Heavy and light chain variable regions of a bisphenol a monoclonal antibody and uses thereof

Abstract

The application discloses a bisphenol A monoclonal antibody and application thereof, and belongs to the technical field of biology. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region are both composed of a complementarity determining region and a framework region, and the complementarity determining region is composed of CDR1, CDR2 and CDR3. The monoclonal antibody can be used for preparing a bisphenol A detection product. The bisphenol A colloidal gold detection test strip provided by the application has the characteristics of high sensitivity, good specificity and strong stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the heavy and light chain variable regions of a bisphenol A monoclonal antibody and their applications. Background Technology

[0002] Bisphenol A is a widely used industrial chemical with the chemical formula C6H2O. 15 H 16 O2. Its primary use is as a raw material in the production of polycarbonate plastics and epoxy resins. The main route of bisphenol A (BPA) entry into the human body is through dietary intake, such as migration from the coating of plastic containers or the inner walls of food cans into food.

[0003] The most significant health risk of bisphenol A (BPA) lies in its role as an endocrine disruptor, mimicking estrogen and interfering with normal hormonal function. Animal studies have shown that it may impair fertility, affect the neurological and gonadal development of fetuses and children, and is associated with precocious puberty. The European Union has classified it as a reproductive toxicant. Research suggests a possible link to obesity, diabetes, cardiovascular disease, and immune system dysfunction. Therefore, establishing a safe and efficient biological detection method for BPA is imperative.

[0004] Immunoassay methods offer advantages such as low cost, high efficiency, high sensitivity, and relatively low skill requirements, making them suitable for rapid detection of large numbers of samples. The purpose of this invention is to provide a monoclonal antibody with extremely high affinity and specificity for bisphenol A, laying the foundation for establishing a colloidal gold test strip detection method and developing and promoting colloidal gold test strip products. Summary of the Invention

[0005] Therefore, the present invention provides a heavy chain and light chain variable region of a bisphenol A monoclonal antibody and its application.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] In a first aspect, the present invention provides a bisphenol A monoclonal antibody, characterized in that: the monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0008] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1;

[0009] The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2;

[0010] Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3.

[0011] The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5;

[0012] The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6;

[0013] The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7;

[0014] The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8;

[0015] The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9;

[0016] The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 10.

[0017] Secondly, the present invention provides the bisphenol A monoclonal antibody described above, characterized in that:

[0018] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3;

[0019] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 4.

[0020] Thirdly, the present invention provides the application of a bisphenol A monoclonal antibody in the preparation of a test strip for detecting bisphenol A.

[0021] Preferably, the test strip is a colloidal gold test strip, a fluorescent microsphere test strip, or a latex microsphere test strip.

[0022] The present invention has the following advantages:

[0023] This invention provides a bisphenol A monoclonal antibody and its applications. The prepared monoclonal antibody exhibits high specificity, high sensitivity, and good stability, and can be used as a raw material for enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay. The purpose of this invention is to provide the heavy and light chain variable regions of a bisphenol A monoclonal antibody and their detection applications, laying the foundation for the research and development and promotion of ELISA, colloidal gold test strips, and fluorescent immunoassay strips. Attached Figure Description

[0024] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0025] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0026] Figure 1 The results of SDS-PAGE identification of purified monoclonal antibodies;

[0027] Figure 2 This is a schematic diagram for interpreting the results of colloidal gold test strips. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Synthesis of Bisphenol A Artificial Antigen

[0030] 1. Preparation of Bisphenol A Immunogen

[0031] (1) Bisphenol A solution: Weigh 10 mg of bisphenol A hydrochloride, dissolve it in 0.5 mL of anhydrous DMSO, and vortex until completely clear.

[0032] (2) Protein solution: Weigh 73 mg BSA and dissolve it in 5.0 mL of pre-cooled 0.1 mol / L MES buffer (pH 6.0). Mix gently to ensure complete dissolution.

[0033] (3) Take a small centrifuge tube and quickly weigh 510 mg EDC and 306 mg NHS. Add 2.0 mL of pre-cooled 0.1 mol / L MES buffer (pH 6.0) and vortex to dissolve completely. Add the bisphenol A solution dropwise to the EDC / NHS MES solution while gently vortexing. Activate at room temperature (25°C) in the dark for 30 minutes with gentle stirring or vortexing.

