A triiodothyronine immunoassay antibody and its preparation method and application

Abstract

The present invention provides a triiodothyronine immunodetection antibody and a preparation method and application thereof. The triiodothyronine immunodetection antibody comprises a scFv recombinant antibody and a binding antibody; the scFv recombinant antibody can specifically bind to triiodothyronine, and the sequence of the scFv recombinant antibody is shown in SEQ.ID No.1; the binding antibody can specifically bind to a complex formed by the scFv recombinant antibody and triiodothyronine, and the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the binding antibody are shown in SEQ.ID No.6, SEQ.ID No.7, and SEQ.ID No.8, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the binding antibody are shown in SEQ.ID No.11, SEQ.ID No.12, and SEQ.ID No.13, respectively.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological immunology, and in particular relates to a triiodothyronine immunodetection antibody and a preparation method and application thereof. Background Art

[0002] Triiodothyronine (T3) is a hormone secreted by the thyroid gland. Approximately 65% ​​is produced directly by the thyroid gland, and 35% is formed by deiodination of thyroxine (T4) in peripheral tissues. T3 has a molecular weight of approximately 650 and is a small molecule hapten. T3 is involved in nearly every physiological process in the body, including growth and development, metabolism, and regulation of body temperature and heart rate.

[0003] Approximately 99.7% of T3 in the human body is specifically bound to thyroid hormone-binding protein (TBG), with the remaining 0.3% existing in a free form. Only free T3 (FT3) is metabolically active and can more accurately reflect the functional status of the thyroid gland and its effects on human function. The sum of free and bound T3 is called total T3 (TT3). Currently, the combined measurement of FT3, free T4 (FT4), and high-sensitivity thyroid-stimulating hormone (TSH) is considered the preferred method for assessing thyroid function.

[0004] Currently, the mainstream methodology for detecting T3 in clinical practice is immunoassay. Since T3 is a small molecule compound with a single antigenic determinant, it cannot be detected by sandwich detection using two different antibodies. Therefore, clinical immunoassays generally use a competitive method. However, this method is limited by the methodological defects of the competitive method itself, such as lack of precision, insufficient accuracy, susceptibility to interference, and a narrow linear range.

[0005] The more feasible complex antibody sandwich method, specifically the detection principle, is that one anti-small molecule antibody (the first antibody) forms an immune complex with the small molecule to be detected, and another anti-complex antibody (the second antibody) binds to the aforementioned immune complex to form an immune sandwich complex. The recognition site is the new epitope formed after the first antibody binds to the small molecule, and the anti-complex antibody is required to not recognize the free first antibody or the small molecule. The use of the complex antibody sandwich method can greatly improve the sensitivity and accuracy of small molecule detection.

[0006] However, screening for anti-complex antibodies is difficult. Summary of the Invention

[0007] The present invention provides a triiodothyronine immunoassay antibody and a preparation method and application thereof, which can effectively solve the above problems.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a triiodothyronine immunodetection antibody, comprising a scFv recombinant antibody and a binding antibody;

[0010] The scFv recombinant antibody can specifically bind to triiodothyronine, and the sequence of the scFv recombinant antibody is shown in SEQ.ID No.1;

[0011] The binding antibody can specifically bind to the complex formed by the scFv recombinant antibody and triiodothyronine. The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the binding antibody are shown as SEQ.ID No.6, SEQ.ID No.7, and SEQ.ID No.8, respectively. The sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the binding antibody are shown as SEQ.ID No.11, SEQ.ID No.12, and SEQ.ID No.13, respectively.

[0012] In a second aspect, the present invention provides a scFv recombinant antibody that can specifically bind to triiodothyronine, and its sequence is shown in SEQ.ID No.1.

[0013] In a third aspect, the present invention provides a method for preparing the above-mentioned scFv recombinant antibody, comprising: using triiodothyronine as an immunogen to immunize mice to obtain antibodies, and then connecting the heavy chain variable region and light chain variable region of the antibody.

