CN103048445A - Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开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及其应用，旨在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，该试剂盒具有前处理简单、快速准确、成本低廉，可用于现场大批量检测；其技术要点是：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；应用双酚A包被抗原制备双酚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；属于酶联免疫技术领域。

The invention discloses a method for preparing bisphenol A coated antigen with polylysine as a carrier and its application and application, aiming to provide a bisphenol A with polylysine as a carrier with a simple synthesis method Coated antigen preparation method and its application, and finally established an enzyme-linked immunosorbent assay kit for bisphenol A detection. The kit has the advantages of simple pretreatment, fast accuracy, low cost, and can be used for on-site large-scale detection; its technical points are: Using polylysine as a carrier protein, coupled with bisphenol acid to prepare bisphenol A coated antigen; using bisphenol A coated antigen to prepare a bisphenol A enzyme-linked immunoassay kit, the bisphenol A enzyme-linked The immunoassay kit consists of a solid-phase carrier coated with antigen, antibody working solution, bisphenol A standard solution, enzyme-labeled secondary antibody solution, antibody diluent, washing concentrate, substrate solution and stop solution; it belongs to enzyme-linked immunosorbent technology field.

Description

Bisphenol-A envelope antigen preparation method and application thereof take poly-D-lysine as carrier

Technical field

The present invention relates to poly-D-lysine is bisphenol-A envelope antigen preparation method and the application thereof of carrier, belongs to the Enzyme-multiplied immune technique field.

Background technology

Bisphenol-A (Bisphenol A, BPA) be 2, two (4-hydroxy phenyl) propane of 2-imitate or disturb endogenous estrogen action by being combined with estrogen receptor or affecting the mode such as cellular signal transduction approach in vivo, and Person's skin, respiratory tract, alimentary canal and cornea are had pungency.When the bisphenol-A in the body reaches a certain amount of, will destroy liver cell and nephrocyte, cause slow poisoning, make the people in various degree symptoms such as giddy, headache, fash, spiritual uneasiness and diarrhoea occur.In recent years, the bisphenol-A of all finding the estrogen similar effect all over the world in barreled drinking water, baby bottles and the various water body exists that (Science. 2007,317 (5840): 884-885), because instrument analytical method is not suitable with on-the-spot and fast bisphenol-A in barreled drinking water and the baby bottles is detected, and immunoassay reacts based on the specific binding between Ag-Ab, selectivity is good, by selecting suitable mark system, can realize the rapid sensitive of trace materials in the complex system is detected.Enzyme-linked immune analytic method is highly sensitive, high specificity, quick, economic, is fit to very much the residual screening of BPA and detects.

Study the enzyme-linked immune analytic method of bisphenol-A, at first must prepare the antibody of bisphenol-A.Bisphenol-A is micromolecular compound, and is simple in structure, can not produce antibody by the direct immunization animal, must first itself and carrier protein coupling be prepared comlete antigen, utilizes immunizing antigen injection animal to bring out generation antibody.Method commonly used is to utilize classical carbodlimide method or glutaraldehyde method at present, and selecting bovine serum albumin(BSA) or ovalbumin is that carrier comes the synthesis of bisphenol A artificial antigen.Application number is in 200810234850.2 the patent, and the petty official is transmitted etc. and to have been introduced take diphenolic acid as haptens and the OVA coupling prepares the method for envelope antigen.Poly-l-lysine (PLL) is a kind of artificial synthetic polypeptide, autoimmunity originality is very poor but can increase haptenic immunity, and has more free amino group, can greatly improve carrier protein and haptenic coupling rate, so recent years is the disclosed method for preparing melamine complete antigen take poly-D-lysine as carrier in 200810051488.5 by in the increasing preparation in order to artificial immunity antigen such as China Patent No.; The Chinese patent patent No. 200610041918.6 provides the new synthetic method of ivermectin artificial antigen.But be showed no the patent for preparing the artificial envelope antigen of bisphenol-A take poly-D-lysine as carrier.

