Monoclonal antibody mutant for T4 detection and preparation method and application thereof

Abstract

The present invention relates to a monoclonal antibody mutant for T4 detection and a coding nucleic acid molecule thereof, the monoclonal antibody mutant comprises complementary determining regions CDR-VL1, CDR-VL2 and CDR-VL3, the CDR-VL1 is represented by QNIYSN, the CDR-VL2 is represented by RAS, and the fifth amino acid Y, represented by QSYYYASGSTYANA, of the CDR-VL3 is deleted or substituted by a non-Y amino acid residue. According to the monoclonal antibody mutant, the cross reactivity with a free anti-T4 antibody is remarkably reduced while high-affinity binding of the monoclonal antibody mutant to a T4-T4 antibody compound is maintained. When the kit is used for T4 detection, a target immune complex and a free antibody component can be clearly and accurately distinguished in a complex clinical sample, and the kit has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically the field of immunoassay technology, and more specifically relates to a monoclonal antibody mutant that can be used for T4 detection, its preparation method, and its application. Background Technology

[0002] In clinical diagnosis, accurate assessment of thyroid function relies heavily on precise measurement of relevant hormone levels. Free thyroxine (FT4), as the main active form of thyroid hormone, has a blood concentration that is a key indicator for assessing thyroid physiological status. However, achieving high-precision detection of FT4 presents inherent technical challenges: firstly, FT4 is present in extremely low concentrations in the blood, accounting for approximately 0.03% of total thyroxine (T4); secondly, it exists in dynamic equilibrium with various binding proteins, making direct detection unaffected by interference from these proteins exceptionally difficult.

[0003] Currently, T4 / FT4 detection in clinical laboratories mainly relies on two types of technologies. The first is mass spectrometry, which, although considered the benchmark method, is limited in its widespread application in routine clinical testing due to its high equipment cost, complex sample pretreatment, and low throughput. The second is immunoassay, which has become the mainstream method due to its ease of operation, speed, and automation. However, for small molecule haptens like T4, traditional immunoassay methods often employ competitive methods. These methods have inherent limitations, including low sensitivity, susceptibility to interference from structural analogs in the sample leading to insufficient specificity, and a narrow dynamic range, prone to signal saturation in high concentration ranges.

[0004] To overcome these shortcomings of competitive methods, the industry has proposed an innovative strategy using "anti-immune complex antibodies" to achieve sandwich detection. This technical approach aims to transform small-molecule detection into a sandwich detection mode similar to that of large molecules by recognizing novel spatial epitopes formed after T4 binds to its capture antibody, thereby potentially achieving higher sensitivity and specificity. However, this technical approach has revealed a key flaw in practical applications: the obtained anti-complex antibodies often fail to completely distinguish between the "T4-T4 antibody complex" and the "free capture antibody (i.e., T4 antibody)" itself. The high structural similarity between the two leads to a significant cross-reactivity between the anti-complex antibody and the free antibody, resulting in a high background signal in complex clinical sample matrices, severely affecting the specificity and accuracy of the detection, and potentially leading to false positive results. The high cross-reactivity of anti-T4 immune complex antibodies in coexisting with free anti-T4 antibodies results in strong background signals, poor specificity, and an increased risk of false positives in immunodetection.

[0005] Therefore, based on the successful acquisition of antibodies against the T4-T4 antibody complex, the inventors need to further modify the existing antibodies to minimize their cross-reactivity with free anti-T4 antibodies while retaining their high affinity for the T4-T4 antibody complex. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a monoclonal antibody mutant that can be used for T4 detection, which, while maintaining high affinity binding to the T4-T4 antibody complex, can significantly reduce non-specific binding to free anti-T4 antibody.

[0007] In a first aspect, the present invention provides a monoclonal antibody mutant that can be used for T4 detection, characterized in that the monoclonal antibody mutant includes a light chain variable region and a heavy chain variable region, the heavy chain variable region includes complementarity-determining regions CDR-VH1, CDR-VH2, and CDR-VH3, and the light chain variable region includes complementarity-determining regions CDR-VL1, CDR-VL2, and CDR-VL3, wherein CDR-VL1 is shown as QNIYSN, CDR-VL2 is shown as RAS, and the fifth amino acid Y in CDR-VL3 is shown as QSYYYASGSTYANA, wherein the fifth amino acid Y is deleted or replaced by a non-Y amino acid residue, CDR-VH1 is shown as GFSLSSYP, CDR-VH2 is shown as INRDGNA, and CDR-VH3 is shown as ARGDDNYGDLLFNL.