[0014] In a fourth aspect, the present invention provides a binding antibody that can specifically bind to the complex formed by the scFv recombinant antibody and triiodothyronine, and the sequences of CDR1, CDR2, and CDR3 of its heavy chain variable region are shown as SEQ.ID No.6, SEQ.ID No.7, and SEQ.ID No.8, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the binding antibody are shown as SEQ.ID No.11, SEQ.ID No.12, and SEQ.ID No.13, respectively.

[0015] In a fifth aspect, the present invention provides a method for preparing the aforementioned binding antibody, comprising: using the complex formed by the aforementioned scFv recombinant antibody and triiodothyronine as an immunogen to immunize mice.

[0016] In a sixth aspect, the present invention provides a triiodothyronine immunoassay kit comprising the above-mentioned scFv recombinant antibody and the above-mentioned binding antibody.

[0017] The beneficial effects of the present invention are:

[0018] The present invention screens and obtains a triiodothyronine immunodetection antibody, which includes a scFv recombinant antibody and a binding antibody, and can be used in a complex antibody sandwich method to greatly improve the sensitivity and accuracy of triiodothyronine detection.

[0019] Furthermore, the present invention improves the screening efficiency of the second antibody by optimizing the screening strategy, constructs the anti-T3 antibody into a single-chain antibody 27F5-scFv-Avi-His, reduces the interference of the first antibody in the complex immunization, and adds an Avi tag at the C-terminus to facilitate the subsequent site-specific labeling of biotin at the C-terminus, making the immune complex binding site more easily exposed and less likely to be masked during detection.

[0020] Furthermore, the Elisa used in the present invention further determines whether the cloned wells of the hybridoma cells after fusion produce anti-complex antibodies through two-step detection, thereby more efficiently screening and ultimately screening out an anti-complex antibody 82C11. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is an SDS-PAGE protein gel electrophoresis diagram of the antibody 27F5-scFv expressed in 293F cells according to an embodiment of the present invention.

[0023] Figure 2 This is an SDS-PAGE protein gel electrophoresis diagram of the recombinant anti-complex antibody 82C11 expressed in 293F cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention for which protection is sought, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] Sources of reagents in the embodiments of the present invention:

[0026] The T3-KLH was purchased from Creative Diagnostics;

[0027] The T3-BSA was purchased from Creative Diagnostics;

[0028] The Freund's complete adjuvant was purchased from Sigma;

[0029] The myeloma cells were obtained from Xiamen University;

[0030] The high-purity plasmid mini-extraction kit model is DP107, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0031] The 293F cells were obtained from Xiamen University;

[0032] The molecular amplification reagents and cloning ligation kit were purchased from Takara;

[0033] The amplification primers were synthesized by Guangzhou Qingke Biotechnology Co., Ltd.

[0034] The Escherichia coli DH5α competent strain was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0035] The OPM-293CD05 culture medium was purchased from Shanghai Aopuma Biotechnology Co., Ltd.

[0036] The OPM-293 ProFeed culture medium was purchased from Shanghai Aopuma Biotechnology Co., Ltd.

[0037] The nickel column and the Protein G column were purchased from Huiyan Biotechnology Co., Ltd.

[0038] The eluent reagent was purchased from Sinopharm Group;

[0039] The goat anti-mouse IgG-HRP was purchased from Sigma;

[0040] T3 natural standard was purchased from Sigma;

[0041] EDC was purchased from Thermofish;

[0042] SA was purchased from Tiandirenhe Biotechnology Co., Ltd.

[0043] Example 1

[0044] The present invention provides a T3 antibody 27F5-scFv (first antibody, Ab1), which is a single-chain antibody that can specifically bind to the small molecule T3 to form a complex.