Summary of the invention

For above-mentioned deficiency, the object of the present invention is to provide simply bisphenol-A envelope antigen preparation method and the application thereof take poly-D-lysine as carrier of a kind of synthetic method, and finally set up the enzyme linked immunological kit that bisphenol-A detects.

For solving the problems of the technologies described above, last technical scheme of the present invention is such: a kind of bisphenol-A envelope antigen preparation method and application thereof take poly-D-lysine as carrier take poly-D-lysine as carrier protein, prepares the bisphenol-A envelope antigen with the diphenolic acid coupling.

Above-mentioned bisphenol-A envelope antigen preparation method and application thereof take poly-D-lysine as carrier comprise the steps: 1 successively) be dissolved in diphenolic acid in the organic solvent fully; 2) be dissolved in respectively NHS (N-hydroxy-succinamide) and EDCHCL in the machine solvent fully; 3) with step 2) solution that makes drops in the solution that step 1) makes, and the room temperature lucifuge stirs 1-24h, and centrifuging is got supernatant and is got A liquid; 4) poly-D-lysine is dissolved in one-tenth B liquid in the phosphate buffer solution; 5) the A drop with the step 3) preparation adds in the B liquid, and the room temperature lucifuge stirs 1-24h, centrifuging, supernatant is transferred to bag filter, with the phosphate buffer solution 24-96h that dialyses, per 12 h change dislysate one time, and at last dislysate being obtained the artificial envelope antigen of bisphenol-A through freeze drying is BVA-PLL;

Wherein: the mol ratio of diphenolic acid and NHS, EDCHCL, poly-D-lysine is: 30～200 ﹕, 30～400 ﹕, 30～400 ﹕ 1.

Further, above-mentioned bisphenol-A envelope antigen preparation method and application thereof take poly-D-lysine as carrier, described poly-D-lysine is poly-l-lysine or poly dextrorotation lysine or poly-l-lysine and poly dextrorotation lysine mixing use.

Further, above-mentioned bisphenol-A envelope antigen preparation method and application thereof take poly-D-lysine as carrier, it is characterized in that step 1) and step 2) described organic solvent is one of them of tetrahydrofuran or DMF or dimethyl sulfoxide (DMSO).

Further, above-mentioned bisphenol-A envelope antigen preparation method and application thereof take poly-D-lysine as carrier, the pH value of step 4) and the described phosphate buffer solution of step 5) is 6-8.

A technical scheme is such after of the present invention: use the bisphenol-A envelope antigen as the bisphenol-A enzyme-linked immunologic detecting kit, described bisphenol-A enzyme-linked immunologic detecting kit is comprised of solid phase carrier, antibody working fluid, bisphenol-A standard solution, ELIAS secondary antibody solution, antibody diluent, concentrated solution for washing, substrate solution and the stop buffer of envelope antigen;

Wherein: described solid phase carrier is the 96 hole polystyrene ELISA Plate that are coated with diphenolic acid and poly-l-lysine conjugate, and has sealed the site that micropore surface does not adsorb bisphenol-A antigen; 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.

Further, the application of the above-mentioned bisphenol-A envelope antigen take poly-D-lysine as carrier is characterized in that described antibody working fluid is the antibody of rabbit source Anti-TNF-α bisphenol-A.

Further, the application of the above-mentioned bisphenol-A envelope antigen take poly-D-lysine as carrier is characterized in that, described substrate solution is the pH5.0 phosphoric acid citric acid solution that contains tetramethyl benzidine and hydrogen peroxide; Described stop buffer is the 2mol/L sulfuric acid solution.

Further, the application of the above-mentioned bisphenol-A envelope antigen take poly-D-lysine as carrier is characterized in that, described concentrated solution for washing is that 10X contains 5% Tween-20, pH7.0, and concentration is the phosphate buffer of 0.01mol/L.

Further, the application of the above-mentioned bisphenol-A envelope antigen take poly-D-lysine as carrier is characterized in that described ELIAS secondary antibody solution is goat-anti rabbit-horseradish peroxidase.

