Anti-thyroid stimulating hormone monoclonal antibody and its preparation method and application

Abstract

The invention discloses an anti-thyroid stimulating hormone monoclonal antibody and a preparation method and application thereof, and relates to the technical field of monoclonal antibody preparation, comprising: heavy chain variable region amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4; light chain variable region amino acid sequences SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; the rabbit monoclonal antibody involved in the invention is used as an important raw material for diagnosis, has higher affinity than traditional mouse monoclonal antibodies, can greatly improve the sensitivity of detection reagents, has a higher clinical compliance rate, and has important clinical significance for the diagnosis and treatment of diseases such as primary hyperthyroidism, thyroid cancer, and thyroid nodules.

Description

Technical Field

[0001] The present invention belongs to the technical field of monoclonal antibody preparation, and in particular is an anti-thyroid stimulating hormone monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Thyroid stimulating hormone (TSH), also known as thyroid stimulating hormone, thyroid stimulating hormone, and thyrotropin, is a glycoprotein hormone secreted by basophils of the pituitary gland.

[0003] Thyroid hormone is a very sensitive and specific indicator for testing thyroid function and is widely used in the screening, diagnosis, treatment effect evaluation and prognosis of the following diseases.

[0004] Screening for thyroid diseases: TSH has important clinical significance in the diagnosis of diseases such as primary hyperthyroidism, thyroid cancer, chronic lymphocytic thyroiditis, thyroid nodules or nodular goiter.

[0005] Primary hypothyroidism: Clinical manifestations include elevated serum TSH concentrations. Thyroid abnormalities, thyroid inflammation, and thyroid destructive (damage) diseases can all lead to insufficient synthesis and secretion of thyroid hormones, causing primary hypothyroidism and elevated serum TSH. Regardless of the degree of primary hypothyroidism, the first manifestation is an increase in serum TSH. At the same time, the magnitude of the increase in serum TSH is positively correlated to the degree of primary hypothyroidism, that is, the higher the serum TSH, the lower the thyroid function. In subclinical primary hypothyroidism, only serum TSH is elevated, and serum free thyroxine (FT4) is within the normal range. In clinical hypothyroidism, in addition to elevated serum TSH, serum FT4 may be lower than normal.

[0006] TSH-secreting tumors:

[0007] When pituitary TSH cells undergo tumorigenesis, they secrete TSH autonomously, leading to elevated serum TSH levels.

[0008] Monitoring the therapeutic effect of thyroid disease

[0009] Clinically, when patients with hypothyroidism receive thyroid hormone replacement therapy, patients with thyroid nodules receive thyroid hormone suppression therapy, and patients with hyperthyroidism receive antithyroid treatment, the functional status of the pituitary-thyroid axis can be assessed by monitoring changes in serum TSH to adjust the drug dosage.

[0010] Currently, the clinical quantitative detection of this project mainly includes chemiluminescence and fluorescent immunochromatography, but there is a general problem of low sensitivity. In order to improve detection sensitivity and clinical relevance, it is particularly important to develop high-affinity antibodies. Summary of the invention

[0011] The present invention aims to solve at least one of the technical problems existing in the prior art; to this end, the present invention provides an anti-thyroid stimulating hormone monoclonal antibody and a preparation method and application thereof.

[0012] An anti-thyroid stimulating hormone monoclonal antibody, comprising: heavy chain variable region amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4;

[0013] The amino acid sequences of the light chain variable regions are SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0014] A method for preparing an anti-thyroid stimulating hormone monoclonal antibody, the method specifically comprising the following steps:

[0015] (1) Animal immunization: TSH protein was used as an antigen, fully emulsified with an equal amount of Freund's complete adjuvant, and then injected subcutaneously into rabbits for immunization. Three booster immunizations were performed at intervals of two weeks. Blood was collected from the rabbit vein 5-8 days after the last booster immunization, and the serum titer was detected by ELISA. When the serum titer reached the standard of >128K, peripheral blood mononuclear cell suspension was prepared;