[0008] Secondly, the present invention provides a nucleic acid molecule that encodes a monoclonal antibody mutant as described above.

[0009] Thirdly, the present invention provides a recombinant vector comprising the nucleic acid molecules described above.

[0010] Fourthly, the present invention provides a transformed host cell, wherein the transformed cell is a vector containing, as described above, a gene encoding a mutant monoclonal antibody against the anti-T4-T4 antibody complex.

[0011] Fifthly, the present invention provides a method for preparing a mutant that can be used for T4 detection, the method comprising: expressing the monoclonal antibody mutant in host cells transformed as described in the fourth aspect above under conditions suitable for the expression of the monoclonal antibody, and recovering the expressed monoclonal antibody mutant from the culture of the host cells.

[0012] Sixthly, the present invention also provides the use of the monoclonal antibody mutant in the preparation of a detection reagent or kit for detecting T4.

[0013] In a seventh aspect, the present invention provides a reagent or kit for detecting T4, comprising a monoclonal antibody mutant for T4 detection as described in the first aspect above.

[0014] This invention provides the application of the mutant in T4 detection to achieve more accurate and reliable quantitative detection of T4 in samples, fundamentally improving the performance of immunoassay methods.

[0015] This invention offers the following advantages: Based on the obtained wild-type anti-T4-T4 antibody complex monoclonal antibody T4M21, this invention successfully obtained an antibody mutant with a light chain variable region including the sequence SEQ ID NO:3 by saturation mutation or deletion mutation at position 93 of the antibody light chain. This mutant brings significant advantages: while retaining its high affinity binding to the T4-T4 antibody complex, it significantly reduces cross-reactivity with free anti-T4 antibodies. This characteristic effectively solves the key technical challenges of high background signal and insufficient specificity in this type of detection, enabling clear and accurate differentiation between the target T4-T4 antibody complex and free T4 antibody components in complex clinical samples. This greatly improves the accuracy, reliability, and signal-to-noise ratio of immunoassay reagents, and has significant clinical application value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the specific embodiments of the present invention or the prior art, the accompanying drawings used in the description of one or more specific embodiments or the prior art will be briefly introduced below.

[0017] Figure 1 This is a statistical graph showing the correlation between the results of the wild-type and optimal mutant △Y93 anti-T4-T4 antibody complex antibody chemiluminescent immunoassay kit of the present invention and the Roche kit in clinical sample detection in Example 6. In this graph, A represents the clinical correlation of wild-type and B represents the clinical correlation of mutant △Y93. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0020] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0021] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0022] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or," unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described, unless the context clearly specifies otherwise.

[0023] In this article, "affinity" or "affinity" refers to the strength of the binding between an antigen and an antibody, typically referring to the strength of the interaction between a binding site of the antibody and an antigenic determinant. In this invention, the term "affinity" represents the equilibrium constant for the reversible binding of two reagents, denoted as K. D .

[0024] Typically, the variable regions VL / VH of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0025] The term "nucleic acid" in this article includes variants of its conserved substitutions (e.g., substitutions for degenerate codons) and complementary sequences. The terms "nucleic acid" and "polynucleotide" are synonymous and include genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.

[0026] Constructing suitable vectors containing the target coding and regulatory sequences can be performed using standard ligation and restriction techniques known in the art. Isolated plasmids, DNA sequences, or synthetic oligonucleotides are cut, tailed, and re-ligated as needed. Mutations can be introduced into the coding sequence using any method to produce variants of the invention; these mutations can include deletions, insertions, substitutions, etc.

[0027] This invention utilizes structural simulation and molecular docking of antigens and antibodies to analyze binding modes and mutate amino acids that may cause cross-reactivity between antibodies. Through genetic engineering, a mutant anti-T4-T4 antibody complex monoclonal antibody with significantly reduced cross-reactivity to free anti-T4 antibodies was constructed. The invention also includes related nucleic acid and cell products, their preparation methods and applications, and immunoassay reagents based on these products. This reduces cross-signals during detection and greatly improves the specificity and accuracy of T4 detection.

[0028] This invention systematically replaces or deletes the 93rd amino acid of the light chain of the parental antibody against the T4-T4 antibody complex (whose light chain variable region sequence is shown in SEQ ID NO: 1) using saturation mutagenesis or site-directed mutagenesis techniques, thereby obtaining a series of antibody mutants for T4 detection.