Compared with prior art, the present invention has following advantage:

1, the advantage of antigen synthetic method: of the present invention synthetic take poly-D-lysine as carrier, the poly-D-lysine wide material sources, low price, soluble in water; In organic solvent chemical coupling process, poly-D-lysine can keep its structural stability, changeableness not, and the envelope antigen after synthetic has well solubility; The number of amino groups that contains on the poly-D-lysine much larger than albumin class material commonly used (such as chicken ovalbumin, bovine serum albumin(BSA) etc.), these functional groups can with more hapten molecule coupling, and then increased the coupling rate of molecule, be conducive to coupling reaction; Poly-D-lysine is simple in structure, can effectively reduce nonspecific reaction, improves the performance of kit; Coated carrier (chicken ovalbumin) commonly used belongs to biomacromolecule, have complicated three-dimensional structure, when its synthetic envelope antigen contacts with ELISA Plate generation hydrophobicity, deformation easily occurs in envelope antigen, little molecule haptens is shielded by macro-molecular protein, and then be unfavorable for hapten molecule presenting on ELISA Plate, yet synthetic poly-D-lysine belongs to reticulate texture, overcome this shortcoming, can more be conducive to haptens and present in ELISA Plate, improve the specific recognition between envelope antigen and the antibody;

2, be coated on the polystyrene ELISA Plate by finite concentration by synthetic envelope antigen, and with it, use the ovalbumin sealing, prepared the elisa plate bar that can directly apply to enzyme-linked immuno assay; By the bisphenol-A antibody for preparing, set up the kit using method of measuring bisphenol A residues.

3, the present invention can be used for the detection of bisphenol-A in surface water, underground water and the potable water, have pre-treatment simply, quick and precisely, with low cost, can be used for the advantages such as on-the-spot mass detection, and lowest detection is limited to 0.5ng/ml.

Description of drawings

Fig. 1 is the ultraviolet spectrum of BVA, PLL and PLL-BVA

Fig. 2 is that the indirect competitive ELISA method detects bisphenol-A competition inhibition curve;

Bisphenol-A with series concentration (2048ng/mL, 512ng/mL, 128ng/mL, 32ng/mL, 8ng/mL, 2ng/mL, 0.5ng/mL, 0.125ng/mL and 0.0312 ng/mL) carries out indirect competitive ELISA method mensuration, the standard working curve of foundation.

Embodiment

Below in conjunction with the drawings and specific embodiments claim of the present invention is described in further details, but do not consist of any limitation of the invention, the modification of the limited number of time that anyone makes in claim scope of the present invention is still in claim scope of the present invention.

1, the bisphenol-A artificial antigen is synthetic

Embodiment 1

Taking by weighing diphenolic acid (BVA) 61mg is dissolved among the 2mLDMF; Take by weighing again 30.6mgNHS and 53.2mg EDCHCL is dissolved in respectively the 2mL(tetrahydrofuran) among the DMF, slowly be added drop-wise in the BVA solution, after the room temperature lucifuge stirred 24h, centrifuging was got supernatant and is got A liquid.

Take by weighing and become B liquid in the phosphate buffer solution that the 126mg poly-l-lysine is dissolved in 5 mL pH=7, getting A liquid 4mL slowly drips in the B liquid, the room temperature lucifuge stirs 24h, centrifuging, supernatant is transferred to bag filter, with the phosphate buffer solution of pH=7.4 72 h that dialyse, per 12 h change dislysate one time, at last dislysate being obtained the artificial envelope antigen of bisphenol-A through freeze drying is PLL-BVA, identifies its structure through ultraviolet spectrum, consults Fig. 1.

Embodiment 2

Taking by weighing diphenolic acid (BVA) 20.7mg is dissolved among the 1mLDMF; Take by weighing again 6.6mgNHS and 14.2mg EDCHCL is dissolved in respectively the 1mL(tetrahydrofuran) among the DMF, slowly be added drop-wise in the BVA solution, after the room temperature lucifuge stirred 2h, centrifuging was got supernatant and is got A liquid.