[0016] (2) Preparation of PBMC: Add Ficoll, a lymphocyte separation solution, to a centrifuge tube, take anticoagulated peripheral blood and sterile PBS and mix thoroughly at a ratio of 1:1-1:5, use a pipette to slowly stack it on the stratified liquid surface along the tube wall, centrifuge horizontally at 400g×30 minutes, remove part of the upper liquid after centrifugation, and leave about 1mL. Use a pipette to insert into the cloud layer to absorb the mononuclear cells; place in another centrifuge tube, add more than 5 times the volume of PBS, centrifuge at 300g×10 minutes, and wash the cells twice; after the last centrifugation, discard the supernatant, add red blood cell lysis solution, incubate at room temperature for 2 minutes to lyse the red blood cells, add 10mL PBS, centrifuge at 300g×10 minutes, and wash the cells twice; discard the supernatant, add RPMI1640 containing 10% FBS, resuspend the cells, and count;

[0017] (3) Obtaining single B cells: Using the prepared PBMCs, labeling the antigens, and using a single-cell chip screening platform to perform cell screening to obtain B cells with antigen specificity; obtaining the light and heavy chain gene sequences of the antibody by gene amplification of the target B cells.

[0018] Furthermore, after the preparation of the anti-TSH monoclonal antibody, the heavy and light chain gene sequences of the anti-TSH monoclonal antibody need to be obtained, and the specific method is as follows:

[0019] (1) cDNA preparation: RNA is extracted from the sorted B cells, and cDNA is obtained by reverse transcription using RNA as a template, and the genes of the light and heavy chains are amplified by PCR;

[0020] (2) Obtaining light and heavy chain genes: Amplify using rabbit-specific primers, clone the amplified DNA sequences into vectors, and then transform and plate, pick clones, extract plasmids and sequence;

[0021] (3) Analysis of the variable region genes of the light and heavy chains of the antibody: The VH, VL and constant regions are analyzed based on the sequencing results to ultimately obtain the full-length amino acid sequence of the heavy chain and the full-length amino acid sequence of the light chain.

[0022] Furthermore, the method also includes the expression and purification of anti-TSH monoclonal antibodies, specifically in the following manner:

[0023] (1) Cultivate N293 suspension cells until the density reaches 1-3×10 6 / ml, cell transfection was performed when the survival rate was >80-90%;

[0024] (2) transfecting N293 cells with the recombinant plasmid containing the antibody light and heavy chain genes: the mass ratio of PEI / plasmid is 2:1-5:1, and the transfection ratio is 1-3 μg plasmid / 1 ml N293 cells;

[0025] (3) 5-7 days after transfection, the cell supernatant was collected and affinity purified using Protein A resin to obtain high-purity antibody protein. The elution buffer was replaced with PBS by dialysis, and the antibody concentration was measured using Nanodrop.

[0026] A thyroid stimulating hormone detection kit, the kit adopts the monoclonal antibody prepared by the above-mentioned preparation method, or the kit contains the above-mentioned monoclonal antibody.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses TSH eukaryotic recombinant protein as an immunogen, and its structure is closer to the natural TSH protein conformation. Rabbits are immunized, and PBMCs of the rabbits are sorted to obtain cells that specifically bind to the immunogen. Then, the antibody is gene amplified to obtain the light and heavy chain gene sequences of the antibody. The rabbit monoclonal antibody with high purity and high affinity is obtained through subsequent cell recombination expression. The rabbit monoclonal antibody involved in the present invention is an important raw material for diagnosis, and has higher affinity than traditional mouse monoclonal antibodies, can greatly improve the sensitivity of detection reagents, has a higher clinical compliance rate, and has important clinical significance for the diagnosis and treatment of diseases such as primary hyperthyroidism, thyroid cancer, and thyroid nodules. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a chromatogram of the anti-TSH monoclonal antibody of the present invention detected by HPLC;

[0030] Figure 2 This is a graph showing the binding activity of anti-TSH monoclonal antibodies to TSH protein determined by indirect ELISA method of the present invention;

[0031] Figure 3 The test results of the time-resolved fluorescence TSH detection reagent 1 (TSH mAb1+TSH mAb2 paired combination) for clinical sample correlation;

