A murine monoclonal antibody against TPO and its preparation method and application

Abstract

The present application relates to the technical field of in vitro diagnosis, in particular to a mouse monoclonal antibody against TPO and a preparation method and application thereof. The present application discloses a mouse monoclonal antibody against TPO and a preparation method and application thereof, wherein the mouse monoclonal antibody is named TPO-1#, the heavy chain variable region of the mouse monoclonal antibody TPO-1# has an amino acid sequence shown in SEQ ID NO. 1, and the light chain variable region has an amino acid sequence shown in SEQ ID NO. 2. In the present application, recombinant human thyroid peroxidase (TPO) is selected as an immunogen, 5-week-old female Balb / c mice are immunized, and a new specific mouse monoclonal antibody against TPO is obtained. A kit for detecting the concentration of Anti-TPO in serum is established by using the mouse monoclonal antibody prepared by the present application, and compared with the prior art, the detection technology has higher sensitivity, and has better correlation with the same type of detection kit of Roche.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnosis, in particular to a mouse monoclonal antibody against TPO and a preparation method and application thereof. BACKGROUND

[0002] Thyroid peroxidase is abbreviated as TPO, mainly synthesized by thyroid follicular cells, containing 933 amino acids, and the molecular weight is 103kDa. TPOAb is a common autoantibody in the serum of pregnant women with autoimmune thyroid disease, and the content in normal human body is extremely low. The antigen it targets, thyroid peroxidase, is a key enzyme for thyroid kinase biosynthesis, which can catalyze the iodination of tyrosine residues. TPOAb can utilize antibody-dependent cell-mediated cytotoxic effect to destroy thyroid cells, leading to insufficient secretion of thyroid hormones and causing autoimmune-related hypothyroidism. The detection index has high specificity and high sensitivity, which is beneficial to timely find the phenomenon of subclinical hypothyroidism in pregnant women during pregnancy, and is beneficial to provide accurate data for gynecologists.

[0003] The detection of Anti-TPO concentration in serum can use indirect ELISA method or competitive ELISA method. Whether it is indirect ELISA method or competitive ELISA method, the TPO protein used for detection is generally not directly coated on the solid support, but is captured and coated on the solid support by a tag antibody or biotin-avidin. For example, Roche mainly uses biotinized-TPO recombinant antigen and ruthenium-goat anti-TPO as coating and labeling materials and adopts one-step competitive method to build a kit detection method; and Beckman mainly uses indirect method, uses biotinized-TPO recombinant antigen and enzyme-labeled protein A or as coating and labeling materials to build a kit detection method.

[0004] The principles of kits of different manufacturers are inconsistent, and the performance differences between each other are large (sensitivity, specificity, reference interval, detection value, etc.), and the overall coincidence rate is about 90%. However, the current methods for detecting Anti-TPO in serum have low sensitivity. SUMMARY

[0005] Therefore, the present application provides a mouse monoclonal antibody against TPO and a preparation method and application thereof.

[0006] The application provides a mouse monoclonal antibody against TPO and a preparation method and application thereof. The application discloses a mouse monoclonal antibody against TPO, which is named TPO-1#, wherein the mouse monoclonal antibody TPO-1# has a heavy chain variable region with an amino acid sequence shown in SEQ ID NO. 1 and a light chain variable region with an amino acid sequence shown in SEQ ID NO. 2. The application also discloses a preparation method and application of the antibody. The application selects recombinant human thyroid peroxidase (TPO) as an immunogen, immunizes 5-week-old female Balb / c mice, and obtains a new specific mouse monoclonal antibody against TPO. A magnetic particle chemiluminescence method for detecting Anti-TPO in serum is established by using the mouse monoclonal antibody prepared by the application, and the detection technology has higher sensitivity and better correlation with a Roche similar detection kit compared with prior art.

[0007] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions.

[0008] The application provides an anti-TPO mouse monoclonal antibody, which comprises a heavy chain and a light chain.

[0009] (I) the CDR1, CDR2 and CDR3 of the heavy chain have the amino acid sequences shown in SEQ ID NO. 7-9 in turn; and

[0010] the CDR1, CDR2 and CDR3 of the light chain have the amino acid sequences shown in SEQ ID NO. 10-12 in turn; or

[0011] (2) a sequence in which one or more amino acids in the amino acid sequence shown in (1) are substituted, deleted, added and / or replaced; or

[0012] (3) a sequence with more than 75% homology with the amino acid sequence shown in (2).

