CN103048445B - Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof - Google Patents
Preparation method of bisphenol A-coated antigen with polylysine as carrier and application thereof Download PDFInfo
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- CN103048445B CN103048445B CN201210540608.4A CN201210540608A CN103048445B CN 103048445 B CN103048445 B CN 103048445B CN 201210540608 A CN201210540608 A CN 201210540608A CN 103048445 B CN103048445 B CN 103048445B
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- 239000000427 antigen Substances 0.000 title claims abstract description 59
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 108010039918 Polylysine Proteins 0.000 title abstract 4
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- 239000000243 solution Substances 0.000 claims abstract description 34
- 238000002965 ELISA Methods 0.000 claims abstract description 17
- VKOUCJUTMGHNOR-UHFFFAOYSA-N Diphenolic acid Chemical compound C=1C=C(O)C=CC=1C(CCC(O)=O)(C)C1=CC=C(O)C=C1 VKOUCJUTMGHNOR-UHFFFAOYSA-N 0.000 claims abstract description 15
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Abstract
The present invention discloses a preparation method of bisphenol A-coated antigen with polylysine as a carrier and application thereof, is aimed at providing the preparation method of bisphenol A-coated antigen with polylysine as the carrier which is simple in the synthetic method and the application thereof, and finally establishes an enzyme linked immunosorbent assay kit. The kit has the advantages of simple pre-treatment, fast speed and accuracy and low cost, and can be used for on-site testing in large scale. The technical points include: taking polylysine as a carrier protein, preparing bisphenol A-coated antigen by coupling with diphenolic acid, and applying the bisphenol A-coated antigen to prepare the bisphenol A enzyme linked immunosorbent assay kit, wherein the bisphenol A enzyme linked immunosorbent assay kit is composed of a solid phase carrier of envelope antigen, an antibody working solution, a bisphenol A standard solution, an enzyme labeled secondary antibody solution, an antibody diluent, a wash concentrated solution, a substrate solution and a stop solution. The preparation method belongs to the technical field of enzyme linked immunoassay.
Description
Technical field
The present invention relates to bisphenol-A envelope antigen preparation method and application thereof that poly-D-lysine is carrier, belong to Enzyme-multiplied immune technique field.
Background technology
Bisphenol-A (Bisphenol A, BPA) be 2, two (4-hydroxy phenyl) propane of 2-, imitates by being combined with estrogen receptor or affecting the modes such as cell signaling pathway or disturbs endogenous estrogen effect in vivo, irritant to the skin of people, respiratory tract, alimentary canal and cornea.Liver cell and nephrocyte will be destroyed when the bisphenol-A in body reaches a certain amount of, cause slow poisoning, make people occur the symptoms such as the uneasy and diarrhoea of giddy in various degree, headache, fash, spirit.In recent years, there is (Science. 2007 in the bisphenol-A all finding that there is estrogen similar effect all over the world in barreled drinking water, baby bottles and various water body, 317 (5840): 884-885), be not suitable with on-the-spot due to instrument analytical method and fast bisphenol-A in barreled drinking water and baby bottles detected, and immunoassay is based on the specific binding reaction between Ag-Ab, selectivity is good, by selecting suitable mark system, can realize detecting the rapid sensitive of trace materials in complex system.Enzyme-linked immune analytic method is highly sensitive, high specificity, quick, economical, be applicable to very much the residual selective mechanisms of BPA.
The enzyme-linked immune analytic method of research bisphenol-A, first must prepare the antibody of bisphenol-A.Bisphenol-A is micromolecular compound, and structure is simple, can not produce antibody by direct immunization animal, first itself and carrier protein coupling must be prepared comlete antigen, utilizes immunizing antigen to inject animal and brings out generation antibody.Method conventional at present utilizes classical carbodlimide method or glutaraldehyde method, selects bovine serum albumin(BSA) or ovalbumin to be that carrier carrys out synthesis of bisphenol A artificial antigen.Application number is in the patent of 200810234850.2, and petty official to be transmitted etc. and described with diphenolic acid is the method that envelope antigen is prepared in haptens and OVA coupling.Poly-l-lysine (PLL) is a kind of polypeptide of Prof. Du Yucang, autoantigenic is very poor but can increase haptenic immunity, and there is more free amino group, greatly can improve carrier protein and haptenic Conjugate ratio, so recent years is by the increasing preparation in order to artificial immunity antigen, if China Patent No. is for being the method that carrier prepares melamine complete antigen with poly-D-lysine disclosed in 200810051488.5; The Chinese patent patent No. 200610041918.6 provides the new synthetic method of ivermectin artificial antigen.But be showed no with poly-D-lysine is the patent that carrier prepares the artificial envelope antigen of bisphenol-A.