[0032] Figure 4 The test results of the time-resolved fluorescence TSH detection reagent 2 (TSH mAb3+TSH mAb4 paired combination) for clinical sample correlation;

[0033] Figure 5 This is a schematic diagram of the KD results of the four TSH monoclonal antibodies of the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] See also Figure 1-Figure 4 The present application provides an anti-thyroid stimulating hormone monoclonal antibody and a preparation method and application thereof, including:

[0036] 1. Preparation of anti-TSH monoclonal antibodies:

[0037] (1) Animal immunization: TSH protein was used as an antigen, fully emulsified with an equal amount of Freund's complete adjuvant, and then injected subcutaneously into rabbits for immunization. Three booster immunizations were performed at intervals of two weeks. Blood was collected from the rabbit vein 5-8 days after the last booster immunization, and the serum titer was detected by ELISA. When the serum titer reached the standard of >128K, peripheral blood mononuclear cell (PBMC) suspension was prepared;

[0038] (2) Preparation of PBMC: Add Ficoll, a lymphocyte separation solution, to a centrifuge tube, take anticoagulated peripheral blood, i.e. whole blood, and mix thoroughly with sterile PBS at a ratio of 1:1-1:5, and use a pipette to slowly stack it on the stratified liquid surface along the tube wall, centrifuge horizontally at 400g for 30 minutes, remove part of the upper liquid after centrifugation, and leave about 1 mL. Use a pipette to insert into the cloud layer to absorb the mononuclear cells; place in another centrifuge tube, add more than 5 times the volume of PBS, centrifuge at 300g for 10 minutes, and wash the cells twice; after the last centrifugation, discard the supernatant, add red blood cell lysis solution, incubate at room temperature for 2 minutes, lyse the red blood cells, add 10 mL PBS, centrifuge at 300g for 10 minutes, and wash the cells twice; discard the supernatant, add RPMI1640 containing 10% FBS, resuspend the cells, and count;

[0039] (3) Obtaining single B cells: Using the prepared PBMCs, labeling the antigens, and using a single-cell chip screening platform to perform cell screening to obtain antigen-specific B cells; obtaining the light and heavy chain gene sequences of the antibody by gene amplification of the target B cells.

[0040] 2. Obtaining the heavy and light chain gene sequences of anti-TSH monoclonal antibodies;

[0041] (1) cDNA preparation: RNA was extracted from the sorted B cells in Example 1, and reverse transcription was performed using the RNA as a template to obtain cDNA, and the genes of the light and heavy chains were amplified by PCR;

[0042] (2) Obtaining light and heavy chain genes: Amplify using rabbit-specific primers, clone the amplified DNA sequences into vectors, and then transform and plate, pick clones, extract plasmids and sequence;

[0043] (3) Analysis of antibody light and heavy chain variable region genes: VH, VL and constant regions were analyzed based on sequencing results, and finally the heavy chain variable region amino acid sequence (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) and the light chain variable region amino acid sequence (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8) were obtained. The CDR region was underlined.

[0044] TSHmAb1:

[0045] VH (SEQ ID NO: 1):