[0045] A method for preparing the antibody 27F5-scFv, comprising:

[0046] S11, immunization of mice

[0047] Dissolve T3-KLH as the immunogen and emulsify it with an equal volume of Freund's complete adjuvant. Subcutaneously inject 200 μg / mouse at multiple sites into 6-8 week-old SPF-grade Balb / c mice. Two weeks apart, administer 100 μg / mouse subcutaneously at multiple sites with an equal volume of Freund's incomplete adjuvant emulsified with the immunogen. Boost the immune system twice. Three days before cell fusion in step S2, administer a shock injection of 100 μg / mouse intraperitoneally.

[0048] S12. Cell fusion

[0049] The spleen of the mouse immunized in step S11 was obtained, ground and separated to obtain dispersed single spleen cells, and the spleen cells and myeloma cells were fused using an electrofusion instrument. After standing, the cells were added to 1640 culture medium containing 20% ​​FBS and plated in a 96-well plate.

[0050] S13. Subclone screening

[0051] After culturing the hybridoma cells fused in step S12 for one week, the cell supernatant was taken for ELISA (Enzyme-Linked Immunosorbent Assay). T3-BSA was used as the coating antigen and goat anti-mouse IgG-HRP (horseradish peroxidase labeled goat anti-mouse IgG) was used as the enzyme-labeled secondary antibody to evaluate the cell supernatant and screen out the positive wells. After the positive wells were replaced with the medium, they were further subcloned by the limiting dilution method. After culturing for one week, the ELISA test was continued and repeated 3 to 4 times until all the test wells were positive and the cells in the wells were single colonies, and the specific hybridoma cell line 27F5 was obtained by expansion.

[0052] S14. Gene retrieval

[0053] The hybridoma cell line 27F5 in step S13 was expanded, mRNA was extracted, and cDNA products were obtained by reverse transcription. The products were subjected to an A-addition reaction using Taq DNA polymerase and then inserted into the pMD-19T vector. The cells were transformed into DH5α competent cells, and 10 plaques each of the heavy chain and light chain gene clones were collected and sent to a gene sequencing company for sequencing.

[0054] S15. Sequence analysis of antibody genes

[0055] The gene sequence obtained by sequencing in step S14 is placed in the IMGT antibody database for analysis, and analyzed using SnapGene software to determine the correct heavy chain and light chain variable region genes.

[0056] The sequence of the heavy chain variable region of antibody 27F5 is shown in SEQ.ID No.2.

[0057] SEQ.ID No.2:

[0058] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTNYNPSLKSRISITRDTSKNQVLLQLKSVTTEDTATYYCAGNLEYWGQGTTLTVSS

[0059] The sequence of the light chain variable region of antibody 27F5 is shown in SEQ.ID No.3.

[0060] SEQ.ID No.3:

[0061] QIVLTQSPAIMSASPGEKVTITCSASSSVIYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAGTKLEVKRA

[0062] Construction of S16 and scFv recombinant antibody expression plasmids

[0063] A scFv recombinant antibody expression vector was constructed using pcDNA3.1 as a vector using three GGGGS flexible linkers. Based on the antibody variable region gene sequencing results from pMD-19T in step S15, light and heavy chain-specific primer pairs were designed for homologous recombination. Overlapping PCR amplification was used to obtain a fusion fragment of the heavy and light chain variable region genes connected by the linker. Homologous primers were designed for the pcDNA3.1 vector containing a signal peptide, Avi tag, and His tag. After PCR amplification, the vector fragment was recovered by electrophoresis. The gene fragment and vector fragment were homologously ligated and transformed into DH5α competent cells. After positive colony PCR verification, the cells were sequenced. Normal colonies were selected for expansion and culture, and the vector plasmid containing the fusion fragment was extracted and abbreviated as pcDNA3.1-27F5-scFv-Avi-His.

[0064] Colony PCR was verified positive by agarose gel electrophoresis. The target plasmid was extracted using a high-purity plasmid miniprep kit.

[0065] The sequence of the scFv recombinant antibody 27F5-scFv is shown in SEQ.ID No.1.