[0013] In some specific embodiments of the application, the anti-TPO mouse monoclonal antibody comprises a heavy chain variable region and a light chain variable region.

[0014] The heavy chain variable region has:

[0015] (I) the amino acid sequence shown in SEQ ID NO. 1; or

[0016] (II) a sequence in which one or more amino acids in the amino acid sequence shown in (I) are substituted, deleted, added and / or replaced; or

[0017] (III) a sequence with more than 75% homology with the amino acid sequence shown in (I).

[0018] The light chain variable region has:

[0019] (IV) the amino acid sequence shown in SEQ ID NO. 2; or

[0020] (V) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced in the amino acid sequence shown in (IV); or

[0021] (VI) a sequence having more than 75% homology with the amino acid sequence shown in (IV).

[0022] In some embodiments of the present application, the primer set for amplifying the light chain variable region comprises:

[0023] (I) a nucleotide sequence shown in SEQ ID NO. 3; and

[0024] (II) a nucleotide sequence shown in SEQ ID NO. 4; or

[0025] (III) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I) or (II), but different from the nucleotide sequence shown in (I) or (II) due to the degeneracy of the genetic code; or

[0026] (IV) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in any one of (I) to (III), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in any one of (I) to (III); or

[0027] (V) a nucleotide sequence having at least 75% sequence homology with the nucleotide sequence shown in any one of (I) to (IV).

[0028] In some embodiments of the present application, the primer set for amplifying the light chain variable region comprises:

[0029] (I) a nucleotide sequence shown in SEQ ID NO. 3; and

[0030] (II) a nucleotide sequence shown in SEQ ID NO. 4; or

[0031] (III) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (I) or (II), but different from the nucleotide sequence shown in (I) or (II) due to the degeneracy of the genetic code; or

[0032] (IV) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in any one of (I) to (III), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in any one of (I) to (III); or

[0033] (V), a nucleotide sequence having at least 75% sequence homology with any one of the nucleotide sequences of (I)-(IV);

[0034] The PCR reaction conditions for amplifying the light chain variable region or the heavy chain variable region include: 95℃ 5min; 95℃ 15s, 55℃ 45s, 72℃ 30s, 30 cycles; 72℃ 7min.

[0035] In some embodiments of the present application, the titer of the anti-TPO mouse monoclonal antibody includes 1:32000-1:256000.

[0036] In some embodiments of the present application, as preferred, the titer of the anti-TPO mouse monoclonal antibody is 1:256000.

[0037] In some embodiments of the present application, the Kd value of the anti-TPO mouse monoclonal antibody includes 1.001E-9-1.333E-7 mol / L. D

[0038] In some embodiments of the present application, as preferred, the Kd value of the anti-TPO mouse monoclonal antibody is 1.001E-9 mol / L. D

[0039] The present application also provides a preparation method of the anti-TPO mouse monoclonal antibody, which comprises fusing the spleen and myeloma cells of the mouse immunized by the TPO recombinant antigen, screening and purifying to obtain the anti-TPO mouse monoclonal antibody.

[0040] In some embodiments of the present application, the preparation method of the anti-TPO mouse monoclonal antibody includes mouse immunization, hybridoma cell preparation, anti-TPO mouse monoclonal antibody purification, anti-TPO monoclonal antibody titer detection, TPO mouse monoclonal antibody affinity determination, anti-TPO mouse monoclonal antibody identification, and monoclonal antibody sequencing.

[0041] In some embodiments of the present application, the mouse immunization step includes: using an immunogen, fully emulsifying the TPO recombinant antigen with Freund's complete adjuvant, and then immunizing a 5-week-old female mouse through intraperitoneal injection, with an initial immunization dose of 100 μg per mouse; 21 days and 42 days after the first immunization, the second and third immunizations are performed, respectively; about 10 days after the third immunization, tail blood is collected, and the serum titer is detected by indirect method using a 96-well plate coated with TPO recombinant antigen;

[0042] The immunogen is a recombinant human thyroid peroxidase (TPO) containing 828 amino acids, which is purchased from HyTest;

[0043] ​​The mice include Balb / c mice; the second and third immunization dose is 50 μg per mouse.