[0046] QSVGESGGRL VTPGTPLTLT CTAS GFSLSN YH MCWVRQAP GKGLEYIGF IYVSGVT<h2 style=";text-align:left;direction:ltr">YYTNWAKGRFTISK TSTTVDLKIT SPTTEDTATY FC<h2 style=";text-align:left;direction:ltr"> ARSDDNNEFNI <h2 style=";text-align:left;direction:ltr"> WGPGT LVTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:5):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> AIVMTQTPSS ASAAVGGTVT INCQAS<h2 style=";text-align:left;direction:ltr"> QSIN NE <h2 style=";text-align:left;direction:ltr"> LSWYQQKPGQPPKLLIY<h2 style=";text-align:left;direction:ltr"> LAS <h2 style=";text-align:left;direction:ltr"> YLASGVSSRFKGSRSGTQ FTLTISGVQC DDAATYYC<h2 style=";text-align:left;direction:ltr"> QQCYSTEDVHNT <h2 style=";text-align:left;direction:ltr"> FGGGTEVVV K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> TSH mAb2:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:2):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] <h2 style=";text-align:left;direction:ltr"> QSVEESGGRL VTPGTPLTLT CTVS<h2 style=";text-align:left;direction:ltr"> GIDLS RHA <h2 style=";text-align:left;direction:ltr"> MSWVRQAPGKGLEWIGI<h2 style=";text-align:left;direction:ltr"> INAGGST <h2 style=";text-align:left;direction:ltr"> YYASWAKGRFTISK TSTTVDLKIT SPTTEDTATYFC<h2 style=";text-align:left;direction:ltr"> ARVPGY LSYDMDL <h2 style=";text-align:left;direction:ltr"> WGPGTL VTVSS;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0052] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:6):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0053] <h2 style=";text-align:left;direction:ltr"> ALVMTQTPAS VSAAVGDTVT INCQAS<h2 style=";text-align:left;direction:ltr"> QSIG NY <h2 style=";text-align:left;direction:ltr"> LSWYQQ KPGQPPKLLI Y<h2 style=";text-align:left;direction:ltr"> GAS <h2 style=";text-align:left;direction:ltr"> NLESGVPSRFKGSGSG TEYTLTISDL ECDDAATYYC<h2 style=";text-align:left;direction:ltr"> LGEANVANGDVA <h2 style=";text-align:left;direction:ltr"> FGGGTEV VVK;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0054] <h2 style=";text-align:left;direction:ltr"> TSH mAb3:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0055] <h2 style=";text-align:left;direction:ltr"> VH(SEQ ID NO:3):<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0056] <h2 style=";text-align:left;direction:ltr"> QSLEESGGRL VTPGTPLTLT CTAS<h2 style=";text-align:left;direction:ltr"> GFSLSS YA <h2 style=";text-align:left;direction:ltr"> GVSWVRQAP GKGLEWIGY<h2 style=";text-align:left;direction:ltr"> ITYDGGT <h2 style=";text-align:left;direction:ltr"> YYASWAKGRFTISK TSTTVDLKIT SPTTEDTATY FC<h2 style=";text-align:left;direction:ltr"> ARAPLGSPAYMDL <h2 style=";text-align:left;direction:ltr"> WGPGTLVTVS S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0057] <h2 style=";text-align:left;direction:ltr"> VL(SEQ ID NO:7):<h2 style=";text-align:left;direction:ltr">

[0058] AQVLTQTPAS VSAAVGGTVT INCQSS QSVY DNNW LGWYQQ KPGQPPKFLIY SAS TLAPGVPSRFKGSGSG TQFTLTISDL ECVDAATYYC AGYYYSGSAT GFA FGGGTEVVVK;

[0059] TSH mAb4:

[0060] VH (SEQ ID NO:4):

[0061] QEQLEESGGR LVTPGTPLTL TCTVS GFSLSS YA MNWVRQA PGKGLEWIGV INVYGST YYANWAKGRFTIS KTSTTVELKI TSPTTEDTAT YFC ARVGDSYGYAYAYYFNI WGQGTP VTVSS;

[0062] VL (SEQ ID NO:8):

[0063] AYDMTQTPAS VEVAVGGTVT INCQSS QSIS NL LAWYQQKP GQRPKLLIY GAS KLASGVPSRFSGSGSGTD FTLTISGVQC DDAATYYC QS GYESAGVT FGGGTEVVVQ;

[0064] 3. Expression and purification of anti-TSH monoclonal antibodies;

[0065] (1) Cultivate N293 suspension cells until the density reaches 1-3×10 6 / ml, cell transfection was performed when the survival rate was >80%-90%;

[0066] (2) Transfecting N293 cells with the recombinant plasmid containing antibody light and heavy chain genes in Example 2: the mass ratio of PEI / plasmid is 2:1-5:1, and the ratio of transfection is 1-3 μg plasmid / 1 ml N293 cells;

[0067] (3) 5-7 days after transfection, the cell supernatant was collected and affinity purified using Protein A resin to obtain high-purity antibody protein. The elution buffer was replaced with PBS by dialysis, and the antibody concentration was measured using Nanodrop;

