detection of bisphenol A in surface water, ground water and drinking water. It has the advantages of simple pretreatment, fast and accurate, low cost, and can be used for on-site mass detection, and the minimum detection limit is 0.5ng/ml.
附图说明Description of drawings
图1是BVA、PLL和PLL-BVA的紫外光谱Figure 1 is the UV spectrum of BVA, PLL and PLL-BVA
图2是间接竞争ELISA法检测双酚A竞争抑制曲线；Fig. 2 is the competition inhibition curve of bisphenol A detected by indirect competition ELISA method;
以系列浓度（2048ng/mL， 512ng/mL， 128ng/mL，32ng/mL，8ng/mL，2ng/mL，0.5ng/mL，0.125ng/mL和0.0312 ng/mL）的双酚A 进行间接竞争ELISA法测定，建立的标准工作曲线。Indirect competition with BPA at serial concentrations (2048ng/mL, 512ng/mL, 128ng/mL, 32ng/mL, 8ng/mL, 2ng/mL, 0.5ng/mL, 0.125ng/mL and 0.0312ng/mL) Determination by ELISA method, the establishment of the standard working curve.
具体实施方式Detailed ways
下面结合附图和具体实施方式对本发明的权利要求做进一步详细说明，但不构成对本发明的任何限制，任何人在本发明权利要求范围内所做的有限次的修改，仍在本发明的权利要求范围内。The claims of the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments, but this does not constitute any restriction on the present invention. Any limited number of modifications made within the scope of the claims of the present invention are still within the rights of the present invention. within the required range.
1、双酚A人工抗原的合成1. Synthesis of bisphenol A artificial antigen
实施例1Example 1
称取双酚酸（BVA）61mg溶于2mLDMF中；再称取30.6mgNHS与53.2mgEDC·HCL分别溶于2mL（四氢呋喃）DMF中，缓慢滴加到BVA溶液中，室温避光搅拌24h后，离心分离取上清液得A液。Weigh 61 mg of bisphenolic acid (BVA) and dissolve it in 2 mL of DMF; then weigh 30.6 mg of NHS and 53.2 mg of EDC·HCL and dissolve them in 2 mL of (tetrahydrofuran) DMF, slowly add it dropwise to the BVA solution, stir at room temperature in the dark for 24 hours, and then centrifuge Separate the supernatant to obtain liquid A.
称取126mg 多聚左旋赖氨酸溶于5 mL pH= 7的磷酸缓冲溶液中成B液，取A液4mL缓慢滴加B液中，室温避光搅拌24h，离心分离，上清液转移到透析袋，用pH =7.4的磷酸缓冲溶液透析72 h，每12 h换一次透析液，最后将透析液经冷冻干燥得到双酚A人工包被抗原即PLL-BVA，经过紫外光谱鉴定其结构，参阅图1。Weigh 126 mg poly-L-lysine and dissolve it in 5 mL pH=7 phosphate buffer solution to form B solution, take 4 mL of A solution and slowly add dropwise to B solution, stir at room temperature for 24 hours in the dark, centrifuge, and transfer the supernatant to The dialysis bag was dialyzed with a phosphate buffer solution with pH = 7.4 for 72 hours, and the dialysate was changed every 12 hours. Finally, the dialysate was freeze-dried to obtain the artificially coated antigen of bisphenol A, that is, PLL-BVA, and its structure was identified by ultraviolet spectroscopy. See Figure 1.
实施例2Example 2
称取双酚酸（BVA）20.7mg溶于1mLDMF中；再称取6.6mgNHS与14.2mgEDC·HCL分别溶于1mL（四氢呋喃）DMF中，缓慢滴加到BVA溶液中，室温避光搅拌2h后，离心分离取上清液得A液。Weigh 20.7 mg of bisphenolic acid (BVA) and dissolve it in 1 mL of DMF; then weigh 6.6 mg of NHS and 14.2 mg of EDC·HCL and dissolve it in 1 mL (tetrahydrofuran) DMF, slowly add it dropwise to the BVA solution, and stir for 2 hours at room temperature in the dark. The supernatant was collected by centrifugation to obtain liquid A.
