CN116836280B

CN116836280B - Anti-estriol monoclonal antibody, detection reagent and application thereof

Info

Publication number: CN116836280B
Application number: CN202311122843.4A
Authority: CN
Inventors: 巩玉洁; 马溶江; 马燕; 杨晓霞; 赵荣茂; 栾欣雨; 李川敏; 陈娟; 张琼林; 赵方圆; 袁婷婷
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: Beijing Nabai Bio Tech Co ltd
Priority date: 2023-09-01
Filing date: 2023-09-01
Publication date: 2023-10-31
Anticipated expiration: 2043-09-01
Also published as: CN116836280A

Abstract

The invention discloses a monoclonal antibody of antiestriol, a detection reagent and application thereof, wherein the monoclonal antibody contains a peptide V _H Heavy chain variable region of (2) and designated V _L Light chain variable region of (V) _H And V _L Are each composed of a complementarity determining region and a framework region; the complementarity determining regions are each composed of CDR1, CDR2 and CDR 3. Experiments prove that the estriol monoclonal antibody provided by the invention has no obvious cross reaction on other structural analogues of estriol in the preparation of a kit for detecting estriol, and has good specificity and higher sensitivity.

Description

Anti-estriol monoclonal antibody, detection reagent and application thereof

Technical Field

The invention relates to the technical field of rapid biological detection, in particular to an anti-estriol monoclonal antibody, a detection reagent and application thereof.

Background

Estriol (E3) is a metabolite of estradiol and estrone, which has very low values in non-gestational periods and is mainly synthesized by the liver; gestation is mainly synthesized by fetal placenta, and after synthesis, the fetal placenta is metabolized in the liver through maternal blood circulation to form combined E3 by combining with sulfuric acid or glucuronic acid, and the combined E3 is discharged from urine. The environment mainly enters a food chain through the biological enrichment effect, so that food is polluted, then the food indirectly enters a human body through the food chain, the endocrine function of the organism is interfered, endocrine dyscrasia is caused, and the potential hazard to the ecological environment and the health and safety of human beings is not ignored.

Currently, methods for determining estriol residues mainly comprise gas chromatography-mass spectrometry (Gas chromatography-mass spectrometry, GC-MS), high performance liquid chromatography (High performance liquid chromatography, HPLC), enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography test paper method and the like. The GC-MS and the HPLC have the advantages of high sensitivity, good selectivity and the like, but the required instruments and equipment are expensive, and the detection activity is limited to a certain extent. ELISA is one of the more common methods for measuring estriol, has strong specificity, high sensitivity and high analysis speed, can measure a plurality of samples at one time, and is commonly used in actual detection activities. Besides strong specificity, the colloidal gold and fluorescence immunity chromatography has the characteristics of high sensitivity, low cost, simple and convenient operation, wide detection scene and the like, and is a detection method with more practical application. However, due to the similarity of similar molecules, ELISA and colloidal gold immunoassay methods of estriol are easy to generate certain cross reactions on chemical substances with similar structures, and the accuracy of experiments is affected.

Disclosure of Invention

In order to solve the problems, the invention provides an anti-estriol monoclonal antibody, a detection reagent and application thereof, and specifically comprises the following technical scheme:

in a first aspect, the present invention provides a monoclonal antibody against estriol, which monoclonal antibody isThe body contains the name V _H Heavy chain variable region of (2) and designated V _L Light chain variable region of (V) _H And V _L Are each composed of a complementarity determining region and a framework region;

the complementarity determining region consists of CDR1, CDR2 and CDR 3;

v of the monoclonal antibody _H The amino acid sequence of CDR1 of (1) is shown as 85-89 th position in SEQ ID No. 1;

v of the monoclonal antibody _H The amino acid sequence of CDR2 of (1) is shown as 104-120 in SEQ ID No. 1;

v of the monoclonal antibody _H The amino acid sequence of CDR3 of (2) is shown as 153-164 in SEQ ID No. 1;

v of the monoclonal antibody _L The amino acid sequence of CDR1 of (2) is shown as 30-42 in SEQ ID No. 2;

v of the monoclonal antibody _L The amino acid sequence of CDR2 of (2) is shown as 58-64 in SEQ ID No. 2;

v of the monoclonal antibody _L The amino acid sequence of CDR3 of (2) is shown at positions 99-107 of SEQ ID No. 2.