[0066] SEQ.ID No.1:

[0067] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGSTNYNPSLKSRISITRDTSKNQVLLQLKSVTTEDTATYYCAGNLEYWGQGTTLTVSSGGGGS GGGGSGGGGSQIVLTQSPAIMSASPGEKVTITCSASSSVIYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAGTKLEVKRA

[0068] S17, recombinant antibody expression

[0069] The target plasmid obtained in step S16 was transfected into 293F cells by PEI transfection method. After successful transfection, the cells were cultured in an incubator at 37°C, 8% CO2, and 120 rpm for 24 hours. Then, fresh OPM-293CD05 culture medium equal in volume to the culture medium was added and the cells were grown to 4×10 6 When the density of cells was 100 / mL, 1% by volume of OPM-293ProFeed feed medium was added every day, and the cells were returned to the incubator and cultured for 96 h to obtain a 293F cell culture fluid expressing the recombinant antibody 27F5-scFv.

[0070] S18, recombinant antibody purification

[0071] The cell culture fluid obtained in step S17 was centrifuged at 9000 rpm for 15 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and protein purification was performed using a nickel column. The nickel column was equilibrated with 50 column volumes of equilibration buffer before use. The filtered cell supernatant was then passed through the column and eluted with 20 column volumes of eluents containing 0 mM, 50 mM, 150 mM, and 300 mM imidazole concentrations, respectively. The eluents with different imidazole concentrations were collected, and the eluent containing the target protein was screened and dialyzed into pH 6.0 PBS to obtain the antibody 27F5-scFv-Avi-His, referred to as 27F5-scFv.

[0072] The filtered cell supernatant, flow-through and eluate were analyzed by SDS-PAGE protein gel electrophoresis. Figure 1 As shown, it was shown that the antibody 27F5-scFv with good purity was obtained after purification.

[0073] The activity of the antibody 27F5-scFv was detected by ELISA. The results are shown in Table 1, indicating that the prepared antibody 27F5-scFv has good activity.

[0074] Table 1

[0075]

[0076] Example 2

[0077] The present invention provides an anti-complex antibody 82C11 (a second antibody, Ab2). Antibody 82C11 can specifically bind to the complex formed by the T3 antibody 27F5-scFv prepared in Example 1 and T3.

[0078] The preparation method of antibody 82C11 comprises:

[0079] S1. Immunization of mice.

[0080] The T3 natural standard product and the antibody 27F5-scFv prepared in Example 1 were coupled to form a complex as an immunogen.

[0081] In some embodiments, in step S1, the method for preparing the complex comprises:

[0082] T3 native standard was coupled to the antibody 27F5-scFv at a molar ratio of 20:1.

[0083] In some embodiments, in step S1, the method for preparing the complex comprises:

[0084] 1) Dissolve 100 mg of EDC in 2.5 mL of 10 mmol / L PBS (pH 8.0) to obtain Solution I.

[0085] 2) Dissolve 22.5 mg of T3 natural standard in 2 mL of 0.2 mol / L NaOH solution to obtain Solution II.

[0086] 3) Dissolve 50 mg of the antibody 27F5-scFv in 10 mL of pH 8.0, 10 mmol / L PBS to obtain Solution III.

[0087] 4) Mix Solution II and Solution III, and add Solution I dropwise under magnetic stirring, leaving 0.5 mL of Solution I.

[0088] 5) The liquid obtained in step 4) was stirred in the dark at room temperature for 1 hour, and then the remaining solution I was added dropwise.

[0089] 6) The liquid obtained in step 5) was stirred at 4°C for 12 hours and then allowed to stand at 4°C for 10 hours.

[0090] 7) The liquid obtained in step 6) was fully dialyzed against PBS to obtain the immunogen after 48 hours.