称取30.2mg 多聚混合赖氨酸溶于5 mL pH= 7的磷酸缓冲溶液中成B液，取A液3mL缓慢滴加B液中，室温避光搅拌2h，离心分离，上清液转移到透析袋，用pH =7.4的磷酸缓冲溶液透析72 h，每12 h换一次透析液，最后将透析液经冷冻干燥得到双酚A人工包被抗原即PLL-BVA，经过紫外光谱鉴定其结构，参阅图1。Weigh 30.2 mg of poly-mixed lysine and dissolve it in 5 mL of phosphate buffer solution with pH = 7 to form liquid B, take 3 mL of liquid A and slowly add it dropwise to liquid B, stir at room temperature for 2 hours in the dark, centrifuge, and transfer the supernatant To the dialysis bag, dialyze with pH = 7.4 phosphate buffer solution for 72 hours, change the dialysate every 12 hours, and finally freeze-dry the dialysate to obtain bisphenol A artificial coating antigen, namely PLL-BVA, and identify its structure by ultraviolet spectroscopy , see Figure 1.
实施例3Example 3
称取双酚酸（BVA）24mg溶于1mLDMF中；再称取10mg NHS与17.2mgEDC·HCL分别溶于1mL（四氢呋喃）DMF中，缓慢滴加到BVA溶液中，室温避光搅拌12h后，离心分离取上清液得A液。Weigh 24 mg of bisphenolic acid (BVA) and dissolve in 1 mL of DMF; then weigh 10 mg of NHS and 17.2 mg of EDC·HCL and dissolve in 1 mL of (tetrahydrofuran) DMF, slowly add it dropwise to the BVA solution, stir at room temperature in the dark for 12 hours, and centrifuge Separate the supernatant to obtain liquid A.
称取15mg 多聚左旋赖氨酸溶于5 mL pH= 7的磷酸缓冲溶液中成B液，取A液2mL缓慢滴加B液中，室温避光搅拌12h，离心分离，上清液转移到透析袋，用pH =7.4的磷酸缓冲溶液透析72 h，每12 h换一次透析液，最后将透析液经冷冻干燥得到双酚A人工包被抗原即PLL-BVA，经过紫外光谱鉴定其结构，参阅图1。Weigh 15 mg of poly-L-lysine and dissolve it in 5 mL of pH=7 phosphate buffer solution to form B solution, take 2 mL of A solution and slowly add dropwise to B solution, stir at room temperature for 12 hours in the dark, centrifuge, and transfer the supernatant to The dialysis bag was dialyzed with a phosphate buffer solution with pH = 7.4 for 72 hours, and the dialysate was changed every 12 hours. Finally, the dialysate was freeze-dried to obtain the artificially coated antigen of bisphenol A, that is, PLL-BVA, and its structure was identified by ultraviolet spectroscopy. See Figure 1.
选择牛血清白蛋白为载体，利用同法与双酚酸偶联制备免疫抗原。Bovine serum albumin was selected as the carrier, and the immune antigen was prepared by coupling with bisphenolic acid in the same way.
2、抗双酚A的抗体制备2. Antibody preparation against bisphenol A
用PBS溶解免疫抗原BSA-BVA，配制成1mg/mL溶液。取上述溶液0.5mL与等体积弗氏完全佐剂乳化成油包水状态，对2-3kg 雌性新西兰兔进行免疫。第一次免疫一个月后，换用不完全佐剂每两周免疫一次，免疫5次，最后一次免疫15天后取血纯化。The immune antigen BSA-BVA was dissolved in PBS to prepare a 1 mg/mL solution. Take 0.5 mL of the above solution and emulsify it into a water-in-oil state with an equal volume of Freund's complete adjuvant, and immunize 2-3 kg female New Zealand rabbits. One month after the first immunization, they were immunized with incomplete adjuvant every two weeks for 5 times, and blood was collected 15 days after the last immunization for purification.