[0034] (4) Slowly and dropwise add the activated bisphenol A solution to the protein solution, and adjust the final reaction volume to 8-10 mL with MES buffer. Place the reaction system at 4°C, in the dark, and stir (or invert) slowly overnight (12-16 hours).

[0035] (5) After the reaction is complete, add 1 mL of 1 mol / L glycine solution and continue stirring at room temperature for 1 hour.

[0036] (6) Transfer the reaction mixture into a dialysis bag and dialyze to purify it, thus obtaining bisphenol A immunogen (bisphenol A-BSA).

[0037] 2. Preparation of Bisphenol A Detection Antigen

[0038] (1) Bisphenol A solution: Weigh 10 mg of bisphenol A hydrochloride, dissolve it in 0.5 mL of anhydrous DMSO, and vortex until completely clear.

[0039] (2) Protein solution: Weigh 73 mg OVA and dissolve it in 5.0 mL of pre-cooled 0.1 mol / L MES buffer (pH 6.0). Mix gently to ensure complete dissolution.

[0040] (3) Take a small centrifuge tube and quickly weigh 510 mg EDC and 306 mg NHS. Add 2.0 mL of pre-cooled 0.1 mol / L MES buffer (pH 6.0) and vortex to dissolve completely. Add the bisphenol A solution dropwise to the EDC / NHS MES solution while gently vortexing. Activate at room temperature (25°C) in the dark for 30 minutes with gentle stirring or vortexing.

[0041] (4) Slowly and dropwise add the activated bisphenol A solution to the protein solution, and adjust the final reaction volume to 8-10 mL with MES buffer. Place the reaction system at 4°C, in the dark, and stir (or invert) slowly overnight (12-16 hours).

[0042] (5) After the reaction is complete, add 1 mL of 1 mol / L glycine solution and continue stirring at room temperature for 1 hour.

[0043] (6) Transfer the reaction mixture into a dialysis bag and dialyze to purify it, thus obtaining the bisphenol A detection antigen (bisphenol A-OVA).

[0044] Example 2 Preparation of Bisphenol A Monoclonal Antibody

[0045] 1. Mouse immunization

[0046] Three female Balb / c mice aged 6-8 weeks were immunized with 25 μg of bisphenol A-BSA artificial antigen each. For the first immunization, the bisphenol A-BSA artificial antigen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. Immunizations were repeated every two weeks for a total of three immunizations. For the second and third immunizations, Freund's incomplete adjuvant was used for antigen emulsification, and the dosage and method of immunization remained unchanged. One week after the third immunization, blood was collected from the tail vein of the mice, and serum was analyzed using an indirect ELISA method to determine its titer and inhibition. The results are shown in Table 1. Mice #2 showed the highest serum titer after immunization, reaching 1:3.2 × 10⁻⁶. 4 The inhibition rate can reach up to 60.60%. Take 50 μg of bisphenol A-BSA artificial antigen, dilute it with 1×PBS to 200 μL, and inject it intraperitoneally to boost the immunization of mice. Cell fusion can be performed three days later.

[0047] Table 1 Serum titers and inhibition detection in immunized mice

[0048]

[0049] 2. Culture of SP2 / 0 myeloma cells

[0050] One vial of SP2 / 0 myeloma cells, frozen in liquid nitrogen, was immediately transferred to a 37°C water bath. The cryovial was gently agitated periodically until the cells reached a semi-ice crystal state. Under sterile conditions, the SP2 / 0 cells were transferred to a 50 mL sterile centrifuge tube. 10 mL of preheated RPMI 1640 complete culture medium was slowly added dropwise to the centrifuge tube. The tube was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell clumps were gently dispersed, and the cells were resuspended in 5 mL of culture medium and transferred to a T75 cell culture flask. An additional 5 mL of culture medium was added, and the flask was agitated in a cross-hatching motion before being placed in a CO2 cell culture incubator at 37°C. The cell condition was observed under a microscope. When the cell density reached approximately 80%, the SP2 / 0 cells were passaged.

