Triiodothyronine immunodetection antibody as well as preparation method and application thereof

Abstract

The invention provides a triiodothyronine immunodetection antibody as well as a preparation method and application thereof. The triiodothyronine immunodetection antibody comprises an scFv recombinant antibody and a binding antibody, the scFv recombinant antibody can be specifically combined with triiodothyronine, and the sequence of the scFv recombinant antibody is as shown in SEQ. ID No. 1; the binding antibody can be specifically bound with a compound formed by the scFv recombinant antibody and triiodothyronine, the sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the binding antibody are respectively shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, and the sequences of CDR1, CDR2 and CDR3 of a light chain variable region of the binding antibody are respectively shown as SEQ ID No.11, SEQ ID No.12 and SEQ ID No.13. The invention further discloses a kit for detecting the triiodothyronine in the scFv recombinant antibody.

Description

Technical Field

[0001] The 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, about 65% of which is produced directly by the thyroid gland, and 35% is formed by deiodination of thyroxine (T4) in peripheral tissues. The molecular weight of T3 is about 650, and it is a hapten small molecule substance. T3 is involved in almost every physiological process of the body, including growth and development, metabolism, regulation of body temperature and heart rate, etc.

[0003] About 99.7% of T3 in the human body is specifically bound to thyroid hormone binding protein (TBG), and the remaining 0.3% exists in a free form. Only free T3 (FT3) has metabolic activity and can more accurately reflect the functional state of the thyroid gland and other effects on human body functions. The sum of free T3 and bound T3 is collectively called total T3 (TT3). It is currently believed that the combined detection of FT3, free T4 (FT4) and high-sensitivity thyroid-stimulating hormone (TSH) is the preferred option for thyroid function assessment.

[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 cluster, it cannot be detected by sandwich detection using two different antibodies. Therefore, clinical immunoassays generally use the competitive method, but are 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 currently more feasible complex antibody sandwich method has the following specific detection principle: one anti-small molecule antibody (first antibody) forms an immune complex with the small molecule to be detected, and another anti-complex antibody (second antibody) combines with the aforementioned immune complex to form an immune sandwich complex. The recognition site is the new epitope formed after the first antibody combines with the small molecule, and it is required that the anti-complex antibody does not recognize the free first antibody and 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 anti-complex antibodies is difficult. Summary of the invention

[0007] The present invention provides a triiodothyronine immunodetection 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, 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.IDNo.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.

[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 in the heavy chain variable region 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 in 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.

[0015] In a fifth aspect, the present invention provides a method for preparing the above-mentioned binding antibody, comprising: using the complex formed by the above-mentioned 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 invention screens and obtains a triiodothyronine immunodetection antibody, which comprises a scFv recombinant antibody and a binding antibody, and can be used in a complex antibody sandwich method, thereby greatly improving 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 complex immunization, and adds an Avi tag at the C-terminus to facilitate subsequent site-specific labeling of biotin at the C-terminus, making the immune complex binding site easier to expose 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 finally 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 drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It is the SDS-PAGE protein gel electrophoresis diagram of the antibody 27F5-scFv expressed by 293F cells in the example of the present invention.

[0023] Figure 2 It is the SDS-PAGE protein gel electrophoresis diagram of the recombinant anti-complex antibody 82C11 expressed by 293F cells in the example 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, 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 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 claimed for protection, but merely represents the 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 from Xiamen University;

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

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

[0032] The molecular amplification reagents and cloning ligation kits 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-293ProFeed 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 embodiment of the present invention provides a T3 antibody 27F5-scFv (first antibody, Ab1), wherein the antibody 27F5-scFv is a single-chain antibody and can specifically bind to the small molecule T3 to form a complex.

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

[0046] S11, immunization of mice

[0047] T3-KLH was used as the immunogen, and after dissolving, it was emulsified evenly with an equal volume of Freund's complete adjuvant. 6-8 week old SPF grade Balb / c mice were taken and injected subcutaneously at multiple points with 200 μg / mouse. After an interval of 2 weeks, the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points with 100 μg / mouse. The immunization was boosted twice. In step S2, 100 μg / mouse was injected intraperitoneally for shock 3 days before cell fusion.