In a second aspect, the monoclonal antibody V _H The amino acid sequence of (2) is shown as SEQ ID No. 1 in a sequence table;

v of the monoclonal antibody _L The amino acid sequence of (2) is shown as SEQ ID No. 2 in the sequence table.

In a third aspect, V encoding the monoclonal antibody _H The nucleotide sequence of (2) is shown as SEQ ID No. 3 in the sequence table;

v encoding the monoclonal antibody _L The nucleotide sequence of (2) is shown as SEQ ID No. 4 in the sequence table.

In a fourth aspect, the monoclonal antibody is a murine monoclonal antibody, the heavy chain of which is of the IgG1 type and the light chain of which is of the lambda type.

In a fifth aspect, the invention provides an application of a monoclonal antibody against estriol in preparing a test strip or a kit for detecting estriol.

Preferably, the test strip is a colloidal gold test strip, or a fluorescent microsphere test strip, or a latex microsphere test strip;

preferably, the kit is an indirect competition ELISA detection kit.

The embodiment of the invention has the following advantages:

experiments prove that the monoclonal antibody provided by the invention has no obvious cross reaction on other structural analogues of the estriol hormone in the preparation of the kit for detecting the estriol, and has good specificity and higher sensitivity.

Drawings

In order 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 described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.

FIG. 1 shows a RIDA SOFT four-parameter method IC for detecting sensitivity of an anti-estriol monoclonal antibody by an indirect competition ELISA method according to an embodiment of the invention ₅₀ A graph;

FIG. 2 is a diagram showing analysis of nucleotide sequence homology of heavy chain variable region of anti-estriol monoclonal antibody provided by the embodiment of the invention;

FIG. 3 is a diagram showing analysis of nucleotide sequence homology of a light chain variable region of an anti-estriol monoclonal antibody according to an embodiment of the present invention;

FIG. 4 is an analysis chart of amino acid sequence homology of heavy chain variable region of anti-estriol monoclonal antibody provided by the embodiment of the invention;

FIG. 5 is an analysis chart of amino acid sequence homology of a light chain variable region of an anti-estriol monoclonal antibody provided by the embodiment of the invention;

FIG. 6 is a schematic structural diagram of a test strip for detecting estriol according to an embodiment of the present invention;

FIG. 7 is a top view of the structure shown in FIG. 6;

FIG. 8 is a schematic diagram of a microwell reagent according to an embodiment of the present invention.

In the figure: 1-water absorbing paper; a 2-nitrocellulose membrane; 3-sample pad; 4-a quality control line; 5-detecting lines; 6-a bottom plate; 7-test strips; 8-estriol monoclonal antibody-colloidal gold label; 9-a microporous plug; 10-microwell reagent.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 Synthesis of estriol Artificial antigen

The embodiment provides a synthetic route of estriol artificial antigen, which comprises the following specific steps:

4.8 mmol/L (1.4 g) estriol was taken in a 100 ml round bottom flask, 20 ml anhydrous dimethyl sulfoxide (DMSO) was added, stirred at room temperature to dissolve the starting material, and 12.5 mmol/L (0.7 g) KOH was added. 9.6 mmol/L (1.3 g) bromoacetic acid was taken and reacted at room temperature for 2 h. 50 ml ice water was added and unreacted estriol was recovered by extraction with ethyl acetate (repeated three times). The pH of the aqueous phase was adjusted to about 3.0 with 2 mol/L HCl, and a large amount of white precipitate appeared. The reaction was monitored by spot thin layer chromatography on silica gel plates (developing solvent methanol: chloroform=1:5, estriol rf=0.7, product rf=0.1). The water phase is filtered, and filter residue is washed with acidic ice water for 3 times, so that white solid powder is obtained. The dot plate is found to have a small amount of raw materials, 200-300 mesh silica gel is subjected to column chromatography, developing agent methanol/chloroform=1:10, and the solvent is evaporated to dryness to obtain the product with the mass of 0.4 g and the yield of 22%.

Hapten 0.1 mmoL was weighed and dissolved in 0.5 ml DMF, DCC 0.0512 g (0.2 mmoL) and NHS 0.023, 0.023 g (0.2 mmoL) were added with stirring, and reacted overnight with magnetic stirring at 4℃and the supernatant was liquid A after centrifugation. 0.02 g of BSA was weighed out and dissolved in PBS (pH 8.0) having a concentration of 0.1 mol/L at 2ml, and the solution B was prepared by stirring and dissolving. Under the magnetic stirring, the solution A is gradually dripped into the solution B, and the reaction is carried out at 4 ℃ for 12 h. After centrifugation, the supernatant was taken and dialyzed against PBS at 4℃for 3 days, with 4 changes of dialysate per day. The obtained artificial antigen is subpackaged into a 1 ml centrifuge tube at the concentration of 1 mg/ml, and frozen in a refrigerator at-20 ℃ for standby.