[0044] In some embodiments of the present application, the preparation of the hybridoma cells includes: selecting mice for intrasplenic boosting, taking the mouse spleen 3 days after boosting and fusing with mouse myeloma cells, resuspending the fused cells, culturing, detecting the specific antibody content, subcloning, and obtaining 3 hybridoma cell strains against TPO;

[0045] The mice are mice with serum titers greater than 10 4 by indirect method;

[0046] The immunization dose is 100 μg per mouse;

[0047] The ratio of the mouse spleen to the myeloma cells includes 10:1; the myeloma cells include NS1; the resuspension uses HAT DMEM medium (Gibco);

[0048] The culture temperature is 37°C, and the CO2 concentration is 5%;

[0049] The subcloning method is to select positive wells with OD values not less than 0.5 by limited dilution.

[0050] In some embodiments of the present application, the method for purifying the anti-TPO mouse monoclonal antibody includes SPA; the purity of the purified anti-TPO mouse monoclonal antibody is more than 90%.

[0051] In some embodiments of the present application, the step of detecting the titer of the anti-TPO monoclonal antibody includes: diluting the TPO recombinant protein, washing 3 times with PBST after blocking, gradient diluting the purified monoclonal antibody, after reaction, washing the plate 5 times with PBST, adding 1:4000 diluted HRP-goat anti-mouse IgG (SIGMA), 100 μL / well, 37°C reaction for 30 min, washing the plate 5 times with PBST, adding a mixture of enzyme-free substrate and color developing agent, 100 μL / well, 25°C reaction for 10 min in the dark, adding 50 μL of 2 mol / L sulfuric acid to terminate the reaction, and measuring the absorbance of TPO-1#, TPO-2#, and TPO-3# at 450 nm;

[0052] The dilution solution is CB buffer with a concentration of 0.05 mol / L and pH=9.6; the dilution concentration is 1 μg / mL; the blocking reagent is 1% Casein, the temperature is 37°C, and the time is 2 h;

[0053] The concentration of the purified monoclonal antibody is 5 mg / mL;

[0054] The solution used in the gradient dilution includes 0.02 mol / L PBS buffer with pH=7.4;

[0055] The ratio of the gradient dilution includes 1:1K, 1:2K, 1:4K, 1:8K, 1:16K, 1:32K, 1:64K, 1:128K, 1:256K, 1:512K, 1:1024K, 1:2048K;

[0056] The reaction temperature is 37℃, and the time is 30 min;

[0057] The mixing ratio of the mixture is 1:1; the antibody titer of TPO-1# is the highest, which is 1:256000;

[0058] The antibody titer of TPO-2# is 1:64000; and the antibody titer of TPO-3# is 1:32000.

[0059] In some specific embodiments of the present application, the TPO mouse monoclonal antibody affinity determination step includes: after the TPO recombinant antigen is diluted to 180 μL, the chip 2 channel (or 4 or 1 channel) is selected for antigen coupling experiment; after the three TPO antibodies are diluted, they are sequentially diluted to 4000, 2000, 1000, 500, 250, 125, 62.5 nmol / L, the flow rate is set to 25 μL / min, the binding time is 90 s, the dissociation time is 300 s, and the reaction temperature is set to 25℃ by default, and then detection is performed;

[0060] The solution used in the dilution is acetate (pH=4.5); and the concentration after the dilution is 10 μg / mL;

[0061] The solution used in the dilution of the three TPO antibodies includes 1×HBS-EP+(pH7.4) buffer;

[0062] The concentration of the three TPO antibodies after the dilution is 8000 nmol / L;

[0063] The affinity K of the TPO-1# antibody is 1.001E-9 mol / L; the affinity K of the TPO-2# antibody is 1.333E-8 mol / L; and the affinity K of the TPO-3# antibody is 1.333E-7 mol / L. D D D

[0064] On the basis of the above research, the present application also provides the application of any of the following in the preparation of a product for detecting thyroid diseases:

[0065] (I), the anti-TPO mouse monoclonal antibody; and / or​​​

[0066] (II) the anti-TPO mouse monoclonal antibody prepared by the preparation method.

[0067] In some embodiments of the present application, the detection includes Anti-TPO in serum.

[0068] In some embodiments of the present application, the thyroid disease includes autoimmune thyroid disease.

[0069] The autoimmune thyroid disease includes hypothyroidism.