[0048] S12. Cell fusion

[0049] The spleen of the mouse immunized in step S11 is taken, ground and separated to obtain dispersed single spleen cells, and the spleen cells and myeloma cells are fused using an electric fusion instrument. After standing, 1640 culture medium containing 20% ​​FBS is taken out and plated in a 96-well plate.

[0050] S13. Subclone screening

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

[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 inserted into the pMD-19T vector after A addition reaction with TaqDNA polymerase and transformed into DH5α competent cells. Ten plaques of heavy chain and light chain gene clones were taken 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 heavy chain variable region sequence 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] The scFv recombinant antibody expression vector was constructed using pcDNA3.1 as a vector through three GGGGS flexible linkers. According to the sequencing results of the antibody variable region genes in pMD-19T in step S15, a pair of light and heavy chain specific primers for homologous recombination was designed, and the fusion fragments of the heavy chain variable region and light chain variable region genes connected by the linker were obtained by overlapping PCR amplification method. Homologous primers were designed for the pcDNA3.1 vector with existing signal peptides, Avi tags and His tags, and the vector fragments were recovered by electrophoresis after PCR amplification. The gene fragments and the vector fragments were homologously connected and transformed into DH5α competent cells. After the colony PCR verification was positive, they were sent for sequencing. Normal colonies were picked for expansion culture, and the vector plasmid connected to the above fusion fragments was extracted, which was referred to 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 mini-preparation 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 placed in an incubator at 37°C, 8% CO2, and 120 rpm for 24 hours. Then, fresh OPM-293CD05 culture medium with the same volume as the culture medium was added, and the cells were allowed to grow to 4×10 6 When the density of cells was 1 / 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 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 a speed of 9000 rpm for 15 min, the supernatant was collected, filtered with a 0.22 μm filter membrane, and a nickel column was used for protein purification. The nickel column was balanced with 50 column volumes of equilibration buffer before use, and the filtered cell supernatant was passed through the column, and eluted with 20 column volumes of eluents containing 0 mM, 50 mM, 150 mM, and 300 mM imidazole concentrations in sequence, and the eluents with different imidazole concentrations were collected, and the eluent containing the target protein was screened out, 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 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 embodiment of the present invention provides an anti-complex antibody 82C11 (a second antibody, Ab2). The 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 composite comprises:

[0082] The 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 composite comprises:

[0084] 1) Dissolve 100 mg of EDC in 2.5 mL of pH 8.0, 10 mmol / L PBS 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) 50 mg of the antibody 27F5-scFv was fully dissolved in 10 mL of pH 8.0, 10 mmol / L PBS solution 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) After stirring the liquid obtained in step 4) at room temperature in the dark for 1 hour, 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, and the immunogen was obtained after 48 hours.

[0091] Emulsify the immunogen with an equal volume of Freund's complete adjuvant, take 6-8 week old SPF grade Balb / c mice, inject 200 μg / mouse subcutaneously at multiple points, emulsify the immunogen with an equal volume of Freund's incomplete adjuvant at intervals of 2 weeks, and inject 100 μg / mouse subcutaneously at multiple points, boost immunization twice, and inject 100 μg / mouse intraperitoneally for shock 3 days before step S2 cell fusion.

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

[0093] The spleen of the mouse immunized in step S1 is taken, and the dispersed single spleen cells are obtained by grinding and separation. The spleen cells and myeloma cells are fused by an electric fusion instrument. After standing, the culture medium is taken out and plated in 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 for Elisa detection, and confirming the presence of anti-complex antibodies by competition method.

[0097] Streptavidin (SA) was used as the coating antigen. After blocking, biotin-labeled antibody 27F5-scFv (27F5-scFv-bio) was added, and 27F5-scFv-bio and T3 were added at the same time. Then, cell supernatant was added. The color developer 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 to obtain 4 wells, as shown in Table 2.

[0098] Table 2

[0099]

[0100]

[0101] The second step is, S32, reacting the cell supernatant of the clone well 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, and 27F5-scFv-bio and the complex formed by 27F5-scFv-bio and T3 were added after blocking. Then the reaction solution was added, and the color developing agent was goat anti-mouse IgG-HRP secondary antibody. Finally, the positive wells that had no reaction with 27F5-scFv-bio but strongly reacted with the complex formed by 27F5-scFv-bio and T3 were selected.