Summary of the invention
For above-mentioned deficiency, the object of the present invention is to provide a kind of synthetic method to be simply bisphenol-A envelope antigen preparation method and the application thereof of carrier with poly-D-lysine, and finally establish 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 take poly-D-lysine as bisphenol-A envelope antigen preparation method and the application thereof of carrier, take poly-D-lysine as carrier protein, prepares bisphenol-A envelope antigen with diphenolic acid coupling.
Bisphenol-A envelope antigen preparation method and the application thereof of above-mentioned with poly-D-lysine is carrier, comprise the steps: 1 successively) diphenolic acid is dissolved completely in organic solvent; 2) NHS (N-hydroxy-succinamide) and EDCHCL is dissolved completely in machine solvent respectively; 3) by step 2) obtained solution drops in the obtained solution of step 1), and room temperature lucifuge stirs 1-24h, and centrifuging is got supernatant and obtained A liquid; 4) poly-D-lysine is dissolved in phosphate buffer solution becomes B liquid; 5) A drop step 3) prepared adds in B liquid, and room temperature lucifuge stirs 1-24h, centrifuging, bag filter transferred to by supernatant, to dialyse 24-96h with phosphate buffer solution, every 12 h change a dislysate, finally dislysate are obtained the artificial envelope antigen of bisphenol-A and BVA-PLL through freeze drying;
Wherein: the mol ratio of diphenolic acid and NHS, EDCHCL, poly-D-lysine is: 30 ~ 200 ﹕ 30 ~ 400 ﹕ 30 ~ 400 ﹕ 1.
Further, bisphenol-A envelope antigen preparation method and the application thereof of above-mentioned with poly-D-lysine is carrier, described poly-D-lysine is that poly-l-lysine or poly d-lysine or poly-l-lysine and poly d-lysine are used in combination.
Further, bisphenol-A envelope antigen preparation method and the application thereof of above-mentioned with poly-D-lysine is carrier, it is characterized in that, step 1) and step 2) described in organic solvent be one of them of tetrahydrofuran or DMF or dimethyl sulfoxide (DMSO).
Further, bisphenol-A envelope antigen preparation method and the application thereof of above-mentioned with poly-D-lysine is carrier, the pH value of step 4) and the phosphate buffer solution described in step 5) is 6-8.
After of the present invention, a technical scheme is such: application bisphenol-A envelope antigen is as bisphenol-A enzyme-linked immunologic detecting kit, and described bisphenol-A enzyme-linked immunologic detecting kit is made up of the solid phase carrier of envelope antigen, antibody working fluid, bisphenol-A standard solution, ELIAS secondary antibody solution, antibody diluent, concentrated solution for washing, substrate solution and stop buffer;
Wherein: described solid phase carrier is the 96 hole polystyrene ELISA Plate being coated with diphenolic acid and poly-l-lysine conjugate, and enclose the site that micropore surface do 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 above-mentioned with poly-D-lysine the is bisphenol-A envelope antigen of carrier, it is characterized in that, described antibody working fluid is the antibody of rabbit source Anti-TNF-α bisphenol-A.
Further, the application of above-mentioned with poly-D-lysine the is bisphenol-A envelope antigen of carrier, it is characterized in that, described substrate solution is the pH5.0 phosphate citrate acid buffering solution containing tetramethyl benzidine and hydrogen peroxide; Described stop buffer is 2mol/L sulfuric acid solution.