[0070] The present application also provides a product, including any of the following and acceptable adjuvants and / or auxiliaries:

[0071] (I) the anti-TPO mouse monoclonal antibody; and / or

[0072] (II) the anti-TPO mouse monoclonal antibody prepared by the preparation method.

[0073] In some embodiments of the present application, the product includes one or more of magnetic particles, reagents or kits.

[0074] The present application also provides a preparation method of a kit for detecting Anti-TPO concentration in serum, including the following steps:

[0075] Step one: coating the anti-TPO mouse monoclonal antibody according to any one of claims 1 to 4 and / or the anti-TPO mouse monoclonal antibody prepared by the preparation method of claim 5 with magnetic particles to obtain Anti-TPO magnetic particles;

[0076] Step two: forming a complex of the Anti-TPO magnetic particles, recombinant TPO antigen, Anti-TPO-containing serum to be detected and / or HRP-anti-human IgG to prepare the kit for detecting Anti-TPO concentration in serum.

[0077] The present application also provides a kit for detecting Anti-TPO concentration in serum prepared by the preparation method.

[0078] The present application also provides an ELISA method for detecting Anti-TPO concentration in serum, including the following:

[0079] Step one: coating the anti-TPO mouse monoclonal antibody and / or the anti-TPO mouse monoclonal antibody prepared by the preparation method with magnetic particles to obtain Anti-TPO magnetic particles;

[0080] Step two: the Anti-TPO magnetic microparticle, recombinant TPO antigen, Anti-TPO containing serum to be detected and / or HRP-anti-human IgG form a complex, and the Anti-TPO concentration in the serum is detected.

[0081] Specifically, the application provides an ELISA method for detecting the Anti-TPO concentration in serum, which comprises the following steps: coating magnetic microparticles with mouse monoclonal antibody TPO-1#, capturing TPO antigen, and detecting Anti-TPO by indirect method.

[0082] In some specific embodiments of the application, the step of coating magnetic microparticles with mouse monoclonal antibody TPO-1# comprises the following steps: taking 30 μL of mixed magnetic microparticle stock solution, washing 5 times with 300 μL, activating the magnetic microparticles for 1 h, washing the activated magnetic microparticles 2 times, adding monoclonal antibody TPO-1# into the magnetic beads, and coating at 4°C for 2 h, and sealing with BSA sealing liquid for 2 h.

[0083] The solution used for washing comprises PBS buffer solution with pH 7-8; the reagent used for activation comprises EDC and NHS; and the content of TPO-1# is 0.5 μg per human portion.

[0084] In some specific embodiments of the application, the step of capturing TPO antigen and detecting Anti-TPO by indirect method comprises the following steps: capturing recombinant TPO antigen and enzyme-labeled secondary antibody by a solid carrier, forming Anti-TPO magnetic microparticle + recombinant TPO antigen + Anti-TPO containing serum to be detected + HRP-anti-human IgG complex, and detecting the Anti-TPO concentration in serum of patients with autoimmune thyroid disease.

[0085] The solid carrier comprises magnetic beads suspension coated with TPO-1# antibody; and the enzyme-labeled secondary antibody comprises HRP-anti-human IgG.

[0086] The application provides a mouse monoclonal antibody against TPO, a preparation method and application thereof. The application has the advantages that a new mouse monoclonal antibody against TPO is prepared by using a recombinant human thyroid peroxidase (TPO) containing 828 amino acids purchased from HyTest as an immunogen, and the mouse monoclonal antibody prepared by using the application is used as a capture antibody to capture TPO antigen, so that an ELISA method for indirectly detecting the Anti-TPO concentration in serum is established, and the coincidence rate of the capture coating method with Roche is higher than that of direct coating of TPO antigen. BRIEF DESCRIPTION OF DRAWINGS

[0087] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced.

[0088] Figure 1SDS-PAGE analysis of the recombinant TPO antigen in Example 1;

[0089] Figure 2 SDS-PAGE analysis of the TPO mouse monoclonal antibody in Example 1;

[0090] Figure 3 Western blot identification results of the TPO-1# mouse monoclonal antibody in Example 1;

[0091] Figure 4 Relevance of the kit prepared in Example 2 of the application and Roche. DETAILED DESCRIPTION

[0092] The application discloses a mouse monoclonal antibody against TPO and a preparation method and application thereof, and a person skilled in the art can refer to the content of the application and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to a person skilled in the art, and they are regarded as being included in the application. The method and application of the application have been described through preferred embodiments, and a person skilled in the art can obviously change or appropriately change and combine the method and application described in the application without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0093] The application aims to provide a new mouse monoclonal antibody capable of specifically detecting Anti-TPO antibodies in serum on the basis of the prior art, and further provides a preparation method of the antibody and application of the antibody in Anti-TPO kits for detecting serum.