Take by weighing and become B liquid in the phosphate buffer solution that 30.2mg poly mixing lysine is dissolved in 5 mL pH=7, getting A liquid 3mL slowly drips in the B liquid, the room temperature lucifuge stirs 2h, centrifuging, supernatant is transferred to bag filter, with the phosphate buffer solution of pH=7.4 72 h that dialyse, per 12 h change dislysate one time, at last dislysate being obtained the artificial envelope antigen of bisphenol-A through freeze drying is PLL-BVA, identifies its structure through ultraviolet spectrum, consults Fig. 1.

Embodiment 3

Taking by weighing diphenolic acid (BVA) 24mg is dissolved among the 1mLDMF; Take by weighing again 10mg NHS and 17.2mg EDCHCL is dissolved in respectively the 1mL(tetrahydrofuran) among the DMF, slowly be added drop-wise in the BVA solution, after the room temperature lucifuge stirred 12h, centrifuging was got supernatant and is got A liquid.

Take by weighing and become B liquid in the phosphate buffer solution that the 15mg poly-l-lysine is dissolved in 5 mL pH=7, getting A liquid 2mL slowly drips in the B liquid, the room temperature lucifuge stirs 12h, centrifuging, supernatant is transferred to bag filter, with the phosphate buffer solution of pH=7.4 72 h that dialyse, per 12 h change dislysate one time, at last dislysate being obtained the artificial envelope antigen of bisphenol-A through freeze drying is PLL-BVA, identifies its structure through ultraviolet spectrum, consults Fig. 1.

The selection bovine serum albumin(BSA) is carrier, utilizes to prepare immunizing antigen with method and diphenolic acid coupling.

2, the antibody of anti-bisphenol A preparation

With PBS lytic immunity antigen BSA-BVA, be mixed with 1mg/mL solution.Get mentioned solution 0.5mL and the equal-volume Freund's complete adjuvant is emulsified into the Water-In-Oil state, the female new zealand rabbit of 2-3kg is carried out immunity.For the first time immunity was used Freunds incomplete adjuvant per two all immunity instead once after one month, immunity 5 times, and last immunity was got the blood purifying after 15 days.

Indirect elisa method is measured tiring of immune serum.(1) coated: with coated damping fluid CBS envelope antigen (PLL-BVA) is diluted to finite concentration, joins in the 96 hole ELISA Plate with 100 μ L/ holes, 4 ℃ of coated spending the night dry liquid in the hole, and PBST washs once, and thieving paper pats dry.(2) sealing: 30mg/mL ovalbumin (PBS dissolving) 200 μ L/ holes join in the hole 37 ℃ of incubation 1h.(3) dry liquid in the hole, PBST washing three times, thieving paper pats dry.(4) add immune serum: with PBS dilution immune serum, the certain concentration doubling dilution, 100 μ L/ holes are added in the hole, establish simultaneously each 2 hole of negative control and blank.Behind 37 ℃ of incubation 1h, washing pats dry.(5) add ELIAS secondary antibody: to certain concentration, 100 μ L/ holes are added in the hole with PBS dilution ELIAS secondary antibody, and behind 37 ℃ of incubation 1h, washing pats dry.(6) colour developing and termination: preparation substrate buffer solution 10mL adds 10 μ L, 30% hydrogen peroxide, fully mixing.100 μ L/ holes are added in the hole behind the incubation 15min, 2M sulfuric acid 50 μ L/ hole cessation reactions.(7) reading: the optical density value (OD value) of measuring dual wavelength 490nm and 630nm place with microplate reader.If sample well OD value is namely positive more than or equal to 2.1 times of blank hole.

Envelope antigen concentration and optimum antibody working concentration that the screening of chessboard titrimetry is suitable adopt the indirect competitive ELISA method to detect the competition inhibiting rate of immune serum.

Detect by animal immune and serum, obtain the antibody of anti-bisphenol A.