Further, the application of above-mentioned with poly-D-lysine the is bisphenol-A envelope antigen of carrier, it 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 above-mentioned with poly-D-lysine the is bisphenol-A envelope antigen of carrier, it is characterized in that, described ELIAS secondary antibody solution is goat-anti rabbit-horseradish peroxidase.
Compared with prior art, tool of the present invention has the following advantages:
1, the advantage of antigen synthetic method: synthesis of the present invention take poly-D-lysine as carrier, poly-D-lysine wide material sources, low price is soluble in water; In organic solvent chemical coupling process, poly-D-lysine can keep its structural stability, not changeableness, and the envelope antigen after synthesis has solubility well; The number of amino groups that poly-D-lysine contains much larger than conventional albumin class material (as chicken ovalbumin, bovine serum albumin(BSA) etc.), these functional groups can with more hapten molecule coupling, and then add the Conjugate ratio of molecule, be conducive to coupling reaction; Poly-D-lysine structure is simple, effectively can reduce nonspecific reaction, improves the performance of kit; Conventional bag loaded body (chicken ovalbumin) belongs to biomacromolecule, have complicated three-dimensional structure, when the envelope antigen of its synthesis and ELISA Plate generation hydrophobic contact, easily there is deformation in envelope antigen, small haptens is shielded by macro-molecular protein, and then be unfavorable for hapten molecule presenting in ELISA Plate, but the poly-D-lysine of synthesis belongs to reticulate texture, overcome this shortcoming, can more be conducive to haptens to present in ELISA Plate, improve the specific recognition between envelope antigen and antibody;
2, by synthesis envelope antigen, and it is coated in polystyrene ELISA Plate by finite concentration, uses ovalbumin to close, prepared the elisa plate bar that can directly apply to enzyme-linked immuno assay; By the bisphenol-A antibody prepared, establish the kit using method measuring bisphenol A residues.
3, the present invention can be used for the detection of bisphenol-A in surface water, underground water and 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.
Accompanying drawing explanation
Fig. 1 is the ultraviolet spectrum of BVA, PLL and PLL-BVA
Fig. 2 is that indirect competitive ELISA method detects bisphenol-A Competitive assays curve;
Indirect competitive ELISA method mensuration is carried out, the standard working curve of foundation with the bisphenol-A of 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).
Embodiment
Below in conjunction with the drawings and specific embodiments, claim of the present invention is described in further details, but do not form any limitation of the invention, the amendment of anyone limited number of time made within the scope of the claims in the present invention, still in right of the present invention.
1, the synthesis of bisphenol-A artificial antigen
Embodiment 1
Taking diphenolic acid (BVA) 61mg is dissolved in 2mLDMF; Take 30.6mgNHS and 53.2mg EDCHCL again and be dissolved in 2mL(tetrahydrofuran respectively) in DMF, be slowly added drop-wise in BVA solution, after room temperature lucifuge stirs 24h, centrifuging is got supernatant and is obtained A liquid.
Take 126mg poly-l-lysine to be dissolved in the phosphate buffer solution of 5 mL pH=7 and to become B liquid, getting A liquid 4mL slowly drips in B liquid, room temperature lucifuge stirs 24h, centrifuging, bag filter transferred to by supernatant, to dialyse 72 h with the phosphate buffer solution of pH=7.4, every 12 h change a dislysate, finally dislysate is obtained the artificial envelope antigen of bisphenol-A and PLL-BVA through freeze drying, identify its structure through ultraviolet spectrum, consult Fig. 1.
Embodiment 2
Taking diphenolic acid (BVA) 20.7mg is dissolved in 1mLDMF; Take 6.6mgNHS and 14.2mg EDCHCL again and be dissolved in 1mL(tetrahydrofuran respectively) in DMF, be slowly added drop-wise in BVA solution, after room temperature lucifuge stirs 2h, centrifuging is got supernatant and is obtained A liquid.