[0091] Emulsify the immunogen evenly with an equal volume of Freund's complete adjuvant and inject 200 μg / mouse subcutaneously at multiple points in 6-8 week old SPF Balb / c mice. Emulsify the immunogen with an equal volume of Freund's incomplete adjuvant and inject 100 μg / mouse subcutaneously at multiple points 2 weeks apart. Booster immunizations were performed twice. Three days before cell fusion in step S2, 100 μg / mouse was injected intraperitoneally for shock.

[0092] S2. Cell fusion to obtain fused hybridoma cells.

[0093] The spleen of the mouse immunized in step S1 was taken, and the dispersed single spleen cells were obtained by grinding and separation. The spleen cells and myeloma cells were fused using an electrofusion instrument. After standing, the culture medium was taken and plated into a 96-well plate.

[0094] S3. Subclone screening.

[0095] Screening of positive hybridoma cells is divided into two steps.

[0096] The first step is, S31, culturing the hybridoma cells fused in step S2 for one week, taking the cell supernatant and performing ELISA test to confirm the presence of anti-complex antibodies by competitive assay.

[0097] Streptavidin (SA) was used as the coating antigen. After blocking, biotin-labeled antibody 27F5-scFv (27F5-scFv-bio) was added, as well as 27F5-scFv-bio and T3 were added simultaneously. Then, cell supernatant was added. The color developing agent was goat anti-mouse IgG-HRP secondary antibody. Finally, positive wells with low reaction to 27F5-scFv-bio and strong reaction to the complex formed by 27F5-scFv-bio and T3 were selected, and 4 wells were obtained, as shown in Table 2.

[0098] Table 2

[0099]

[0100]

[0101] The second step is, S32, reacting the cell supernatant of the clone wells screened in step S31 with the antibody 27F5-scFv at 37° C. for 1 hour, and then taking the reaction solution for Elisa detection.

[0102] Streptavidin (SA) was used as the coating antigen. After blocking, 27F5-scFv-bio and the complex formed by 27F5-scFv-bio and T3 were added. The reaction solution was then added, and the color development reagent was a goat anti-mouse IgG-HRP secondary antibody. Positive wells that did not react with 27F5-scFv-bio but strongly reacted with the complex formed by 27F5-scFv-bio and T3 were finally selected.

[0103] S33. Subcloning was further performed by limiting dilution. After one week of culture, ELISA was performed again. Positive wells that did not react with 27F5-scFv-bio but strongly reacted with the complex were selected for subcloning. This process was repeated 3-4 times until all wells were positive and the cells in each well were single colonies. The clone well that did not react with 27F5-scFv-bio but had the strongest reaction with the complex was selected. The sample from this clone well was 82C11.

[0104] The cloned well was expanded to obtain the specific hybridoma cell line 82C11.

[0105] Ascites preparation of positive cell lines

[0106] The specific hybridoma cell line 82C11 was injected into mice pre-immunized with Freund's incomplete adjuvant to prepare ascites. The ascites was collected to obtain the anti-complex antibody 82C11, which was affinity purified using a Protein G column.

[0107] Example 3

[0108] This embodiment of the present invention provides a recombinant anti-complex antibody 82C11 of the anti-complex antibody 82C11 prepared in Example 2.

[0109] A method for preparing a recombinant anti-complex antibody 82C11 comprises:

[0110] S4. Gene retrieval

[0111] The hybridoma cell line 82C11 in step S3 was expanded, mRNA was extracted, and cDNA products were obtained by reverse transcription. The products were subjected to an A-addition reaction using Taq DNA polymerase and then inserted into the pMD-19T vector. The cells were transformed into DH5α competent cells, and 10 plaques each of the heavy chain and light chain gene clones were collected and sent to a gene sequencing company for sequencing.

[0112] S5. Sequence analysis of antibody genes

[0113] The gene sequence obtained by sequencing in step S4 is placed in the IMGT antibody database for analysis, and analyzed using SnapGene software to determine the correct heavy chain and light chain variable region genes.