EXAMPLE 2 preparation of anti-estriol monoclonal antibodies

The present example provides a method for preparing a monoclonal antibody against estriol, comprising the steps of:

1. preparation of monoclonal antibody hybridoma cell secreting antiestriol

Primary immunization: the estriol artificial antigen of example 1 was emulsified (1:1) with Freund's complete adjuvant and injected subcutaneously into 6-8 week old BALB/c mice at an immunizing dose of 50. Mu.g/mouse. From the first immunization time, booster immunizations were performed at 14-21 days intervals, 2 total booster immunizations, with Freund's incomplete adjuvant. After 7-10 days of each immunization, the titer can be detected by adopting an indirect ELISA method by fundus vein blood sampling, and the immune effect is verified, and the result is shown in Table 1. After the third immunization, when the inhibition and the titer reach more than 1:10000, 1 impact immunization can be carried out, namely, the direct intraperitoneal injection is carried out, the immunogen is diluted by sterile 1 XPBS, and the injection amount is generally equal to the immune amount or 2 times of the immune amount. Spleen cells were fused with myeloma cells three days later, and positive wells were screened. And cloning the positive hole by utilizing a limiting dilution method to obtain and establish a hybridoma cell strain for stably secreting the estriol monoclonal antibody.

TABLE 1

2. Preparation and purification of anti-estriol monoclonal antibodies

Cell resuscitation: taking out the anti-estriol monoclonal antibody hybridoma cell strain cryopreservation tube, immediately placing into a water bath kettle at 37 ℃ for medium-speed thawing, adding 10 ml of DMEM into a 15 ml centrifuge tube in advance, placing the thawed cells into the DMEM, centrifuging at 1000 rpm for 5min, discarding the supernatant, and transferring the cells into a culture flask for culture.

Preparing ascites: the in-vivo induction method of mice is adopted, healthy BALB/c mice are taken, and 0.2 ml/mouse of sterile liquid paraffin is injected for use for about one week. Preparing positive monoclonal hybridoma cells subjected to expansion culture into cell suspension by using sterile 1×PBS, and counting to obtain total cell amount of 2×10 ⁶ The mice were injected intraperitoneally, 0.5 ml/mouse. Ascites was collected 7-10 days later and 2-5 ml ascites could be obtained in one mouse. 10000 Centrifuging at rpm for 7min, collecting clear liquid, and freezing at-20deg.C or below for purification.

Antibody purification: recording ascites name, centrifuging, and recording volume. 3 ml of sodium acetate buffer with pH value of 4.0.06M is added, evenly mixed for 5min, 10 μl of octanoic acid is added, and stirred for 15 min at 4 ℃. The mixture was filtered through cotton wool once with a syringe. 12000 Centrifuging at 4deg.C for 15 min at rpm, collecting supernatant, adding saturated ammonium sulfate with equal supernatant volume to final concentration of 50%, stirring, and standing at 4deg.C for 3 h.12000 Centrifugation was performed at 4℃for 15 min at rpm, the supernatant was discarded, and 1.8 ml of 0.01M PBS (pH 7.2) was added to the centrifuge tube until the pellet was completely dissolved. The total volume was determined by gun, 1/2 volume of saturated ammonium sulfate was added to a concentration of 33% and stirred overnight at 4 ℃.12000 Centrifuging at 4℃for 15 min at rpm, discarding the supernatant and adding 0.45 ml 0.01M PBS to dissolve the precipitate. Treating the dialysis bag (boiling for 5min, cleaning with pure water, and detecting leakage). The dissolved solution was placed in a dialysis bag and dialyzed overnight against 0.01M PBS. Changing the dialysis solution for 2-3 times, collecting antibody, purifying the crude monoclonal antibody with Protein G pre-packed column for the second time, and storing in-80deg.C refrigerator.