[0051] 3. Cell fusion

[0052] (1) Blood was collected from the orbital cavity of mice after booster immunization and placed in EP tubes. After standing at 37°C for 2 hours, the tubes were centrifuged at 4000 r / min for 10 minutes. The serum was collected as a positive control for subsequent screening of monoclonal antibodies. The mice were euthanized by cervical dislocation and then disinfected by soaking in 75% alcohol.

[0053] (2) Preparation of spleen cells: In a biosafety cabinet, use sterilized scissors and forceps to cut open the mouse skin. Replace with a new set of sterilized scissors and forceps to cut open the mouse abdominal cavity. Then, carefully remove the spleen using a set of sterilized scissors and forceps, and trim away excess fat. Prepare a sterile 15mL centrifuge tube, add 10mL of DMEM culture medium, place the spleen into the centrifuge tube, moisten the spleen, and carefully discard the excess culture medium. Take another 10 mL of DMEM culture medium and place it in a sterile Petri dish. Grind the spleen with a ground glass slide to prepare a single-cell suspension. Filter the suspension through a 200-mesh nylon mesh into a sterile centrifuge tube. Add 30 mL of DMEM to a 50 mL sterile centrifuge tube. Rinse the nylon mesh with a pipette. Centrifuge the centrifuge tube containing the spleen cell suspension at 1500 r / min for 5 minutes. Discard the supernatant. Gently break up the cell clumps by hand. Resuspend the cells in 30 mL of DMEM culture medium and centrifuge again. Discard the supernatant, gently break up the cell clumps by hand, and resuspend the cells in 10 mL of DMEM culture medium.

[0054] (3) Cell fusion: Centrifuge at 1000 r / min for 5 minutes to collect well-grown SP2 / 0 cells into a 50 mL centrifuge tube. Gently break up the SP20 cell clusters, add 30 mL of DMEM medium to resuspend, centrifuge again, add 10 mL of DMEM medium to resuspend, then mix the spleen cell suspension with the SP2 / 0 cell suspension, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, and gently break up the cell clusters. Place in a 37℃ water bath, and add 1 mL of PEG fusion agent to the centrifuge tube within 1 minute. At this time, the cells are red, homogeneous, and quicksand-like, and rotating the tube wall feels like frosted glass.

[0055] (4) Termination of fusion: Take 9 mL of preheated DMEM medium to terminate the fusion, which is divided into three stages. The first stage is to add 1 mL in the first minute, the second stage is to add 1 mL in the first minute, and the third stage is to add the remaining 7 mL of medium in the first 3 minutes. Then, let it stand in a 37°C water bath for 5 minutes to stabilize, and then centrifuge at 800 r / min for 5 minutes.

[0056] (5) Plating: Discard the supernatant, gently break up the cell clumps, add HAT medium (for example, to plate 5 96-well plates, 200 μL / well, remove the feeder layer cells that have been pre-plated at 100 μL / well, then add 50 mL of HAT medium), mix the cells, and then evenly spread the fused cell suspension into the 96-well cell plate with the feeder layer cells added, 100 μL / well, and incubate in a CO2 cell incubator at 37°C.

[0057] 4. Screening of positive hybridoma cells

[0058] Seven days after cell fusion, when the cell clusters were relatively large, the cell supernatant was analyzed using an indirect ELISA method. A 1 μg / mL bisphenol A-OVA artificial antigen was used as the detection antigen; the positive control was serum from fused mice, and the negative control was serum from mice immunized with PBS. Wells with the strongest chromogenic reaction were selected as positive wells. The selected positive hybridoma cells were subcloned using a limiting dilution method. The hybridoma cell lines that stably secreted monoclonal antibodies, identified after subcloning, were expanded and cultured in T75 cell flasks. When the cell count reached approximately 80%, the cells were collected for ascites preparation.

[0059] 5. Preparation of ascites

[0060] Add 10 mL of sterile 1×PBS to the cell culture flask, blow off the cell layer, resuspend it, and transfer it to a 15 mL centrifuge tube. Centrifuge at 1000 r / min for 10 minutes. Discard the supernatant, resuspend the precipitate in 1 mL of sterile 1×PBS, mix well, and aspirate using a 1 mL syringe. Inject approximately 500 μL of the cell suspension into each mouse, monitoring the mouse's growth. Collect ascites fluid one week later when the mouse's abdomen is swollen. Collect the mouse ascites fluid into a centrifuge tube, centrifuge at 8000 r / min for 20 minutes, aspirate the middle ascites layer, and purify the collected ascites fluid.