Take 30.2mg poly mixing lysine to be dissolved in the phosphate buffer solution of 5 mL pH=7 and to become B liquid, getting A liquid 3mL slowly drips in B liquid, room temperature lucifuge stirs 2h, centrifuging, bag filter transferred to by supernatant, to dialyse 72 h with the phosphate buffer solution of pH=7.4, every 12 h change a dislysate, finally dislysate is obtained the artificial envelope antigen of bisphenol-A and PLL-BVA through freeze drying, identify its structure through ultraviolet spectrum, consult Fig. 1.
Embodiment 3
Taking diphenolic acid (BVA) 24mg is dissolved in 1mLDMF; Taking 10mg NHS again and 17.2mg EDCHCL is dissolved in 1mL(tetrahydrofuran respectively) in DMF, be slowly added drop-wise in BVA solution, after room temperature lucifuge stirs 12h, centrifuging is got supernatant and is obtained A liquid.
Take 15mg poly-l-lysine to be dissolved in the phosphate buffer solution of 5 mL pH=7 and to become B liquid, getting A liquid 2mL slowly drips in B liquid, room temperature lucifuge stirs 12h, centrifuging, bag filter transferred to by supernatant, to dialyse 72 h with the phosphate buffer solution of pH=7.4, every 12 h change a dislysate, finally dislysate is obtained the artificial envelope antigen of bisphenol-A and PLL-BVA through freeze drying, identify its structure through ultraviolet spectrum, consult Fig. 1.
Selection bovine serum albumin(BSA) is carrier, utilizes same method and diphenolic acid coupling to prepare immunizing antigen.
2, the antibody preparation of anti-bisphenol A
With PBS lytic immunity antigen BSA-BVA, be mixed with 1mg/mL solution.Get above-mentioned solution 0.5mL and equal-volume Freund's complete adjuvant is emulsified into Water-In-Oil state, immunity is carried out to 2-3kg Female New Zealand rabbit.First time, immunity was after one month, used Freund's incomplete adjuvant immunity in every two weeks instead once, immunity 5 times, and last immunity got blood purifying after 15 days.
Indirect elisa method measures tiring of immune serum.(1) bag quilt: be buffered liquid CBS with bag and envelope antigen (PLL-BVA) is diluted to finite concentration, joins in 96 hole ELISA Plate with 100 μ L/ holes, and 4 DEG C of bags are spent the night, and dries liquid in hole, and once, thieving paper pats dry in PBST washing.(2) close: 30mg/mL ovalbumin (PBS dissolving) 200 μ L/ hole joins in hole, 37 DEG C of incubation 1h.(3) dry liquid in hole, PBST washs three times, and thieving paper pats dry.(4) immune serum is added: dilute immune serum with PBS, certain concentration doubling dilution, 100 μ L/ holes are added in hole, establish negative control and each 2 holes of blank simultaneously.After 37 DEG C of incubation 1h, washing, pats dry.(5) add ELIAS secondary antibody: dilute ELIAS secondary antibody to certain concentration with PBS, 100 μ L/ holes are added in hole, after 37 DEG C of incubation 1h, washing, pats dry.(6) colour developing and termination: preparation substrate buffer solution 10mL, adds 10 μ L 30% hydrogen peroxide, fully mix.100 μ L/ holes are added in hole after incubation 15min, 2M sulfuric acid 50 μ L/ hole cessation reaction.(7) reading: the optical density value (OD value) measuring dual wavelength 490nm and 630nm place by microplate reader.If sample well OD value is more than or equal to 2.1 times of blank control wells and is the positive.
Checkerboard titration method screens suitable envelope antigen concentration and optimum antibody working concentration, adopts indirect competitive ELISA method to detect the Competitive assays rate of immune serum.
By animal immune and Virus monitory, obtain the antibody of anti-bisphenol A.