[0094] To achieve the above object, the application can adopt the following technical scheme:

[0095] The mouse monoclonal antibody against TPO provided by the application is named TPO-1#. The mouse monoclonal antibody TPO-1# has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO. 1 and a light chain variable region with the amino acid sequence shown in SEQ ID NO. 2.

[0096] Specifically, the amino acid sequence of SEQ ID NO. 1 is as follows:

[0097] QVQLQQPGSELVRPGVSVKLSCKASGYTFTSYWMYWVKQRHGQGL

[0098] EWIGNIYPGSGSTNYDEKFKSKVTLTIDTSSSTAYMYLNSLTSEDSAVYYC

[0099] TRRDYWGQGTTLTVSS

[0100] wherein the three CDR regions are CDR1: GYTFTSYW (SEQ ID NO. 7), CDR2: IYPGSGST (SEQ ID NO. 8), and CDR3: TRRDY (SEQ ID NO. 9).

[0101] The amino acid sequence of SEQ ID NO. 2 is as follows:

[0102] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPHLLIYGMSTLASGVPDRFSGSGSGTAFTLRISRVEGEDVGVYYCMQHLEYPLTFGAGTKLELK

[0103] wherein the three CDR regions are CDR1: KSLLHSNGNTY (SEQ ID NO. 10), CDR2: GMS (SEQ ID NO. 11), and CDR3: MQHLEYPLT (SEQ ID NO. 12).

[0104] Further, the murine monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

[0105] Still further, the heavy chain constant region and the light chain constant region are from murine IgG.

[0106] The murine monoclonal antibody against TPO of the present application is prepared by immunizing Balb / c mice with a recombinant human thyroid peroxidase (TPO) containing 828 amino acids purchased from HyTest as an immunogen, then fusing the spleen with myeloma cells NS1, and screening and purifying to obtain the murine monoclonal antibody against TPO. D The affinity K value of the prepared murine monoclonal antibody against TPO to the recombinant antigen is 1.001E-9 mol / L.

[0107] The murine monoclonal antibody against TPO prepared by the present application is used to establish an indirect ELISA detection kit. The murine monoclonal antibody TPO-1# against TPO is coated on magnetic beads as a capture antibody to capture TPO recombinant antigen, and HRP-anti-human IgG is added as an enzyme-labeled secondary antibody. The concentration of Anti-TPO in the serum of patients with autoimmune thyroid disease is detected by indirect method, which has extremely high sensitivity and can be applied to the clinical detection of autoimmune thyroid disease.

[0108] The advantage of the present application is that a new specific anti-TPO mouse monoclonal antibody is prepared by using a recombinant human thyroid peroxidase (TPO) containing 828 amino acids purchased from HyTest as an immunogen, the mouse monoclonal antibody prepared by using the present application is used as a capture antibody to capture the TPO antigen, and a serum Anti-TPO detection kit is constructed, and the capture coating method has a higher Roche coincidence rate than the direct TPO antigen coating method.

[0109] The anti-TPO mouse monoclonal antibody provided by the present application, the preparation method thereof, and the raw materials and reagents used in the application can be purchased from the market.

[0110] The present application is further described below in combination with examples:

[0111] Example 1: Preparation of anti-TPO mouse monoclonal antibody

[0112] 1. Mouse immunization

[0113] The present application uses a recombinant human thyroid peroxidase (TPO) containing 828 amino acids purchased from HyTest as an immunogen, and the electropherogram thereof is shown in Figure 1 The TPO recombinant antigen is fully emulsified with Freund's complete adjuvant, and then 5-week-old female Balb / c mice are intraperitoneally immunized, the initial immunization dose is 100 μg per mouse; the second and third immunizations are performed at intervals of 21 days and 42 days after the first immunization, and the immunization dose is 50 μg per mouse. About 10 days after the third immunization, tail blood is collected, and the serum titer is detected by indirect method using a 96-well plate coated with TPO recombinant antigen.