[0061] 6. Purification of monoclonal antibodies

[0062] The collected ascites fluid was purified. SDS-PAGE was used to assess the purity of the purified monoclonal antibody, which was approximately 95%. The results are shown below. Figure 1 .

[0063] Example 3: Specificity and sensitivity detection of bisphenol A monoclonal antibody

[0064] The sensitivity and specificity of bisphenol A (BPA) monoclonal antibodies were detected using an indirect, competitive ELISA method. Bisphenol A-OVA artificial antigen was coated at a concentration of 1 μg / mL, and 1 mg / mL of monoclonal antibody 3F2-1C5 was diluted to verify the antibody's sensitivity. The results are shown in Table 2. The sensitivity of monoclonal antibody 3F2-1C5 reached a maximum of 64.21% at a dilution of 1:40,000. Nonylphenol artificial antigen, estradiol artificial antigen, and phthalate artificial antigen were coated at a concentration of 1 μg / mL, and 1 mg / mL of monoclonal antibody 3F2-1C5 was diluted 1:5,000 to verify the antibody's specificity. The results are shown in Table 3. The purified monoclonal antibody showed no cross-reactivity with nonylphenol artificial antigen, estradiol artificial antigen, or phthalate artificial antigen, indicating that the purified monoclonal antibody had good specificity.

[0065] Table 2. Monoclonal antibody sensitivity validation

[0066]

[0067] Table 3. Validation of Monoclonal Antibody Specificity

[0068]

[0069] Example 4: Cloning of Bisphenol A Monoclonal Antibody Gene

[0070] 1. Hybridoma cell culture and total RNA extraction

[0071] Hybridoma cells 3F2-1C5 were cultured in RPMI 1640 complete medium at 37°C and 5% CO2 until the cell number reached 1×10⁻⁶. 7 Total RNA was extracted from cells using a total RNA extraction kit (purchased from Tiangen).

[0072] 2. Synthesis of the first strand of cDNA

[0073] The first strand of cDNA was synthesized using a reverse transcription kit (purchased from TAKARA) with the total RNA extracted in step 1 as the amplification template.

[0074] 3. Gene amplification

[0075] Design downstream primers and upstream universal primers for Lambda, Kappa, and Heavy chains.

[0076] Primer: F (SEQ ID No. 11): AAGCGTGGTATCAACGCAGA

[0077] Rκ (SEQ ID No. 12):AACATTGATGTCTTTGGGGTAGAA

[0078] Rλ (SEQ ID No.13):AATCGTACACACCAGTGTGTGGG

[0079] R H (SEQ ID No. 14):AGGGATCCAGAGTTCCAGGT.

[0080] PCR amplification was performed using the first strand of cDNA as a template in a 50 μL reaction volume. The reaction volume consisted of 3 μL template, 2.5 μL upstream primer (10 μM), 2.5 μL downstream primer (10 μM), 25 μL 2×Taq enzyme, and 17 μL sterile water.

[0081] The landing PCR reaction conditions were as follows: 98℃ for 30 seconds; 98℃ for 15 seconds, 64℃~58℃ for 30 seconds, decreasing by 0.5℃ each time until reaching 58℃, for 10 cycles; 72℃ for 30 seconds; 98℃ for 15 seconds, 56℃ for 30 seconds, 72℃ for 30 seconds, for 15 cycles; and the program ended at 72℃ for 7 minutes.

[0082] 4. Cloning and screening of PCR amplification products

[0083] The PCR products were subjected to 1% agarose gel electrophoresis. The Kappa, Lambda and Heavy chain amplification fragments were recovered using a PCR product recovery kit (purchased from Tiangen). The recovered and purified target fragments were inserted into the pLB vector using a pLB zero-background rapid cloning kit (purchased from Tiangen). The vector was then transformed into DH5α competent cells (ampicillin resistant). Recombinant positive clones were screened and sequenced.