3, the foundation of bisphenol-A ELISA standard working curve and the determination of detectability
Wrap by 96 hole ELISA Plate with the PLL-BVA of suitable concentration, 100 μ L/ holes, 4 DEG C of refrigerator bags are spent the night, and after closing with confining liquid, washing pats dry; To in enzyme mark bar, first add each concentration BPA standard solution (0ng/mL, 3ng/mL, the 8ng/mL of 50 μ L, 25ng/mL, 75ng/mL, 224ng/mL, 673ng/mL, 2018ng/mL), add the antibody of 50 μ L suitable concentrations again, mix, in blank control wells, add 100 μ L10% methyl alcohol PBS damping fluids; 37 DEG C of constant temperature incubation 40min; Wash three times, pat dry; Add 100 μ L ELIAS secondary antibody working fluids, 37 DEG C of constant temperature incubation 40min; Wash three times, pat dry; Every hole adds 100 μ L substrate solution colour developings, adds 50 μ L stop buffer cessation reactions, shake up, read optical density value in 15 minutes after 37 DEG C of incubation lucifuge reaction 15min.
With inhibiting rate B/B
0for ordinate, bisphenol-A concentration BPA (ngmL
-1) make typical curve, its IC for horizontal ordinate
50for 14.5ng/ml, lowest detection is limited to 0.5ng/ml, consults Fig. 2.
4, the specific mensuration of bisphenol-A ELISA kit
Indirect competitive ELISA method is adopted 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 ELISA Plate of having wrapped and being closed with antibody respectively simultaneously.Utilize antiserum to the IC of bisphenol-A
50value and antiserum are to the IC of each analog
50the ratio of value obtains cross reacting rate (CR%), and cross reaction measurement result is as follows:
Bisphenol-A-100%, diphenolic acid-121%, the equal < 0.01 of phenol, benzene, m-cresol and p-dihydroxy-benzene.Experimental result shows, 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 process: aqueous sample is made into 10% methyl alcohol-PBS solution be used for detect, example: get 0.8 mL water sample, add the methyl alcohol of 0.1 mL, then add 0.1 mL concentrate after 10X PBS solution.(time as muddy in water sample, can centrifugal 10 minutes of 15000rpm, get supernatant to be measured).
(2) kit is used to detect:
1. from refrigerator, take out kit, room temperature rewarming, notice that often kind of liquid reagent must shake up before using.Get microwell plate, mark the position of blank control wells, standard model, sample in advance, recommend to carry out three hole Parallel testings.
2. in enzyme mark bar, first add each concentration BPA standard solution of 50 μ L, then add 50 μ L antibody working fluids, mix, in blank control wells, add 100 μ L10% methyl alcohol PBS damping fluids.
3. in enzyme mark bar, first add 50 μ L sample solutions, then add 50 μ L antibody working fluids, mix, with 2. in enzyme mark bar jointly in 37 DEG C of constant temperature incubation 40min, microwell plate applies film.
4. incubation complete after, remove film and the solution in micropore got rid of in tank fast, clean microwell plate with cleansing solution (10X concentrate distilled water diluting).
5. 100 μ L ELIAS secondary antibody working fluids are added in all well, 37 DEG C of constant temperature incubation 40min.
6. step is with 4., pats dry after washing three times in thieving paper, and every hole adds 100 μ L substrate solution colour developings, 37 DEG C of incubation lucifuge reaction 15min.
7. every hole adds 50 μ L stop buffer cessation reactions, shakes up, reads optical density value in 15 minutes.
8. in microplate reader, measure the optical density value (OD value) at dual wavelength 450nm and 630nm place.
6, bisphenol-A ELISA kit accuracy determination
In tap water, Pearl River water water sample, add the bisphenol-A of 15 ng/mL, 100 ng/mL and 250 ng/mL, in triplicate, do at every turn three parallel, adopt indirect competitive ELISA method to measure B/B0, calculate the recovery, result is respectively: 109.2%, 90.5%, 95.9% and 104.3%, 87.6% and 100%.