EXAMPLE 3 characterization of monoclonal antibodies against estriol

This example provides characterization of monoclonal antibodies against estriol, including sensitivity, specificity, subclass identification:

1. monoclonal antibody sensitivity identification of antiestriol

The sensitivity of the monoclonal antibody against estriol was detected by indirect competition ELISA. The indirect competition ELISA procedure was: estriol artificial antigen was coated at 100 μl/well overnight at 4deg.C; adding 200 μl/hole of sealing solution, sealing at 37deg.C for 2 hr, and drying; adding 50 mu l of gradient diluted estriol artificial antigen into each hole, and fully and uniformly mixing; adding 50 μl of anti-estriol monoclonal antibody into each well, mixing well, and incubating at 37deg.C for 1 hr; discarding the liquid in the ELISA plate, beating to dry, washing the plate with 300 μl/hole of washing liquid, repeating for 4-5 times, and thoroughly beating the liquid after the plate is washed for the last time; adding 100 μl of enzyme-labeled antibody into each well, mixing well, and incubating at 37deg.C for 1 hr; discarding the liquid in the ELISA plate, beating to dry, washing the plate with 300 μl/hole of washing liquid, repeating for 4-5 times, and thoroughly beating the liquid after the plate is washed for the last time; adding 100 μl of substrate solution into each well, and incubating at room temperature in dark place for 10min; adding 50 μl of stop solution into each well to stop the reaction; the microplate reader reads the OD at 450nm wavelength. Calculation of half Inhibition Concentration (IC) ₅₀ ），IC ₅₀ =0.198 ppb, and the results are shown in table 2 and fig. 1.

TABLE 2

2. Monoclonal antibody specificity identification of antiestriol

Estriol and structural analogues thereof are selected for cross reaction, the specificity of the estriol monoclonal antibody is evaluated, no obvious cross reaction exists on other structural analogues under the coating of the estriol artificial antigen, the specificity is good, and the results are shown in table 3.

TABLE 3 Table 3

3. Identification of monoclonal antibody subclass of antiestriol

The subclass of the monoclonal antibody against estriol was detected using an ELISA kit for murine monoclonal antibody IgG class/subclass identification, and the results showed that the heavy chain of the monoclonal antibody against estriol was of IgG1 type and the light chain was of Lambda type.

EXAMPLE 4 monoclonal antibody variable region Gene PCR amplification and sequence identification of anti-estriol

The embodiment provides the monoclonal antibody variable region gene PCR amplification and sequence identification of the antiestriol, and the specific steps are as follows:

1. culturing hybridoma secreting estriol monoclonal antibody with RPMI 1640 complete culture medium at 37deg.C under 5% carbon dioxide to 1×10 ⁷ Total RNA in the cells was then extracted using the total RNA extraction kit (purchased from Tiangen).

2. The first strand of cDNA was synthesized using the reverse transcription kit (purchased from TAKARA) using the total RNA extracted in step 1 as an amplification template.

3. The Lambda strand, kappa strand, downstream primer and upstream universal primer of the Heavy strand were designed.

Primer: f AAGCAGTGGTATCAACGCAGA

Rκ:AACATTGATGTCTTTGGGGTAGAA

Rλ:AATCGTACACACCAGTGTGTGGG

RH:AGGGATCCAGAGTTCCAGGT

PCR amplification was performed using the first strand of cDNA as a template, and the reaction system was 50. Mu.l. 3. Mu.l of template, 2.5. Mu.l of upstream primer (10. Mu.M), 2.5. Mu.l of downstream primer (10. Mu.M), 25. Mu.l of 2 XTaq enzyme and 17. Mu.l of sterile water.

The drop PCR reaction conditions were: 98 ℃ for 30s; 15s at 98 ℃, 64-58 ℃ for 30s, each time decreasing by 0.5 ℃ until 58 ℃, and circulating for 10 times; 30s at 72 ℃; 15 times of circulation are carried out at 98 ℃ for 15s,56 ℃ for 30s and 72 ℃ for 30s; the procedure was ended at 72℃for 7 min.

4. The PCR products were subjected to 1.5% agarose gel electrophoresis, kappa, lambda and Heavy chain amplified fragments were recovered using a PCR product recovery kit (purchased from heaven), the recovered purified target fragments were inserted into pLB vector using a pLB zero background rapid cloning kit (purchased from heaven), transformed into DH 5. Alpha. Competent cells (ampicillin resistance), and the recombinant positive clones were selected and sequenced.

The PCR products were electrophoresed on a 1.5% agarose gel, and the result showed that Kappa chains were not striped.