[0084] 5. The variable region gene sequence and amino acid sequence of the bisphenol A monoclonal antibody in this embodiment are as follows:

[0085] (1) Heavy chain variable region gene sequence (SEQ ID No. 3):

[0086] GACGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCTACAGCATCACCAGCGGCTACTTCTGGAACTGGTTCAGGCAGTTCCCCGGCTACAAGCTGGAGTGGCTGGCCTACATCAGCTACGACGGCAGCTACGGCTACAA CCCCAGCCTGAAGAACAGGATCAGCATCACCAGGGACACCAGCACCAACCAGTTCTTCCTGAAGCTGGAGAGCGTGACCACCGAGGACACCGCCACCTACTACTGCGGCTACTACTACATCACCAGGTACAACTACGAGATGGACTACTCGGCCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0087] (2) Amino acid sequence of the heavy chain variable region (SEQ ID No. 1):

[0088] DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYFWNWFRQFPGYKLEWLAYISYDGSYGYNPSLKNRISITRDTSTNQFFLKLESVTTEDTATYYCGYYYITRYNYEMDYSAQGTSVTVSS.

[0089] (3) Light chain variable region gene sequence (SEQ ID No. 4):

[0090] GAGATCGTGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAGGGTGACCATCACCTGCAGCGCCACCAGCAGCGTGAGCTACATGCACTGGTTCCAGCAGAAGCCCGGCACCAGCCCCAAGCTGTGGATCTACAGGACCAGCAACCTG GCCAGCGGCGTGCCCGTGAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGAGGGGCAGCCACCCCCCACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG.

[0091] (4) Amino acid sequence of the light chain variable region (SEQ ID No. 2)

[0092] EIVLTQSPAIMSASPGERVTITCSATSSVSYMHWFQQKPGTSPKLWIYRTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRGSHPPTTFGAGTKLELK.

[0093] 6. Variable region amino acid sequence and homology analysis

[0094] The heavy and light chain gene sequences were compared and analyzed in the NCBI database. The results showed that the heavy chain variable region gene sequence of monoclonal antibody 3F2-1C5 showed the highest homology with the mouse immunoglobulin heavy chain variable region gene sequence (Sequence ID: AJ784030.1), with a homology of 40 / 42 (95%). The amino acid sequence of the heavy chain variable region of monoclonal antibody 3F2-1C5 showed the highest homology with the mouse immunoglobulin heavy chain variable region amino acid sequence (Sequence ID: P18531.1), with a homology of 87 / 97 (90%). The light chain variable region gene sequence of monoclonal antibody 3F2-1C5 showed the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: AJ784038.2), with a homology of 290 / 318 (91%). The amino acid sequence of the light chain variable region of the monoclonal antibody 3F2-1C5 showed the highest homology with the amino acid sequence of the light chain variable region of mouse immunoglobulin (Sequence ID: CAB46135.1), with a homology of 88 / 95 and a homology percentage of 93%.

[0095] 7. CDR Area Analysis

[0096] The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody 3F2-1C5 were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR region.

[0097] Antibody heavy chain CDR region:

[0098] CDR-H1 (SEQ ID No.5): SGYFWN

[0099] CDR-H2 (SEQ ID No.6): YISYDGSYGYNPSLKN

[0100] CDR-H3 (SEQ ID No. 7): YYITRYNYEMDY.

[0101] Antibody light chain CDR region:

[0102] CDR-L1 (SEQ ID No.8): SATSSVSYMH

[0103] CDR-L2 (SEQ ID No.9):RTSNLAS

[0104] CDR-L3 (SEQ ID No. 10): QQRGSHPPT.

[0105] Example 5: Preparation of Bisphenol A Colloidal Gold Test Strip

[0106] 1. Preparation of chloroauric acid

[0107] Weigh 800 mL of ultrapure water into a 1000 mL Erlenmeyer flask, add 8 mL of 1% chloroauric acid to the ultrapure water, and place the flask on a magnetic heating rod stirrer to mix and heat at 500℃ and speed setting 1. After the water boils, increase the speed to speed setting 6. Once the liquid is stirring, quickly add 1 mL of 1.2% reducing agent and adjust the speed to speed setting 5. After heating for 6 minutes and 30 seconds, stop heating and allow it to cool to room temperature. The prepared colloidal gold is pure, transparent, and free of precipitates and floating matter.