[0068] 4. Detection of anti-TSH monoclonal antibodies

[0069] (1) Purity test of anti-TSH antibodies:

[0070] Take 10 μl of the purified antibody and add it to the inner tube, exhaust all bubbles and wait for loading; prepare 150 mM phosphate buffer for HPLC, adjust the pH to 7.0, and sterilize the buffer by vacuum filtration, then place it in an ultrasonic instrument for ultrasonic degassing and set it aside; turn on the instrument, turn on each module of the HPLC, and preheat the instrument. First, exhaust the bubbles of the instrument system, then use the mobile phase to replace the solution with the instrument, connect the SEC column to the instrument, exhaust the bubbles, and equilibrate the column with the equilibration buffer, and set it aside; sample loading: open the software, perform methodology editing, generate a sequence, and then load the sample; result analysis and processing: open the software, import the results, perform analysis, and generate an analysis report.

[0071] like Figure 1-4 As shown, the purity of the four TSH monoclonal antibodies reached 99%;

[0072] (2) Anti-TSH antibody activity detection:

[0073] ① Antigen coating: dilute the antigen to an appropriate concentration, then add it to an empty ELISA plate at a volume of 100 u l / well; place the ELISA plate in a 4°C refrigerator and incubate overnight;

[0074] ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry;

[0075] ③ Incubate the primary antibody: dilute the antibody to a suitable concentration gradient with 1xPBS; add the diluted antibody to the ELISA plate, 100 μl per well, reaction time: 37 degrees / 1h; wash the plate 5 times with a plate washer and dry;

[0076] ④ Incubate the secondary antibody: dilute the enzyme-labeled secondary antibody in an appropriate ratio, then add it to the ELISA plate, 100 μl per well, reaction time: 37 degrees / 30 minutes; wash the plate 5 times with a plate washer and dry;

[0077] ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37 degrees / 15 minutes;

[0078] ⑥ Stop and read: add stop solution, volume 100u l / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the test results into Excel table;

[0079] ⑦Data analysis: Use software to analyze data, draw graphs, and fit EC50.

[0080] like Figure 5As shown, the KD of the four TSH monoclonal antibodies all reached 10 -11 M, all have high affinity. Antibody detection conditions are as follows:

[0081] The raw material information is as follows:

[0082] name Part Number batch number Concentration: mg / ml Remark TSH antigen DRA17 0332309MF102 1.93 -- TSH mAb1 R567e9 7537733-1 3 Rabbit Source TSH mAb2 R568e3 7955679-1 4 Rabbit Source TSH mAb3 R516c6 7535693-1 5 Rabbit Source TSH mAb4 R517t4 6265188-1 3 Rabbit Source

[0083] The operation points are as follows:

[0084]

[0085] The results are shown in the following table:

[0086]

[0087]

[0088] 5. Preparation of TSH detection reagent by time-resolved fluorescence method

[0089] (1) Preparation of fluorescent microsphere-anti-TSH monoclonal antibody complex

[0090] ① Pretreatment of time-resolved fluorescent microspheres

[0091] Take 25ul of time-resolved fluorescent microspheres, add 400ul of PB buffer solution at pH 6.0, mix well, centrifuge at 14000rpm, 10℃ for 15min, remove the supernatant, and resuspend the precipitate in 400ul of PB buffer solution at pH 6.0; prepare 2.5mg / ml carbodiimide and succinimide, add 2.5ul each, react at room temperature for 30min, centrifuge at 14000rpm, 10℃ for 15min, and resuspend the precipitate in 200ul of PB solution at pH 6.0 to obtain an activated microsphere solution;

[0092] ②Microspheres coupled to TSH monoclonal antibody

[0093] 10-50ug TSH monoclonal antibody was added to the above-mentioned time-resolved fluorescent microsphere solution, mixed, reacted at room temperature for 3 hours, blocked with 1-5% BSA at room temperature for 0.5 hour, centrifuged at 14000rpm, 10°C for 15min, the supernatant was removed, and the precipitate was resuspended with 50ul preservation solution to obtain a fluorescent microsphere-anti-TSH monoclonal antibody complex; the preservation solution contained 10-100mMTris buffer at pH7.5-8.5, 1%-5% sucrose, 1%-5% trehalose, and 1%-5% BSA;