5. The variable region gene sequence and amino acid sequence of the anti-estriol monoclonal antibody of this example are as follows:

(1) Heavy chain variable region nucleotide sequence (SEQ ID No. 3) of anti-estriol monoclonal antibody:

GCAGTTGGGTTCAACGCAGAGTACATGGGGTCTGACAGAGGAGCCAAGCCCTGGATTCCCAGGTCCTCACATTCAGTGATCAGCACTGAACACAGACCACTCACCATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTATTTTAAAAGGTGTCCAGTGTGATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATTCACTGGGTTCGTCAGGCTCCAGAAAAGGGGCTGGAGTGGATCGCTTACATTAGTGATGACAGTAGTACCATCTACTATTCAGACACAGTGAGGGGCCGATTCACCATTTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTACAAGAAGATCATGGGCCCCCCATTACTTTGCTATGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGTCAAGGGCTATGCCTGCACGT; FIG. 2 shows a nucleotide sequence homology analysis chart of a heavy chain variable region of a monoclonal antibody of the estriol provided by the embodiment of the invention.

(2) Anti-estriol monoclonal antibody light chain variable region nucleotide sequence (SEQ ID No. 4):

AATTTGACTGTTTTCAACATGACATCGACTCTATTATTCCTTGCTGTTCTTCATCACTTAACAGGAAGCACAGTCAAACTGTCTTGCAAGCGCAGCACTGGTAACATTGGAAGCAGCTATGTGTACTGGTACCAGCAGCATGAGGGAAGATCTCCCACCACTATGATTTATGATGATGATAAGAGACCAGATGGAGTTCCTGATAGGTTCTCTGGCTCCATTGACAGCTCTTCCAACTCAGCCTTCCTGACAATCAATAATGTGCAGATTGAAGATGAAGCTATCTACTTCTGTCAGTCTTACAGTAGTGGTATTAATGTTTTCGGTGGTGGAACCAAGCTCACTGTCCTAGGTCAGCCCAAGTCCACTCCCACACTCACAGTATTTCCACCTTCAACTGAGGAGCTCCAGGGAAACAAAGCCACACTGGTGTGTCTGATTTCTGATTTCTACCCGAGTGATGTGGAAGTGGCCTGGAAGGCAAAT; as shown in FIG. 3, the nucleotide sequence homology analysis chart of the light chain variable region of the anti-estriol monoclonal antibody provided by the embodiment of the invention.

(3) Anti-estriol monoclonal antibody heavy chain variable region amino acid sequence (SEQ ID No. 1):

AVGFNAEYMGSDRGAKPWIPRSSHSVISTEHRPLTMDSRLNLVFLVLILKGVQCDVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGIHWVRQAPEKGLEWIAYISDDSSTIYYSDTVRGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCTRRSWAPHYFAMDSWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLSRAMPAR; as shown in FIG. 4, the amino acid sequence homology analysis chart of the heavy chain variable region of the anti-estriol monoclonal antibody provided by the embodiment of the invention.

Monoclonal antibody V of anti-estriol of the embodiment of the invention _H And V _L Are each composed of a complementarity determining region and a framework region; the complementarity determining regions consist of CDR1, CDR2 and CDR 3. As shown in Table 4, V which is a monoclonal antibody against estriol _H Is a complementary determining region amino acid sequence of (a).

TABLE 4 Table 4

(4) Amino acid sequence of the light chain variable region of the monoclonal antibody against estriol (SEQ ID No. 2):

NLTVFNMTSTLLFLAVLHHLTGSTVKLSCKRSTGNIGSSYVYWYQQHEGRSPTTMIYDDDKRPDGVPDRFSGSIDSSSNSAFLTINNVQIEDEAIYFCQSYSSGINVFGGGTKLTVLGQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKAN; as shown in FIG. 5, the amino acid sequence homology analysis chart of the light chain variable region of the anti-estriol monoclonal antibody provided by the embodiment of the invention. As shown in Table 5, V, which is a monoclonal antibody against estriol _L Is a complementary determining region amino acid sequence of (a).

TABLE 5

Example 5 preparation of estriol colloidal gold test strip

As shown in fig. 6 to 7, the test strip 7 is composed of a water absorbing paper 1, a nitrocellulose membrane (NC membrane) 2, a sample pad 3, and a bottom plate 6; two parallel strips were cut on a nitrocellulose membrane (NC membrane) 2 with a film cutter at a distance of 6.5mm, and the strip widths were all about 1mm. The first one is a quality control line 4 near the end of the absorbent paper 1, and the second one is a detection line 5 near the end of the sample pad 3. Spraying goat anti-mouse (IgG) and one ten thousandth blue pigment on the quality control line 4, wherein the concentration is 1-1.5 mg/ml; and spraying the estriol artificial antigen synthesized in the example 1 with the concentration of 0.1-0.5 mg/ml on the detection line 5.