Claims (9)
1. be a bisphenol-A envelope antigen preparation method for carrier with poly-D-lysine, it is characterized in that, take poly-D-lysine as carrier protein, prepare bisphenol-A envelope antigen with diphenolic acid coupling;
The method comprises the steps: 1 successively) diphenolic acid is dissolved completely in organic solvent; 2) NHS and EDCHCL is dissolved completely in respectively in machine solvent; 3) by step 2) obtained solution drops to step 1) in obtained solution, room temperature lucifuge stirs 1-24h, and centrifuging is got supernatant and obtained A liquid; 4) poly-D-lysine is dissolved in phosphate buffer solution becomes B liquid; 5) by step 3) the A drop prepared adds in B liquid, room temperature lucifuge stirs 1-24h, centrifuging, bag filter transferred to by supernatant, to dialyse 24-96h with phosphate buffer solution, every 12h changes a dislysate, finally dislysate is obtained the artificial envelope antigen of bisphenol-A and BVA-PLL through freeze drying;
Wherein: the mol ratio of diphenolic acid and NHS, EDCHCL, poly-D-lysine is: 30 ~ 200 ﹕ 30 ~ 400 ﹕ 30 ~ 400 ﹕ 1.
2. according to claim 1 take poly-D-lysine as the bisphenol-A envelope antigen preparation method of carrier, it is characterized in that, described poly-D-lysine is that poly-l-lysine or poly d-lysine or poly-l-lysine and poly d-lysine are used in combination.
3. according to claim 1 take poly-D-lysine as the bisphenol-A envelope antigen preparation method of carrier, it is characterized in that, step 1) and step 2) described in organic solvent be one of them of tetrahydrofuran or DMF or dimethyl sulfoxide (DMSO).
4. according to claim 1 take poly-D-lysine as the bisphenol-A envelope antigen preparation method of carrier, it is characterized in that, step 4) and step 5) described in the pH value of phosphate buffer solution be 6-8.
5. according to claim 1 take poly-D-lysine as the application of the bisphenol-A envelope antigen of carrier, it is characterized in that, application bisphenol-A envelope antigen is as bisphenol-A enzyme-linked immunologic detecting kit, and described bisphenol-A enzyme-linked immunologic detecting kit is made up of the solid phase carrier of envelope antigen, antibody working fluid, bisphenol-A standard solution, ELIAS secondary antibody solution, antibody diluent, concentrated solution for washing, substrate solution and stop buffer;
Wherein: described solid phase carrier is the 96 hole polystyrene ELISA Plate being coated with diphenolic acid and poly-l-lysine conjugate, and enclose the site that micropore surface do 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.
6. according to claim 5 take poly-D-lysine as the application of the bisphenol-A envelope antigen of carrier, and it is characterized in that, described antibody working fluid is the antibody of rabbit source Anti-TNF-α bisphenol-A.
7. according to claim 5 take poly-D-lysine as the application of the bisphenol-A envelope antigen of carrier, and it is characterized in that, described substrate solution is the pH5.0 phosphate citrate acid buffering solution containing tetramethyl benzidine and hydrogen peroxide; Described stop buffer is 2mol/L sulfuric acid solution.
8. according to claim 5 take poly-D-lysine as the application of the bisphenol-A envelope antigen of carrier, and it 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.
9. according to claim 5 take poly-D-lysine as the application of the bisphenol-A envelope antigen of carrier, and it is characterized in that, described ELIAS secondary antibody solution is goat-anti rabbit-horseradish peroxidase.
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| CN104101702B (en) * | 2014-07-04 | 2016-04-20 | 中国海洋大学 | A kind of indirect competitive enzyme-linked immunosorbent detection method of tetrabromobisphenol A |
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| CN106153928A (en) * | 2016-07-06 | 2016-11-23 | 广东工业大学 | A kind of bisphenol-A immunity test strip containing bisphenol-A immunological probe and application thereof |
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| CN111239383A (en) * | 2020-01-23 | 2020-06-05 | 武汉伊莱瑞特生物科技股份有限公司 | Coating liquid and application thereof in ELISA |
| CN111521775A (en) * | 2020-04-14 | 2020-08-11 | 天津科技大学 | Method for preparing paper-based micro-fluidic chip for bisphenol A detection based on wax-spraying printing technology |
| CN112213481A (en) * | 2020-08-13 | 2021-01-12 | 茅台学院 | Hapten directly coated bisphenol A ABC enzyme-linked immunosorbent assay method based on monoclonal antibody |
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