[0114] The 82C11 heavy chain sequence is shown in SEQ.ID No.4.

[0115] SEQ.ID No.4:

[0116] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSTNQVFLKIISVDTADTATYYCARKCLLRWVDYWGQGTS VTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIC The sequence of the heavy chain variable region of TVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK is shown in SEQ.ID No.5.

[0117] SEQ.ID No.5:

[0118] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQSSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSTNQVFLKIISVDTADTATYYCARKCLLRWVDYWGQGTSVTVSS

[0119] The CDR1 sequence of the heavy chain variable region is shown in SEQ.ID No.6.

[0120] SEQ.ID No.6:

[0121] TSGMGVS

[0122] The CDR2 sequence is shown as SEQ.ID No.7.

[0123] SEQ.ID No.7:

[0124] HIYWDDDKRYNPSLKS

[0125] The CDR3 sequence is shown as SEQ.ID No.8.

[0126] SEQ.ID No.8:

[0127] KCLLRWVDY

[0128] The 82C11 light chain sequence is shown in SEQ.ID No.9.

[0129] SEQ.ID No.9:

[0130] QIVLTQSPAIMSASPGETVTMTCSASSSVSYMHWFQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0131] The light chain variable region sequence is shown in SEQ.ID No.10.

[0132] SEQ.ID No.10:

[0133] QIVLTQSPAIMSASPGETVTMTCSASSSVSYMHWFQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGAGTKLELKRA

[0134] The CDR1 sequence of the light chain variable region is shown in SEQ.ID No.11.

[0135] SEQ.ID No.11:

[0136] SASSSVSYMH

[0137] The CDR2 sequence is shown as SEQ.ID No.12.

[0138] SEQ.ID No.12:

[0139] DTSKLAS

[0140] The CDR3 sequence is shown in SEQ.ID No.13.

[0141] SEQ.ID No.13:

[0142] FQGSGYPLT

[0143] S6. Construction of recombinant antibody expression plasmid

[0144] A recombinant antibody expression vector was constructed using pTT5, which already contained a signal peptide and constant region. Based on the antibody variable region gene sequencing results from pMD-19T in step S5, light and heavy chain-specific primer pairs were designed for homologous recombination. The light and heavy chain gene fragments were amplified by PCR. Homologous primers were designed for the pTT5 vector, which already contained a signal peptide and constant region. After PCR amplification, the vector fragments were recovered. The gene and vector fragments were homologously ligated and transformed into DH5α competent cells. After positive colony PCR verification, the cells were sequenced. Normal colonies were selected and expanded, and the vector plasmids containing the ligated heavy and light chain variable regions were extracted, referred to as pTT5-82C11 H chain and pTT5-82C11 L chain.

[0145] The sequences of the designed light and heavy chain specific primer pairs are shown in Table 3.

[0146] Table 3

[0147] Primer name Primer sequence (5' to 3') 82C11-HF gggtgcccggatccaccggcCAAGTGACCCTGAAGGAGAG 82C11-HR gatgggcccttggtgctagcGGAGCTGACTGTCACGC 82C11-LF gggtgcccggatccaccggcCAGATCGTGCTGACACAGA 82C11-LR gatggtgcagccaccgtacgGGCTCTCTTCAGCTCCAG PTT5-HF gctagcaccaagggcccatc PTT5-HR gccggtggatccgggcaccc PTT5-LF cgtacggtggctgcaccatc PTT5-LR gccggtggatccgggcaccc Remark The lowercase sequence is the homologous connecting part, H is the heavy chain, and L is the light chain

[0148] S7. Recombinant Antibody Expression

[0149] The target plasmid obtained in step S6 was transfected into 293F cells by PEI transfection method. After successful transfection, the cells were cultured in an incubator at 37°C, 8% CO2, and 120 rpm for 24 hours. Then, fresh OPM-293CD05 culture medium equal in volume to the culture medium was added and the cells were grown to 4×10 6 When the density of cells / mL was reached, 1% by volume of OPM-293ProFeed feed medium was added every day, and the cells were returned to the incubator and cultured for 96 h to obtain a 293F cell culture fluid expressing the recombinant anti-complex antibody 82C11.