The preparation method comprises the following steps:

and (3) drying the chromatographic membrane with the quality control line 4 and the detection line 5 at 40 ℃ for 16 hours, sealing, and preserving at normal temperature. The water absorbing paper 1, the NC film 2 and the sample pad 3 are sequentially adhered on the bottom plate 6, the initial end of the sample pad 3 is connected with the tail end of the NC film 2, the initial end of the NC film 2 is connected with the water absorbing pad 1, the tail end of the sample pad 3 is aligned with the tail end of the bottom plate 6, and the initial end of the water absorbing pad 1 is aligned with the initial end of the bottom plate 6. The glued plastic plate was cut longitudinally with a slitter into test strips 7 of 4.5mm width.

As shown in fig. 8, the microwell reagent 10 has a microwell plug 9, and the monoclonal antibody-colloidal gold conjugate 8 is lyophilized in the microwell reagent 10.

8 cut test strips 7 and 1 microporous reagent 10 are taken and put into a reagent barrel to be sealed, and the test strips are preserved at the temperature of 2-8 ℃.

Example 6 detection of estriol content in milk powder

1g (accurate to 0.01 g) of milk powder is weighed, poured into a centrifuge tube, added with 8ml of purified water and uniformly mixed, a sample 1 to be tested (estriol 1 ppb), a sample 2 to be tested (estriol 0.5 ppb) and a sample 3 to be tested (estriol 0 ppb) are prepared for standby. The sample to be tested must be uniform liquid, and cannot have caking and sedimentation blocks of fermentation deterioration, otherwise, the detection result is affected.

200 μl of the sample solution to be measured is pipetted into the microwell reagent, slowly aspirated and thoroughly mixed with the reagent in the microwell, and timing is started. After incubation at 40℃for 3min, the test strip prepared in example 5 was inserted into the microwells, allowed to fully immerse in the solution, and timing was started. After incubation for 5min at 40 ℃ again, the test strips were removed and judged to be invalid at other times according to the schematic judgment result.

Result determination

Negative (-). The color of the C line and the T line is developed, and the color development of the T line is far stronger than that of the C line, which indicates that the sample does not contain estriol or is far lower than the detection limit.

Positive (+): developing color by a C line; the color development of the T line is the same as that of the C line, the color development of the T line is weaker than that of the C line or the T line does not develop, and the concentration of the estriol in the sample is equal to or higher than the detection limit.

Invalidation: the absence of the C line indicates an incorrect operation or that the test card has failed.

The results show that: sample 1 to be tested (estriol 1 ppb) was positive, sample 2 to be tested (estriol 0.5 ppb) was negative, and sample 3 to be tested (estriol 0 ppb) was negative. This result indicates that the test strip detection limit is 1ppb.

Therefore, the colloidal gold detection test strip prepared by the anti-estriol monoclonal antibody can improve the detection sensitivity of estriol.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.

Claims

1. An anti-estriol monoclonal antibody comprising a polypeptide designated V _H Heavy chain variable region of (2) and designated V _L Light chain variable region of (V) _H And V _L Are each composed of a complementarity determining region and a framework region;

the complementarity determining region consists of CDR1, CDR2 and CDR 3;

the monoclonal antibodyV of antibody _L The amino acid sequence of CDR1 of (2) is shown as 30-42 in SEQ ID No. 2;

2. The monoclonal antibody of claim 1,

v of the monoclonal antibody _H The amino acid sequence of (2) is shown as SEQ ID No. 1 in a sequence table;

v of the monoclonal antibody _L The amino acid sequence of (2) is shown as SEQ ID No. 2 in the sequence table;

v encoding the monoclonal antibody _H The nucleotide sequence of (2) is shown as SEQ ID No. 3 in the sequence table;

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a murine monoclonal antibody.

4. Use of a monoclonal antibody according to any one of claims 1-3 for the preparation of a test strip or kit for detecting estriol.

5. The use according to claim 4, wherein,

the test paper is a colloidal gold detection test paper, or a fluorescent microsphere detection test paper, or a latex microsphere detection test paper;

the kit is an indirect competition ELISA detection kit.