[0103] S33, further subcloning by limiting dilution method, culturing for one week, continuing to perform Elisa detection, continue to select positive wells that have no reaction with 27F5-scFv-bio but strongly react with the complex for subcloning, repeat 3 to 4 times, until all the detection wells are positive and the cells in the wells are single colonies, select the clone well that has no reaction with 27F5-scFv-bio but has the strongest reaction with the complex. The sample of this clone well is 82C11.

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

[0105] Ascites preparation of positive cell lines

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

[0107] Example 3

[0108] The 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, comprising:

[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 inserted into the pMD-19T vector after A addition reaction with TaqDNA polymerase and transformed into DH5α competent cells. Ten plaques of heavy chain and light chain gene clones were taken 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 sequence of the light chain variable region 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 as shown in SEQ.ID No.12.

[0138] SEQ.ID No.12:

[0139] DTSKLAS

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

[0141] SEQ.ID No.13:

[0142] FQGYP

[0143] S6. Construction of recombinant antibody expression plasmid

[0144] The recombinant antibody expression vector was constructed using pTT5 with existing signal peptide and constant region as a vector. According to the sequencing results of the antibody variable region gene in pMD-19T in step S5, a pair of light and heavy chain specific primers for homologous recombination was designed, and the light chain and heavy chain gene fragments were obtained by PCR amplification. Homologous primers were designed for the pTT5 vector with existing signal peptide and constant region, and the vector fragment was recovered after PCR amplification. The gene fragment and the vector fragment were homologously connected, and DH5α competent cells were transformed. After the colony PCR verification was positive, it was sent for sequencing, and normal colonies were picked for expansion culture, and the vector plasmids connected to the heavy chain and light chain variable regions were extracted, which were 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 placed 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 allowed to grow to 4×10 6 When the density of cells was 1 / mL, 1% by volume of OPM-293ProFeed supplement 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 a speed of 9000 rpm for 15 minutes, the supernatant was collected, filtered with a 0.22 μm filter membrane, and a Protein G column was used for protein purification. Before use, the Protein G column was balanced with 5 column volumes of an equilibrium buffer containing 0.02M PB and 0.15M NaCl, pH 7.4, and then the filtered cell supernatant was passed through the column, and then the column was washed with 5 column volumes of an equilibrium buffer containing 0.02M PB and 0.15M NaCl, pH 7.4, and then eluted with 5 column volumes of an eluent containing 0.1M Glycine-HCl, pH 2.7, and the eluent was immediately neutralized with a neutralizing solution of 1.0M Tris-HCl, pH 9.0, and finally the purified recombinant antibody was dialyzed into PBS at pH 8.0 to obtain the 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] The 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] The antibody 27F5-scFv is used as the first antibody, and the antibody 82C11 or its recombinant antibody 82C11 is used as the second antibody for T3 detection using a complex antibody sandwich method.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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, 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.

2. The triiodothyronine immunodetection antibody according to claim 1, characterized in that: 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, characterized in that: 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 described in claim 4 and triiodothyronine, and the sequences of CDR1, CDR2, and CDR3 in its heavy chain variable region 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 in its light chain variable region are shown in SEQ.ID No.11, SEQ.ID No.12, and SEQ.ID No.13, respectively.

6. A method for preparing the scFv recombinant antibody according to claim 4, characterized in that: include: The antibody is obtained by immunizing mice with triiodothyronine as an immunogen, and then connecting the heavy chain variable region and the light chain variable region of the antibody.

7. The preparation method according to claim 6, characterized in that: The method comprises: constructing a scFv recombinant antibody expression plasmid, wherein the plasmid is obtained by homologously connecting a gene fragment of the scFv recombinant antibody and a vector fragment, and the vector fragment contains an Avi tag.

8. A method for preparing the binding antibody according to claim 5, characterized in that: include: The complex formed by the scFv recombinant antibody of claim 4 and triiodothyronine is used as an immunogen to immunize mice.

9. The preparation method according to claim 8, characterized in that: include: The scFv recombinant antibody and the conjugate formed by the scFv recombinant antibody and triiodothyronine are respectively used as coating antigens, and Elisa detection is used to perform subclone screening to screen out binding antibodies that have no reaction with the scFv recombinant antibody but strongly react with the conjugate.

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

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