[0029] In some embodiments, a monoclonal antibody mutant for T4 detection is disclosed, the monoclonal antibody mutant comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity-determining regions CDR-VH1, CDR-VH2, and CDR-VH3, and the light chain variable region comprising complementarity-determining regions CDR-VL1, CDR-VL2, and CDR-VL3, wherein CDR-VL1 is shown as QNIYSN, CDR-VL2 as RAS, and CDR-VL3 as QSYYYASGSTYANA, wherein the fifth amino acid Y is deleted or substituted by a non-Y residue, CDR-VH1 as GFSLSSYP, CDR-VH2 as INRDGNA, and CDR-VH3 as ARGDDNYGDLLFNL.

[0030] In some implementations, the CDR-VL3 is QSYYASGSTYANA, or QSYYAASGSTYANA, or QSYYGASGSTYANA.

[0031] In some embodiments, the light chain variable region is based on the sequence shown in SEQ ID NO: 1, in which the amino acid at position 93 is deleted or replaced by a non-Y residue.

[0032] In some embodiments, the light chain variable region is a sequence based on the sequence shown in SEQ ID NO: 1, with the 93rd amino acid deleted, or with a Y93A mutation, or with a Y93G mutation.

[0033] In some embodiments, the heavy chain variable region is as shown in SEQ ID NO: 2.

[0034] In some embodiments, the light chain variable region of the monoclonal antibody mutant is shown as SEQ ID NO: 3.

[0035] Through screening, we unexpectedly discovered that the mutant containing the sequence shown in SEQ ID NO:3 in the light chain variable region maintained high binding activity with the T4-T4 antibody complex while exhibiting the lowest cross-reactivity with free anti-T4 antibody.

[0036] Some embodiments of the present invention involve a class of nucleic acid molecules that encode monoclonal antibody mutants as described above.

[0037] Some embodiments of the present invention relate to a recombinant vector comprising a nucleic acid molecule encoding a monoclonal antibody mutant as described above.

[0038] The recombinant vector can be a cloning vector or an expression vector.

[0039] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0040] Some embodiments of the present invention relate to a transformed host cell, wherein the transformed cell is converted with a vector containing a gene encoding a mutant, as described above.

[0041] The host cells encompass both prokaryotic and eukaryotic systems. Prokaryotic cells may include Escherichia coli or Bacillus; eukaryotic cells include yeast, insect cells, and mammalian cells (such as commonly used cell lines like CHO and HEK293).

[0042] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0043] The construction of mutants for T4 detection described in this invention is not limited to PCR amplification using the designed primers used in this invention, but also includes methods such as gene synthesis.

[0044] The expression of antibody mutants for T4 detection described in this invention is not limited to the expression system and expression form used in this invention. The system for expressing antibodies also includes prokaryotic cells, eukaryotic cells, mammalian cells, etc. The expression forms of antibodies also include Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)2, biantibodies, peptides containing CDRs, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies, etc.

[0045] Some embodiments of the present invention relate to a method for preparing the above-described monoclonal antibody mutant for T4 detection, the method comprising: expressing the monoclonal antibody mutant in host cells transformed as described in the fourth aspect above under conditions suitable for the expression of the monoclonal antibody, and recovering the expressed monoclonal antibody mutant from the culture of the host cells.

[0046] Some embodiments of the present invention relate to the use of the monoclonal antibody mutant for T4 detection in the preparation of detection reagents or kits for detection.

[0047] Some embodiments of the present invention relate to a detection reagent or kit for detecting T4, which includes monoclonal antibodies as described above.

[0048] This invention provides the application of the monoclonal antibody mutant in T4 detection to achieve more accurate and reliable quantitative detection of T4 in samples, fundamentally improving the performance of immunoassay methods.

[0049] Preferably, the detection reagent is a double-antibody sandwich immunoassay kit, comprising: a) binding an anti-T4 antibody to the T4 antigen in the test sample to form a T4-T4 antibody complex under conditions sufficient for antigen-antibody binding reaction; and b) binding the monoclonal antibody mutant thereon to the T4-T4 antibody complex from step a) to form an immune sandwich complex; and c) determining the amount of the T4 antigen by detecting the amount of the immune sandwich complex.

[0050] Some embodiments of the present invention relate to a method for detecting the presence or level of T4 in a sample, the method comprising using a monoclonal antibody or an antigen-binding fragment thereof as described above.

[0051] The detection methods may include enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, colloidal gold immunoassay, immunochromatography, and similar methods.

[0052] Preferably, the method employs a chemiluminescent immunoassay sandwich method.

[0053] The general methods for using monoclonal antibodies to detect the presence or level of a target antigen in a sample are well known to those skilled in the art.

[0054] The present invention will be further described in detail below with reference to specific embodiments.