间接ELISA法测定免疫血清的效价。（1）包被：用包被缓冲液CBS将包被抗原（PLL-BVA）稀释至一定浓度，以100μL/孔加入到96孔酶标板中，4℃包被过夜，甩干孔中液体，PBST洗涤一次，吸水纸拍干。（2）封闭：30mg/mL卵清蛋白(PBS溶解)200μL/孔加入到孔中，37℃温育1h。（3）甩干孔中液体，PBST洗涤三次，吸水纸拍干。（4）加入免疫血清：以PBS稀释免疫血清，特定浓度倍比稀释，100μL/孔加到孔中，同时设阴性对照和空白对照各2孔。37℃温育1h后，洗涤，拍干。（5）加入酶标二抗：以PBS稀释酶标二抗至特定浓度，100μL/孔加到孔中，37℃温育1h后，洗涤，拍干。（6）显色和终止：配制底物缓冲液10mL，加入10μL 30%过氧化氢，充分混匀。100μL/孔加到孔中温育15min后，2M硫酸50μL/孔终止反应。（7）读数：用酶标仪测定双波长490nm和630nm处的光密度值（OD值）。若样品孔OD值大于或等于空白对照孔的2.1倍即为阳性。The titer of the immune serum was determined by indirect ELISA. (1) Coating: Dilute the coating antigen (PLL-BVA) to a certain concentration with the coating buffer CBS, add 100 μL/well to the 96-well microtiter plate, coat at 4°C overnight, and dry the liquid in the well , washed once with PBST, and patted dry with absorbent paper. (2) Blocking: 200 μL/well of 30 mg/mL ovalbumin (dissolved in PBS) was added to the wells, and incubated at 37° C. for 1 hour. (3) Dry the liquid in the well, wash with PBST three times, and pat dry with absorbent paper. (4) Adding immune serum: Dilute the immune serum with PBS, dilute the specific concentration doubly, add 100 μL/well to each well, and set 2 wells each for negative control and blank control. After incubation at 37°C for 1 h, wash and pat dry. (5) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody to a specific concentration with PBS, add 100 μL/well to the well, incubate at 37°C for 1 hour, wash, and pat dry. (6) Color development and termination: Prepare 10 mL of substrate buffer, add 10 μL of 30% hydrogen peroxide, and mix well. After adding 100 μL/well to the wells and incubating for 15 min, 50 μL/well of 2M sulfuric acid was used to terminate the reaction. (7) Reading: use a microplate reader to measure the optical density value (OD value) at the dual wavelengths of 490nm and 630nm. If the OD value of the sample well is greater than or equal to 2.1 times that of the blank control well, it is positive.
棋盘滴定法筛选合适的包被抗原浓度和最佳抗体工作浓度，采用间接竞争ELISA法检测免疫血清的竞争抑制率。The appropriate coating antigen concentration and optimal antibody working concentration were screened by checkerboard titration, and the competitive inhibition rate of immune serum was detected by indirect competition ELISA.
通过动物免疫和血清检测，得到抗双酚A的抗体。Through animal immunization and serum detection, the anti-bisphenol A antibody is obtained.