[0108] 2. Preparation of Bisphenol A Monoclonal Antibody-Colloidal Gold Label

[0109] (1) Add bare gold: Mix the colloidal bare gold well and take 1L of colloidal gold solution;

[0110] (2) Add K2CO3: Add 0.2M K2CO3 at a ratio of 5μL / mL colloidal gold and vortex mix well;

[0111] (3) Add antibody: Mix 1 mL of 1 mg / mL bisphenol A3F2-1C5 antibody with 4 mL of 10% BSA (IgG), and then add the mixed antibody dropwise to the gold using a 1 mL pipette, and equilibrate for 5 minutes;

[0112] (4) Leveling: Reduce the rotation speed to stabilize the vortex at a drop of about 1 cm, and then rebalance for 1 hour;

[0113] (5) Add BSA: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10% BSA (Cebao), balance for 3 minutes, reduce the rotation speed to stabilize the vortex at about 1cm, and then balance for another 30 minutes;

[0114] (6) Add PEG: Increase the rotation speed until the vortex drops by about 3cm, pour in 10mL of 10% PEG, and let it balance for 5 minutes;

[0115] (7) Centrifugation: Using a benchtop high-speed centrifuge, place the centrifuge tubes in pairs at opposite corners of the rotor, 12000 r / min, 8 minutes, 4℃;

[0116] (8) Discard the supernatant: After centrifugation, gently remove the centrifuge bucket and place it on the experimental table of the negative pressure aspiration device. Do not shake or bump it to avoid disturbing the gold precipitate in the centrifuge bucket; stop aspiration when it is close to the bottom of the centrifuge bucket (about 5~6 mL remaining), and carefully remove the remaining supernatant with a 1 mL pipette to avoid aspirating the gold precipitate;

[0117] (9) Resuspension: Stop aspirating when the liquid is close to the bottom of the centrifuge bucket (about 2~3 mL remaining), gently shake the centrifuge bucket to resuspend the gold precipitate, and transfer the resuspended gold precipitate to a blue cap bottle;

[0118] (10) Rinsing the container: Add about 20 mL of reconstitution solution to the centrifuge container, shake gently to wash away the residual gold precipitate, transfer the liquid to the other three centrifuge containers in turn, and finally transfer it to the blue cap bottle. Repeat once.

[0119] (11) Volume adjustment: Finally, adjust the volume to 1 / 10 of the gold solution, i.e. 100 mL, with the blue cap bottle, mix well and set aside.

[0120] 3. Preparation of microporous reagents

[0121] (1) Prepare lyophilization buffer: 0.05M phosphate buffer, 0.5% BSA (Science), 3% trehalose;

[0122] (2) Preparation of microwell reagent solution: Prepare according to the ratio of 10 μL / well bisphenol A colloidal gold and 50 μL / well lyophilized buffer; after preparation, stir and mix for 20 minutes.

[0123] (3) Microwell reagent coating: Dispense 60 μL / well of microwell reagent solution into 96-well plates;

[0124] (4) Drying of micro-well reagents: The coated 96-well microplates are placed in a vacuum freeze dryer with a preset freeze drying program for processing;

[0125] (5) Microporous reagent packaging: After the freeze-drying process is completed, take out the microporous reagent, put on the rubber cap, put it in an aluminum foil bag with desiccant, and store it for later use.

[0126] 4. Preparation of sample absorption pads

[0127] The sample absorption pad was immersed in 0.1 mol / L phosphate buffer containing 0.5% BSA, pH=7.2 for 2 hours and then dried at 37℃ for 2 hours to obtain the sample absorption pad.

[0128] 5. Preparation of nitrocellulose membranes

[0129] Bisphenol A artificial antigen was diluted to 10 mg / mL with phosphate buffer and coated onto the detection line T of a nitrocellulose membrane using a Biodot coating instrument, with a coating amount of 1.0 μg / cm. Goat anti-mouse IgG antibody was diluted to 200 μg / mL with 0.01 mol / L, pH 7.2 PBS buffer and coated onto the control line C of a nitrocellulose membrane using a Biodot coating instrument. The coated reaction membrane was then dried at 37°C for 16 hours to obtain the nitrocellulose membrane.