[0055] Example 1: Preparation of anti-T4 monoclonal antibody

[0056] 1) Animal immunization

[0057] ① Immunogen preparation: Dissolve the purchased T4-BSA immunogen (Abgree, T7045) in sterile PBS, mix it with an equal volume of Freund's complete adjuvant (Sigma) and emulsify thoroughly.

[0058] ② Experimental animals: 6-8 week old, SPF-grade female Balb / c mice were selected.

[0059] ③ Primary immunization: Each mouse was injected with the above-mentioned emulsified antigen containing 300 μg of T4-BSA via subcutaneous multi-point injection.

[0060] ④ Booster immunization: Starting from the 3rd week after the primary immunization, booster immunization is performed by emulsifying Freund's incomplete adjuvant with the alloantigen (150 μg / animal) using the same injection method as before. This step is repeated twice, with an interval of 3 weeks between each time.

[0061] ⑤ Pre-fusion shock: Three days before the planned cell fusion operation, mice were intraperitoneally injected with 50 μg of purified T4-BSA antigen dissolved in PBS to complete the final shock immunization.

[0062] 2) Preparation, screening and cloning of hybridoma cells

[0063] ① Cell fusion and initial culture: Spleen cells from immunized mice were fused with myeloma cells using the PEG method, resuspended in HAT selective medium, and then plated in 96-well plates for culture.

[0064] ② Preliminary screening (competitive ELISA): After one week of culture, cell supernatant was collected and preliminary screening was performed using a competitive ELISA method. The cells were coated with a secondary antibody (rabbit anti-mouse IgG) and screened for positive wells with strong T4 binding ability (significant competitive inhibition) using the T4-specific enzyme-labeled antigen as the competitive agent.

[0065] ③ Specificity screening (cross-reactivity assessment): Cross-reactivity tests are performed on the supernatant of the initial screening positive wells to assess their specificity for non-target antigens (such as T3), and wells with low cross-reactivity rates are selected.

[0066] ④ Subcloning and Identification: Subcloning is performed on highly specific candidate wells using a limiting dilution method. After culturing, the supernatant from each subcloning well is analyzed in steps two and three. This process is repeated 3-4 times until all wells are positive and the cell population within each well is confirmed to be monoclonal.

[0067] ⑤ Establishment: Expand and culture the above monoclonal cell population to obtain a hybridoma cell line that stably secretes anti-T4 monoclonal antibody, named 6E2C8.

[0068] 3) Antibody preparation

[0069] ① Cell expansion and animal inoculation: After expanding the monoclonal hybridoma cell line (6E2C8), inoculate each animal with (5-10) × 10⁶ cells / animals. 6 A dose of 1 cell was administered intraperitoneally to compatible mice that had been pre-stimulated with Freund's incomplete adjuvant (IFA).

[0070] ②Ascites induction and collection: After inoculation, when the mice's abdomens are significantly distended, ascites is collected using aseptic puncture or after sacrifice. The ascites is centrifuged, and the clear supernatant is collected.

[0071] ③ Antibody affinity purification: The antibodies in the ascites fluid were purified using protein A (SPA) affinity chromatography. The specific process was as follows: the ascites supernatant was loaded into a pre-equilibrated Protein A chromatography column, thoroughly washed, and the bound specific antibodies were eluted with a low pH buffer and then rapidly neutralized to physiological pH.

[0072] ④ Product acquisition: The elution peak solution is desalted or replaced (e.g., replaced with PBS buffer), and then filtered through a 0.22μm filter membrane for sterilization to obtain a high-purity monoclonal antibody (6E2C8) that specifically binds to T4.

[0073] Example 2: Construction of an antibody mutation library for detecting T4

[0074] This embodiment illustrates the process of obtaining a monoclonal antibody mutant against T4 using site-directed mutagenesis.

[0075] 1) Parental antibodies and site selection

[0076] Based on the parental monoclonal antibody (T4M21 antibody) developed by Guangzhou Wondfo Biotech Co., Ltd. that can bind to the T4-T4 antibody complex, its light chain variable region (VL) amino acid sequence is shown in SEQ ID NO: 1. Through structural analysis and sequence alignment, position 93 (by Kabat number) of the light chain was identified as a critical position affecting the complementary conformation. To optimize its specificity, we decided to perform saturation mutation and deletion at this site.

[0077] Light chain variable region (VL) SEQ ID NO: 1 (underlined parts are complement determination regions CDR-VL1, CDR-VL2, and CDR-VL3, respectively):

[0078] DLDMTQTPSSVEAAVGGTVTIKCQAS QNIYSN LAWYQQKPGQPPKLLIS RAS NLASGVPSRFKGSGSGTQFTLTISDLECADAATYYC TO THE END FGGGTELEIL.