3、双酚A ELISA标准工作曲线的建立和检测限的确定3. Establishment of bisphenol A ELISA standard working curve and determination of detection limit
用适宜浓度的 PLL-BVA包被96孔酶标板，100μL/孔，4℃冰箱包被过夜，用封闭液封闭后洗涤拍干；向酶标条中，先加入50μL的各浓度BPA标准溶液（0ng/mL， 3ng/mL， 8ng/mL，25ng/mL，75ng/mL，224ng/mL，673ng/mL，2018ng/mL），再加入50μL适宜浓度的抗体，混合均匀，在空白对照孔中加入100μL10%甲醇PBS缓冲液；37℃恒温温育40min；洗涤三次，拍干；加入100μL酶标二抗工作液，37℃恒温温育40min；洗涤三次，拍干；每孔加入100μL底物溶液显色，37℃温育避光反应15min后加50μL 终止液终止反应，摇匀，15分钟内读取光密度值。Coat 96-well ELISA plate with appropriate concentration of PLL-BVA, 100 μL/well, overnight in 4°C refrigerator, seal with blocking solution, wash and pat dry; add 50 μL of BPA standard solution of each concentration to the enzyme label strip (0ng/mL, 3ng/mL, 8ng/mL, 25ng/mL, 75ng/mL, 224ng/mL, 673ng/mL, 2018ng/mL), then add 50μL of appropriate concentration of antibody, mix well, put in the blank control well Add 100 μL of 10% methanol in PBS buffer; incubate at 37°C for 40 minutes; wash three times and pat dry; add 100 μL enzyme-labeled secondary antibody working solution, incubate at 37°C for 40 minutes; wash three times and pat dry; add 100 μL of substrate solution to each well For color development, incubate at 37°C in the dark for 15 minutes, then add 50 μL of stop solution to terminate the reaction, shake well, and read the optical density within 15 minutes.
以抑制率B/B0为纵坐标，双酚A 浓度BPA(ng·mL-1)为横坐标作标准曲线，其IC50为14.5ng/ml，最低检测限为0.5ng/ml，参阅图2。Take the inhibition rate B/B0 as the ordinate, and the concentration of bisphenol A BPA (ng·mL-1 ) as the abscissa to draw a standard curve. Its IC50 is 14.5ng/ml, and the minimum detection limit is 0.5ng/ml, see Fig. 2.
4、双酚A ELISA试剂盒特异性的测定4. Determination of specificity of bisphenol A ELISA kit
采用间接竞争ELISA法测定双酚A结构类似物（双酚酸、苯酚、苯、间甲苯酚和对苯二酚）与抗体混合物的交叉反应。将系列浓度（1 ng/mL，10 ng/mL，100 ng/mL，1 ug/mL， 10 ug/mL，100 ug/mL，1mg/mL，10 mg/mL）的上述物质分别与抗体同时加入到已包被封闭好的酶标板中。利用抗血清对双酚A的IC50值与抗血清对各类似物的IC50值的比值得到交叉反应率（CR%）,交叉反应测定结果如下：Cross-reactivity of bisphenol A structural analogues (bisphenolic acid, phenol, benzene, m-cresol, and hydroquinone) with antibody mixtures was determined by indirect competition ELISA. A series of concentrations (1 ng/mL, 10 ng/mL, 100 ng/mL, 1 ug/mL, 10 ug/mL, 100 ug/mL, 1 mg/mL, 10 mg/mL) of the above substances were mixed with the antibody at the same time Add to the coated and blocked microtiter plate. The ratio of the IC50 value of the antiserum to bisphenol A and the IC50 value of the antiserum to each analog was used to obtain the cross-reaction rate (CR%), and the cross-reaction determination results were as follows:
双酚A-100%，双酚酸-121%，苯酚、苯、间甲苯酚和对苯二酚均＜0.01。实验结果表明，该法对双酚A具有良好的特异性。Bisphenol A-100%, bisphenolic acid-121%, phenol, benzene, m-cresol and hydroquinone are all <0.01. The experimental results show that the method has good specificity for bisphenol A.
5、该试剂盒的使用方法5. How to use the kit
步骤如下：Proceed as follows:
（1）样品前处理；(1) Sample pretreatment;
水样处理：将水性样品配成10%甲醇-PBS溶液用于检测，例：取0.8mL水样，加0.1 mL的甲醇，再加0.1 mL浓缩后的10X PBS溶液。（如水样浑浊时，可15000rpm离心10分钟，取上清待测）。Water sample treatment: Prepare water-based samples into 10% methanol-PBS solution for detection, for example: take 0.8mL water sample, add 0.1 mL methanol, and add 0.1 mL concentrated 10X PBS solution. (If the water sample is turbid, it can be centrifuged at 15000rpm for 10 minutes, and the supernatant is taken for testing).