[0150] S8. Recombinant Antibody Purification

[0151] The cell culture fluid obtained in step S8 was centrifuged at 9000 rpm for 15 minutes, and the supernatant was collected and filtered through a 0.22 μm filter membrane. Protein G column was then used for protein purification. Before use, the Protein G column was equilibrated with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl, pH 7.4. The filtered cell supernatant was passed through the column and then rinsed with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl, pH 7.4. The column was then eluted with 5 column volumes of eluent containing 0.1 M Glycine-HCl, pH 2.7. The eluent was immediately neutralized with 1.0 M Tris-HCl, pH 9.0. Finally, the purified recombinant antibody was dialyzed into PBS, pH 8.0, to obtain recombinant anti-complex antibody 82C11.

[0152] The filtered cell supernatant, flow-through and eluate were analyzed by SDS-PAGE protein gel electrophoresis. Figure 2 As shown, it was shown that the recombinant anti-complex antibody 82C11 with good purity was obtained after purification.

[0153] The activity of the recombinant anti-complex antibody 82C11 was detected by ELISA. The results are shown in Table 4, indicating that the prepared recombinant anti-complex antibody 82C11 has good activity.

[0154] Table 4

[0155]

[0156] Example 4

[0157] An embodiment of the present invention provides a triiodothyronine immunoassay kit, comprising the T3 antibody 27F5-scFv prepared in Example 1 and the anti-complex antibody 82C11 prepared in Example 2, or comprising the T3 antibody 27F5-scFv prepared in Example 1 and the recombinant anti-complex antibody 82C11 prepared in Example 3.

[0158] Antibody 27F5-scFv is used as the first antibody, and antibody 82C11 or its recombinant antibody 82C11 is used as the second antibody for T3 detection using a complex antibody sandwich method.

[0159] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A triiodothyronine immunoassay antibody, characterized in that: including a scFv recombinant antibody and a binding antibody; The scFv recombinant antibody can specifically bind to triiodothyronine, and the sequence of the scFv recombinant antibody is shown in SEQ.ID No.1; The binding antibody can specifically bind to the complex formed by the scFv recombinant antibody and triiodothyronine. The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the binding antibody are shown as SEQ.ID No.6, SEQ.ID No.7, and SEQ.ID No.8, respectively. The sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the binding antibody are shown as SEQ.ID No.11, SEQ.ID No.12, and SEQ.ID No.13, respectively.

2. The triiodothyronine immunodetection antibody according to claim 1, wherein The sequence of the heavy chain variable region of the binding antibody is shown in SEQ.ID No.5, and the sequence of the light chain variable region is shown in SEQ.ID No.

10.

3. The triiodothyronine immunodetection antibody according to claim 1, wherein The sequence of the heavy chain of the binding antibody is shown in SEQ.ID No.4, and the sequence of the light chain is shown in SEQ.ID No.

9.

4. A scFv recombinant antibody, characterized in that It can specifically bind to triiodothyronine, and its sequence is shown in SEQ.ID No.

1.

5. A binding antibody, characterized in that It can specifically bind to the complex formed by the scFv recombinant antibody according to claim 4 and triiodothyronine, and the sequences of CDR1, CDR2, and CDR3 of its heavy chain variable region are shown as SEQ.ID No.6, SEQ.ID No.7, and SEQ.ID No.8, respectively, and the sequences of CDR1, CDR2, and CDR3 of its light chain variable region are shown as SEQ.ID No.11, SEQ.ID No.12, and SEQ.ID No.13, respectively.

6. A triiodothyronine immunoassay kit, characterized in that: The invention comprises the scFv recombinant antibody according to claim 4 and the binding antibody according to claim 5.

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