[0114] 2. Hybridoma cell preparation

[0115] Mice with an indirect detection serum titer greater than 10 4 are selected for intrasplenic booster immunization, and the immunization dose is 100 μg per mouse. Three days after booster immunization, the mouse spleen is taken and fused with mouse myeloma cells NS1 at a ratio of 10:1, and the fused cells are resuspended in DMEM medium (Gibco) containing HAT, and cultured in a 37℃, 5% CO2 carbon dioxide incubator.

[0116] About 6-7 days after fusion, the specific antibody content in the cell culture supernatant is detected by indirect method using a 96-well plate coated with TPO, and positive wells with an OD value not less than 0.5 are selected for 3 rounds of subcloning by limiting dilution method, and finally 3 hybridoma cell strains stably secreting anti-TPO are obtained.

[0117] 3. Purification of anti-TPO mouse monoclonal antibody

[0118] The obtained mouse hybridoma cells which can stably secrete anti-TPO were injected into the abdominal cavity of mice, and the ascites were collected and purified by SPA to obtain mouse monoclonal antibodies against TPO with a purity of more than 90% (such as Figure 2 ).

[0119] 4. Titer detection of anti-TPO monoclonal antibodies

[0120] TPO recombinant protein was diluted to 1 μg / mL with CB buffer of 0.05 mol / L and pH = 9.6, and 50 μL was added to each well of a 96-well enzyme immunoassay plate (Corning), which was coated at 4°C overnight. The next day, the plate was washed with PBST for 3 times, and then blocked with Casein of 1% concentration, 100 μL / well, at 37°C for 2 h. The purified monoclonal antibodies were respectively (all at a concentration of 5 mg / mL) diluted with PBS buffer of 0.02 mol / L and pH = 7.4 according to the gradient of 1:1K, 1:2K, 1:4K, 1:8K, 1:16K, 1:32K, 1:64K, 1:128K, 1:256K, 1:512K, 1:1024K, and 1:2048K. The diluted antibodies were added to the enzyme immunoassay plate coated with TPO recombinant protein, and 0.05 mol / L PBS buffer of pH = 7.4 was added to the negative control wells, 50 μL / well. After reaction at 37°C for 30 min, the plate was washed with PBST for 5 times, dried, and then 1:4000 diluted HRP-goat anti-mouse IgG (SIGMA) was added, 100 μL / well, and reacted at 37°C for 30 min. The plate was washed with PBST for 5 times, dried, and then a mixture of enzyme substrate and color developing agent (mixed according to a ratio of 1:1) was added, 100 μL / well, and reacted at 25°C in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was measured. The detection results are shown in Table 1, and the titer of TPO-1# antibody among the 3 antibodies was the highest, which was 1:256000.

[0121] Table 1. Titer detection of TPO monoclonal antibodies

[0122] Antibody No. OD value Titer TPO-1# 1.5 1:256K TPO-2# 1.4 1:64K TPO-3# 1.9 1:32K

[0123] 5. Affinity determination of TPO mouse monoclonal antibodies

[0124] The application adopts Biacore technology to analyze the interaction force between antigen and antibody. Biacore T200 instrument is selected. The TPO recombinant antigen is diluted to 10 μg / mL with acetate (pH = 4.5), 180 μL, and then the antigen coupling experiment is carried out in channel 2 (or 4 or 1) of the chip. Then the three TPO antibodies are diluted to 8000 nmol / L with 1xHBS-EP+ (pH = 7.4) buffer, and then sequentially diluted to 4000, 2000, 1000, 500, 250, 125, 62.5 nmol / L, and then set the flow rate to 25 μL / min, the binding time to 90 s, the dissociation time to 300 s, and the reaction temperature to 25 °C by default, and then the machine is detected. The results are shown in Table 2, k a The value represents the binding rate of antigen and antibody, K d The value represents the dissociation rate of antigen and antibody, the affinity K D of the three antibodies is 1.001E-9 (mol / L), 1.333E-8 (mol / L), and 1.333E-7 (mol / L), respectively, and the K D value of TPO-1# antibody is the smallest, that is, the affinity is the highest. Therefore, TPO-1# antibody is preferred in the subsequent.