[0094] (2) Gold spraying

[0095] The final concentration of the fluorescent microsphere-anti-TSH monoclonal antibody complex was diluted to 0.5-2 mg / mL, and the gold spraying volume was set to 1-4 μL / cm, and then the sprayed marking pad was placed in a 37°C oven to dry for 1-4 hours, and then the marking pad was placed in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36%, and sealed for storage;

[0096] (3) Coating quality control line C, detection line T

[0097] ① Preparation of coating solution for quality control line C and test line T: Take goat anti-rabbit antibody and dilute it with 10-100mM PBS buffer at pH 7.4 to 0.5-1.0mg / mL antibody solution; Take anti-TSH monoclonal antibody and dilute it with 10-100mM PBS buffer at pH 7.4 to 0.5-1.0mg / mL antibody solution;

[0098] ② Coating: Coat the control line C and the detection line T coating solution to the control line and the detection line of the nitrocellulose membrane respectively, with the interval between the C and T lines being 5-8 mm, and the coating amount being 0.8-1.5 μL / cm; place the coated PVC base plate in a 37°C oven for 12-24 hours, and then place the PVC base plate in an aluminum foil bag pre-added with desiccant at a humidity not higher than 36%, seal and store for later use;

[0099] (4) Pretreatment of sample pad

[0100] Clean the drying net, soak the sample pad with the sample pad pretreatment solution, and then drain it until no water drops. Then, dry it in a 37°C oven for 4-6 hours, and then seal it in an aluminum foil bag with desiccant added in advance at a humidity of no more than 36% for storage;

[0101] The sample pad pretreatment solution contains 10-100 mM Tris buffer at pH 7.5-8.5, 0.1%-0.5% PVP10, 0.1%-0.5% PEG20000, 0.1%-0.5% Tween20 and 0.1%-0.5% casein sodium;

[0102] (5) Assembly and cutting of membrane materials

[0103] At a humidity not higher than 36%, take out the absorbent pad, the treated sample pad and the marking pad, cut them according to the size of (15-20mm)*300mm, take out the coated PVC bottom plate, and assemble them according to the following process: first stick the marking pad on the PVC bottom plate so that the marking pad presses the nitrocellulose membrane by 1-2mm, then stick the sample pad so that the sample pad part presses the marking pad by 3-5mm, and finally stick the absorbent pad so that the absorbent pad presses the nitrocellulose membrane by 1-2mm; cut the assembled PVC bottom plate into 3-5mm time-resolved fluorescent immunochromatographic test strips for detecting TSH.

[0104] The antibody pairing of test strip 1 is: TSH mAb2 (for preparing microsphere-antibody complex) + TSH mAb1 (for T-line coating); the antibody pairing of test strip 2 is: TSH mAb3 (for preparing microsphere-antibody complex) + TSH mAb4 (for T-line coating).

[0105] (6) Test strip detection

[0106] Take the Roche assigned clinical sample and add 75 μl directly to the sample addition point. Start testing with the fluorescent immunoassay analyzer at 15 minutes and process the data.

[0107] Correlation between two different antibody paired TSH detection reagents 2 The values ​​were all greater than 0.97, which had good clinical consistency with Roche reagent.

[0108] The above embodiments are only used to illustrate the technical method of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical method of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical method of the present invention.

Claims

1. An anti-thyroid stimulating hormone monoclonal antibody, characterized in that: comprising the following amino acid sequences: heavy chain variable region SEQ ID NO: 1 and light chain variable region SEQ ID NO: 5; or a heavy chain variable region of SEQ ID NO: 2 and a light chain variable region of SEQ ID NO: 6; or a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of SEQ ID NO: 7; Or a heavy chain variable region of SEQ ID NO:4 and a light chain variable region of SEQ ID NO:

8.

2. A thyroid stimulating hormone detection kit, characterized in that: The kit contains the monoclonal antibody according to claim 1.

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