3, the foundation of bisphenol-A ELISA standard working curve and detectability determines

With the coated 96 hole ELISA Plate of the PLL-BVA of suitable concentration, 100 μ L/ holes, 4 ℃ of coated spending the night of refrigerator pat dry with washing after the confining liquid sealing; In enzyme mark bar, each concentration BPA standard solution (0ng/mL, 3ng/mL, 8ng/mL of adding first 50 μ L, 25ng/mL, 75ng/mL, 224ng/mL, 673ng/mL, 2018ng/mL), add again the antibody of 50 μ L suitable concentrations, mix, in the blank hole, add 100 μ L10% methyl alcohol PBS damping fluids; 37 ℃ of constant temperature incubation 40min; Wash three times, pat dry; Add 100 μ L ELIAS secondary antibody working fluids, 37 ℃ of constant temperature incubation 40min; Wash three times, pat dry; Every hole adds the colour developing of 100 μ L substrate solutions, adds 50 μ L stop buffer cessation reactions behind 37 ℃ of incubation lucifuge reaction 15min, shakes up, and reads optical density value in 15 minutes.

With inhibiting rate B/B₀Be ordinate, bisphenol-A concentration BPA (ngmL^-1) make typical curve, its IC for horizontal ordinate₅₀Be 14.5ng/ml, lowest detection is limited to 0.5ng/ml, consults Fig. 2.

4, the specific mensuration of bisphenol-A ELISA kit

Adopt the indirect competitive ELISA method to measure the cross reaction of structure of bisphenol A analog (diphenolic acid, phenol, benzene, m-cresol and p-dihydroxy-benzene) and mixtures of antibodies.The above-mentioned substance of series concentration (1 ng/mL, 10 ng/mL, 100 ng/mL, 1 ug/mL, 10 ug/mL, 100 ug/mL, 1mg/mL, 10 mg/mL) is joined in the good ELISA Plate of coated sealing simultaneously with antibody respectively.Utilize antiserum to the IC of bisphenol-A₅₀Value and the IC of antiserum to each analog₅₀The ratio of value obtains cross reacting rate (CR%), and the cross reaction measurement result is as follows:

Bisphenol-A-100%, diphenolic acid-121%, phenol, benzene, m-cresol and p-dihydroxy-benzene are all＜0.01.Experimental result shows that this method has good specificity to bisphenol-A.

5, the using method of this kit

Step is as follows:

(1) sample pre-treatments;

Water sample is processed: aqueous sample is made into 10% methyl alcohol-PBS solution for detection of, example: get 0.8 mL water sample, add the methyl alcohol of 0.1 mL, add the 10X PBS solution of 0.1 mL after concentrated again.(when muddy such as water sample, but centrifugal 10 minutes of 15000rpm, it is to be measured to get supernatant).

(2) use kit to detect:

1. take out kit in refrigerator, the room temperature rewarming is noted must shaking up before every kind of liquid reagent uses.Get microwell plate, three hole Parallel testings are recommended to carry out in the position of the blank control wells of mark, standard model, sample in advance.

2. in enzyme mark bar, add first each concentration BPA standard solution of 50 μ L, add again 50 μ L antibody working fluids, mix, in the blank hole, add 100 μ L10% methyl alcohol PBS damping fluids.

3. in enzyme mark bar, add first 50 μ L sample solutions, add again 50 μ L antibody working fluids, mix, with 2. in enzyme mark bar jointly in 37 ℃ of constant temperature incubation 40min, microwell plate applies film.

4. incubation complete after, remove film the solution in the micropore got rid of in the tank fast, with cleansing solution (10X concentrate distilled water diluting) cleaning microwell plate.

5. add 100 μ L ELIAS secondary antibody working fluids in whole holes, 37 ℃ of constant temperature incubation 40min.

6. step is with 4., pats dry in thieving paper after wash three times, and every hole adds the colour developing of 100 μ L substrate solutions, and 37 ℃ of incubation lucifuges are reacted 15min.

7. every hole adds 50 μ L stop buffer cessation reactions, shakes up, and reads optical density value in 15 minutes.