[0125] Table 2: Affinity determination of TPO mouse monoclonal antibody

[0126] Antibody No. k a (1 / Ms)]]> k d (1 / s) K D (mol / L) TPO-1# 1.132E+4 1.133E-5 1.001E-9 TPO-2# 4.543E+4 6.057E-4 1.333E-8 TPO-3# 4.679E+3 4.758E-4 1.333E-7

[0127] 6. Identification of anti-TPO mouse monoclonal antibody

[0128] The TPO recombinant protein is diluted to 0.1 mg / mL with CB buffer of 0.05 mol / L and pH = 9.6, 20 μL of the diluted recombinant protein is subjected to 10% SDS-PAGE electrophoresis, and after the electrophoresis is completed, the gel is placed in the transfer buffer for 10 min, and the membrane is transferred at 200 mA for 30 min. The membrane is placed in the blocking solution and blocked at 25 °C overnight, the blocking solution is discarded, the purified TPO-1# monoclonal antibody with a concentration of 5 μg / mL is added, and incubated at 4 °C for 2 h. The membrane is washed with TBST for 3 times, each time for 3 min. The horseradish peroxidase-labeled goat anti-mouse IgG diluted by 1 / 4000 is added, and incubated at 37 °C for 2 h. The membrane is washed with TBST for 4 times, each time for 3 min. The A liquid and B liquid of the DAB kit are mixed in equal proportions and colored. From Figure 3 The identification results show that a clear band can be seen at a molecular weight of about 100 kD.

[0129] 7. Sequencing of monoclonal antibody

[0130] According to the constant region sequence of the antibody gene, the following primers are synthesized:

[0131] 4C-LF 5'-GACATTGTGATGACCCAGTCTCCT-3' (as shown in SEQ ID NO. 3)

[0132] 4C-LR 5'-TGGACACTGTTGGGGCCGCATCGGCCCT-3' (as shown in SEQ ID NO. 4)

[0133] 4C-HF 5'-CAGGTGCAGCTGCAGGAGTCAGGA-3' (as shown in SEQ ID NO. 5)

[0134] 4C-HR 5'-GATAGACAGATGGGGGTGTCGTTTTGGC-3' (as shown in SEQ ID NO. 6)

[0135] Total RNA of TPO-1# 3 x 10 6 hybridoma cells was extracted by Trizol Reagent, and the total RNA was reversely transcribed into cDNA. The heavy chain variable region of the monoclonal antibody was amplified by PCR using 4C-HF and 4C-HR as primers, and the light chain variable region of the monoclonal antibody was amplified by PCR using 4C-LF and 4C-LR as primers. The PCR reaction was hot-start, and the reaction conditions were as follows: 95 °C for 5 min; 95 °C for 15 s, 55 °C for 45 s, 72 °C for 30 s, 30 cycles; 72 °C for 7 min. The PCR product was separated by 1% agarose gel electrophoresis, and the target fragment was recovered and purified. The fragment was cloned into PM18-T vector, transformed into E. coli DH5α cells, and screened on LB solid plates. White colonies were inoculated in LB liquid medium containing ampicillin for amplification. Positive clones were screened, and the plasmid was extracted by QIAGEN plasmid extraction kit and sequenced to determine the heavy chain and light chain variable region sequences of the murine monoclonal antibody TPO-1#.

[0136] The heavy chain variable region of the murine monoclonal antibody TPO-1# has the amino acid sequence shown in SEQ ID NO. 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO. 2.

[0137] Example 2: Establishment of a detection kit for Anti-TPO in serum

[0138] 1. Magnetic microparticle coated murine monoclonal antibody TPO-1#

[0139] Take 30 μL mixed Merck M8945 magnetic bead stock solution with 300 μL pH 7-8 PBS buffer solution to wash 5 times, then use 50 μL 20 mg / mL EDC and NHS solution to activate the magnetic microparticles for 1 h, then wash the activated magnetic microparticles with pH 7-8 PBS buffer solution for 2 times. The monoclonal antibody TPO-1# is added to the magnetic beads at an amount of 0.5 μg per person, and coated at 4°C for 2 h. Finally, the blocking solution containing BSA is used for blocking for 2 h.

[0140] 2, TPO antigen capture and indirect method detection Anti-TPO

[0141] The coated magnetic bead suspension containing TPO-1# antibody is used as a solid phase carrier to capture the recombinant TPO antigen, and HRP-anti-human IgG is used as an enzyme-labeled secondary antibody to form an Anti-TPO magnetic microparticle + recombinant TPO antigen + to-be-detected serum (containing Anti-TPO) + HRP-anti-human IgG complex, thereby constructing a kit for detecting the concentration of Anti-TPO in the serum of patients with autoimmune thyroid disease.