[0130] 6. Assembly of Bisphenol A Colloidal Gold Test Strips

[0131] The sample absorption pad, nitrocellulose membrane, and absorption pad are sequentially attached to the base plate. The end of the sample absorption pad is connected to the beginning of the nitrocellulose membrane, and the end of the nitrocellulose membrane is connected to the beginning of the absorption pad. The beginning of the sample absorption pad is aligned with the beginning of the base plate, and the end of the absorption pad is aligned with the end of the base plate. This assembly forms a colloidal gold test strip. The strip is then cut into strips at a size of 4.50 mm / strip to obtain the colloidal gold test strip.

[0132] Example 6: Application of Bisphenol A Colloidal Gold Test Strips

[0133] 1. Test strip detection

[0134] The required test strips and sample diluent should be brought to room temperature (20-25°C). Using a micropipette, pipette 200µL of the sample solution into each well, slowly aspirating and thoroughly mixing it with the reagent in the well. Incubate at 40°C for 3 minutes. Insert the labeled test strip into the well, ensuring it is fully immersed in the solution. Incubate at 40°C for 5 minutes. Remove the test strip and follow the instructions in the diagram (…). Figure 2 The judgment result is valid at other times; judgments made at other times are invalid.

[0135] 2. Interpretation of test results

[0136] Negative (﹣): Both C and T lines show color, with the T line showing stronger color than the C line, indicating that the concentration of bisphenol A in the sample is below the detection limit.

[0137] Positive (+): C line shows color, T line shows the same color as C line, T line shows weaker color than C line, or T line shows no color, indicating that the concentration of bisphenol A in the sample is equal to or higher than the detection limit.

[0138] Invalid: No C line appears, indicating incorrect operation or that the test strip has deteriorated and become ineffective. In this case, carefully read the instructions again and retest with a new test strip.

[0139] If the test strips need to be archived, the lower sponge pad should be cut off immediately after interpretation, and the strips should be dried before archiving.

[0140] In addition to naked-eye interpretation, NBReader can be used for result interpretation.

[0141] 3. Sensitivity testing of bisphenol A colloidal gold test strips

[0142] The concentration of bisphenol A standard was diluted to 6 ppb and 3 ppb, and raw milk samples were spiked and tested according to the test strip detection method to verify the product's limit of detection. The results are shown in Table 4. The test strip provided by this invention has a detection sensitivity of 6 ppb for bisphenol A standard in raw milk.

[0143] Table 4 Sensitivity of Bisphenol A Colloidal Gold Test Strips

[0144]

[0145] 4. Specificity detection of bisphenol A colloidal gold test strips

[0146] The nonylphenol, estradiol, and phthalates at 1000 ppb were tested according to the detection method, and the results are shown in Table 5. All results were negative, indicating that the test strip provided by this invention does not cross-react with other toxins and drugs, and has good specificity.

[0147] Table 5. Specificity of Bisphenol A Colloidal Gold Test Strips

[0148]

[0149] 5. Stability testing of bisphenol A colloidal gold test strips

[0150] The prepared test strips were subjected to accelerated testing at 4℃ and 37℃. Bisphenol A standard (6 ppb) was detected using the strips at 0, 7, 14, and 28 days. Error analysis was performed on the measured concentration and the actual sample concentration, and the results are shown in Table 6. The CV values ​​were all <10%, indicating that the test strips provided by this invention have good stability.

[0151] Table 6. Stability of Bisphenol A Colloidal Gold Test Strips

[0152]

[0153] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A bisphenol A monoclonal antibody, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 2; Both the heavy chain variable region and the light chain variable region are composed of complementary determination regions and framework regions, and the complementary determination regions are composed of CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 5; The amino acid sequence of CDR2 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 6; The amino acid sequence of CDR3 in the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7; The amino acid sequence of CDR1 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8; The amino acid sequence of CDR2 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9; The amino acid sequence of CDR3 in the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

10.

2. The bisphenol A monoclonal antibody according to claim 1, characterized in that: The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 3; The gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

4.

3. The application of the bisphenol A monoclonal antibody according to claim 1 in the preparation of bisphenol A detection products.

4. A bisphenol A detection product, characterized in that: The test product consists of a bisphenol A test strip and a microwell reagent; the test strip is a colloidal gold test strip, and the microwell reagent contains the bisphenol A monoclonal antibody as described in claim 1 or 2.

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