（2）使用试剂盒进行检测：(2) Use the kit for detection:
①从冰箱里取出试剂盒，室温复温，注意每种液体试剂使用前均须摇匀。取微孔板，预先标记空白对照孔、标准样品、样品的位置，推荐进行三孔平行检测。①Take out the kit from the refrigerator and rewarm at room temperature. Note that each liquid reagent must be shaken well before use. Take the microplate, and pre-mark the position of the blank control well, standard sample, and sample. It is recommended to perform three-hole parallel detection.
②向酶标条中，先加入50μL的各浓度BPA标准溶液，再加入50μL抗体工作液，混合均匀，在空白对照孔中加入100μL10%甲醇PBS缓冲液。②Add 50 μL of BPA standard solution of each concentration to the enzyme labeling strip, then add 50 μL of antibody working solution, mix well, and add 100 μL of 10% methanol PBS buffer to the blank control well.
③向酶标条中，先加入50μL样品溶液，再加入50μL抗体工作液，混合均匀，与②中的酶标条共同于37℃恒温温育40min，微孔板敷上薄膜。③ Add 50 μL of sample solution to the enzyme labeling strip first, then add 50 μL of antibody working solution, mix well, and incubate with the enzyme labeling strip in ② at 37°C for 40 minutes at a constant temperature, and cover the microwell plate with a film.
④温育完后，去掉薄膜将微孔中的溶液快速甩入水槽中，用洗涤液（10X浓缩液用蒸馏水稀释）清洗微孔板。④ After the incubation, remove the film and quickly throw the solution in the microwell into the sink, and wash the microplate with washing solution (10X concentrated solution diluted with distilled water).
⑤加入100μL酶标二抗工作液于全部孔中，37℃恒温温育40min。⑤ Add 100 μL enzyme-labeled secondary antibody working solution to all wells, and incubate at 37°C for 40 minutes.
⑥步骤同④，洗涤三次后于吸水纸拍干，每孔加入100μL底物溶液显色，37℃温育避光反应15min。⑥The procedure is the same as ④. After washing three times, pat dry on absorbent paper, add 100 μL of substrate solution to each well for color development, and incubate at 37°C for 15 minutes in the dark.
⑦每孔加50μL 终止液终止反应，摇匀，15分钟内读取光密度值。⑦ Add 50 μL of stop solution to each well to stop the reaction, shake well, and read the optical density value within 15 minutes.
⑧在酶标仪上测定双波长450nm和630nm处的光密度值（OD值）。⑧ Measure the optical density value (OD value) at dual wavelengths of 450nm and 630nm on a microplate reader.
6、双酚A ELISA试剂盒准确度测定6. Determination of the accuracy of bisphenol A ELISA kit
向自来水、珠江水水样中添加15 ng/mL、100 ng/mL和250 ng/mL的双酚A，重复三次，每次做三个平行，采用间接竞争ELISA法测定B/B0，计算回收率，结果分别为：109.2%，90.5%，95.9%和104.3%，87.6%及100%。Add 15 ng/mL, 100 ng/mL and 250 ng/mL bisphenol A to tap water and Pearl River water samples, repeat three times, do three parallels each time, use indirect competitive ELISA method to determine B/B0, calculate recovery rate, the results were: 109.2%, 90.5%, 95.9% and 104.3%, 87.6% and 100%.
| Application Number | Priority Date | Filing Date | Title |
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| CN201210540608.4ACN103048445B (en) | 2012-12-14 | 2012-12-14 | Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof |
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| CN201210540608.4ACN103048445B (en) | 2012-12-14 | 2012-12-14 | Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof |
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| CN201210540608.4AActiveCN103048445B (en) | 2012-12-14 | 2012-12-14 | Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof |
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