[0142] 3, Compared with Roche kit

[0143] (1) Clinical sample positive and negative coincidence rate

[0144] Collect 26 clinical samples from a hospital, and detect them by using the capture coating (indirect method) of the application, the commonly used direct coating antigen (indirect method), and the Roche kit, respectively. The results are shown in Table 3. The Roche kit detects 19 positive samples and 7 negative samples. The direct coating (indirect method) detects 15 positive samples and 11 negative samples. The capture (indirect method) detects 17 positive samples and 9 negative samples. Compared with the Roche kit, the direct coating (indirect method) has 4 missed samples, and the capture coating (indirect method) has 2 missed samples. Therefore, the capture coating can improve the positive and negative coincidence rate of the kit, improve the problem of missed samples, and improve the sensitivity. The direct coating indirect method couples the TPO recombinant antigen on the magnetic microparticles by a chemical method. The antigen may be partially inactivated, or the antigen conformation may change, resulting in reduced binding capacity with the TPO antibody in the sample, leading to a high missed detection rate. The capture indirect method established by the present application uses TPO-1# antibody as an intermediate bridge to avoid the problems in the direct coating method, thereby improving the missed detection rate. (Samples greater than the critical value indicate positive samples, and samples less than the critical value indicate negative samples)

[0145] Table 3 Positive and negative coincidence rate of clinical samples

[0146]

[0147]

[0148] (2) Random clinical comparison

[0149] Randomly collected 205 clinical samples from a hospital, respectively, with the capture coated antigen indirect method and direct coated antigen method for detection, and Roche kit detection results as shown in Figure 4 directly coated (indirect method) and Roche kit overall correlation is only close to 62%, and the overall correlation of the kit of the present application and Roche kit reaches R 2 = 0.84, compared with the overall correlation of direct coating (indirect method) and Roche kit, which is improved by more than 20%.

[0150] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A mouse monoclonal antibody against thyroid peroxidase, characterized in that, It includes heavy chains and light chains: The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are shown in SEQ ID NO. 7-9, respectively; and The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown in SEQ ID NO.10, GMS and SEQ ID NO.12, respectively.

2. The anti-thyroid peroxidase mouse monoclonal antibody as described in claim 1, characterized in that, It includes the heavy chain variable region and the light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

2.

3. The anti-thyroid peroxidase mouse monoclonal antibody as described in claim 2, characterized in that, The titer of the anti-thyroid peroxidase mouse monoclonal antibody is 1:32000~1:256000.

4. The anti-thyroid peroxidase mouse monoclonal antibody as described in claim 3, characterized in that, The K of the anti-thyroid peroxidase mouse monoclonal antibody D The values ​​range from 1.001E-9 to 1.333E-7 mol / L.

5. The use of the anti-thyroid peroxidase mouse monoclonal antibody as described in any one of claims 1 to 4 in the preparation of products for detecting thyroid diseases: The detection method is to detect antibodies against thyroid peroxidase in serum; The thyroid disease mentioned is an autoimmune thyroid disease; The autoimmune thyroid disease mentioned is hypothyroidism.

6. The product, characterized in that, Includes the anti-thyroid peroxidase mouse monoclonal antibody as described in any one of claims 1 to 4, and acceptable excipients and / or adjuvants.

7. The product as described in claim 6, characterized in that, This includes one or more of magnetic microparticles, reagents, or kits.

8. A method for preparing a kit for detecting the concentration of anti-thyroid peroxidase antibodies in serum, characterized in that, Includes the following steps: Step 1: Coat the magnetic microparticles with the anti-thyroid peroxidase mouse monoclonal antibody as described in any one of claims 1 to 4 to obtain anti-thyroid peroxidase antibody magnetic microparticles; Step 2: The antibody magnetic microparticles against thyroid peroxidase, recombinant thyroid peroxidase antigen, test serum containing antibody against thyroid peroxidase, and / or HRP-anti-human IgG form a complex to prepare the kit for detecting the concentration of antibody against thyroid peroxidase in serum.

9. A kit for detecting the concentration of anti-thyroid peroxidase antibody in serum, prepared by the method described in claim 8.

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