8. measure the optical density value (OD value) at dual wavelength 450nm and 630nm place in microplate reader.

6, bisphenol-A ELISA kit accuracy determination

Add the bisphenol-A of 15 ng/mL, 100 ng/mL and 250 ng/mL in tap water, the Pearl River water water sample, triplicate, do at every turn three parallel, adopt the indirect competitive ELISA method to measure B/B0, calculate recovery rate, the result is respectively: 109.2%, 90.5%, 95.9% and 104.3%, 87.6% and 100%.

Claims (10)

Translated fromChinese

1.一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，其特征在于，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。1. A method for preparing a bisphenol A-coated antigen with polylysine as a carrier and its application, characterized in that, polylysine is used as a carrier protein, coupled with bisphenol Acid to prepare bisphenol A coated antigen Antigen.2.根据权利要求1所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，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；2. The preparation method and application of bisphenol A-coated antigen with polylysine as a carrier according to claim 1, characterized in that it comprises the following steps in sequence: 1) completely dissolving bisphenol-acid in organic 2) Dissolve NHS and EDC·HCL in the organic solvent completely; 3) Add the solution prepared in step 2) dropwise to the solution prepared in step 1), stir at room temperature for 1-24 hours in the dark, and centrifuge Separate and take the supernatant to obtain liquid A; 4) Dissolve polylysine in phosphate buffer solution to form liquid B; 5) Add liquid A prepared in step 3) to liquid B dropwise, and stir at room temperature for 1-24 hours in the dark , centrifuged, the supernatant was transferred to a dialysis bag, dialyzed with phosphate buffer solution for 24-96 hours, and the dialysate was changed every 12 hours, and finally the dialysate was freeze-dried to obtain the artificially coated antigen of bisphenol A, 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: 30-200:30-400:30-400:1.3.根据权利要求1或2所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，其特在在于，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。3. The method for preparing bisphenol A coated antigen with polylysine as carrier and its application according to claim 1 or 2, characterized in that said polylysine is poly-L-lysine Amino acid or poly-D-lysine or a mixture of poly-L-lysine and poly-D-lysine.4.根据权利要求2所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，其特在在于，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。4. The preparation method and application of bisphenol A-coated antigen with polylysine as a carrier according to claim 2, which is characterized in that the organic solvent described in step 1) and step 2) is tetrahydrofuran or One of N,N-dimethylformamide or dimethyl sulfoxide.5.根据权利要求2所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，其特在在于，步骤4）和步骤5）所述的磷酸缓冲溶液的pH值为6-8。5. The method for preparing bisphenol A-coated antigen with polylysine as a carrier and its application according to claim 2, which is characterized in that the pH of the phosphate buffer solution described in step 4) and step 5) The value is 6-8.6.权利要求1所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，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；6. the application of the bisphenol A coated antigen with polylysine as the carrier according to claim 1 is characterized in that, the bisphenol A coated antigen is used as the bisphenol A enzyme-linked immunoassay kit, said The bisphenol A ELISA kit is composed of solid phase carrier coated with 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.7.根据权利要求6所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。7. The application of bisphenol A coated antigen with polylysine as carrier according to claim 6, characterized in that, the antibody working solution is polyclonal anti-bisphenol A antibody derived from rabbits.8.根据权利要求6所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。8. the application of the bisphenol A coated antigen with polylysine as carrier according to claim 6, is characterized in that, described substrate liquid is the pH5 that contains tetramethylbenzidine and hydrogen peroxide .0 phosphoric acid citrate buffer solution; the stop solution is 2mol/L sulfuric acid solution.9.根据权利要求6所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。9. the application of the bisphenol A coated antigen taking polylysine as carrier according to claim 6, is characterized in that, described washing concentrate is that 10X contains 5% Tween-20, pH7.0 , a concentration of 0.01mol/L phosphate buffer.10.根据权利要求6所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。10. The application of bisphenol A coated antigen with polylysine as carrier according to claim 6, characterized in that, the enzyme-labeled secondary antibody solution is goat anti-rabbit-horseradish peroxidase .

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