[0079] Heavy chain variable region (VH) SEQ ID NO:2 (underlined parts are complementation determinant regions CDR-VH1, CDR-VH2, and CDR-VH3, respectively):

[0080] QSLEESGGRLVTPGTPLTLTCTAS GFSLSSYP LNWVRQAPGKGLEWIGF INRDGNA YYATWVKGRFTISRTSTTVDLKITSPTTEDTATYFC ARGDDNYGDLLFNL WGQGTLVTISS.

[0081] The above-mentioned antibody is a wild-type antibody against the T4-T4 antibody complex.

[0082] The amino acid sequences of the heavy chain constant region and the light chain constant region are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0083] Heavy chain constant region: SEQ ID NO:8:

[0084] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0085] Light chain constant region: SEQ ID NO:9:

[0086] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0087] 2) Mutant primer design

[0088] Saturation Mutation: A pair of PCR primers was designed and synthesized. The forward primer was a universal sequence, and the reverse primer was a mutant primer containing the degenerate codon "NNK" (where N stands for A / T / C / G and K stands for G / T). This degenerate codon encodes all 20 natural amino acids and covers both TAA and TAG stop codons. Using this primer, the 93rd codon of the parental antibody gene was replaced with "NNK".

[0089] Site deletion mutant: Design and synthesize a pair of PCR primers, deleting amino acid 93 from the primers.

[0090] Table 1 Mutant Primers

[0091]

[0092] 3) Construction of the mutation library:

[0093] Overlap extension PCR was used to replace the corresponding fragment in the parental antibody vector with a mutant fragment containing "NNK" or with the deletion of position 93. After the PCR product was digested with DpnI enzyme to remove the template, it was transformed into *E. coli* competent DH5α cells to construct a mutant library. Single clones were then picked and sequenced to verify that the mutation at this site covered all 20 amino acid substitutions and deletions.

[0094] Example 3: Expression and purification of single-chain antibodies

[0095] This example illustrates the expression and purification of antibody T4M21 in mammalian cells.

[0096] 1) Vector construction and plasmid extraction

[0097] The single clones that were correctly sequenced in Example 1 were expanded and cultured. The plasmids were extracted using an endotoxin-free plasmid extraction kit (such as Tiangen's Endo-Free Maxi Kit) to obtain heavy and light chain plasmids, and their concentration and purity were determined.

[0098] 2) Cell transfection and expression

[0099] Cell line: HEK293F suspension cells.

[0100] Culture conditions: FreeStyle™ 293 Expression Medium was cultured in a shaker at 37°C, 8% CO2, and 125 rpm.

[0101] Transfection: When the HEK293F cell density reached 2.0 x 10⁻⁶ cells / year... 6 Transfection was performed when the cell count was >95% and the cell viability was >95%. For every 1 L of cell suspension, 1 mg of total plasmid (heavy and light chain plasmids mixed at a 1:1 mass ratio) and 3 mg of linear polyethyleneimine transfection reagent were diluted separately with 50 mL of Opti-MEM. After mixing, the mixture was allowed to stand at room temperature for 15-20 minutes to form a complex. The complex was then added dropwise to the cell suspension, and the cells were cultured continuously.

[0102] Harvesting: 5-6 days after transfection, when the viability of HEK293F cells drops to 70%-80%, centrifuge at 4000×g and 4℃ for 30 minutes to collect the cell supernatant.

[0103] 3) Antibody purification

[0104] Cell supernatant was filtered through a 0.45 μm PES membrane. The Protein A affinity chromatography column was equilibrated with 5 column volumes of PBS using the AKTA pure protein purification system. The sample was loaded at a flow rate of 1–2 mL / min. The column was washed with 10 column volumes of PBS until the UV baseline stabilized. Elution was performed with 0.1 M glycine-hydrochloric acid buffer (pH 3.0), and the collected elution peak was immediately neutralized with 1 M Tris-HCl buffer (pH 8.0). The purified antibody solution was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed overnight with 2 L of PBS at 4 °C and magnetic stirring, changing the dialysate 2–3 times during the dialysate process. Finally, the solution was filtered through a 0.22 μm filter for sterilization, aliquoted, and stored at -80 °C.

[0105] Example 4: Activity and affinity assay (ELISA)

[0106] In this embodiment, the binding activity and affinity of wild-type and mutant antibodies to the T4-T4 antibody complex were determined by ELISA.

[0107] Coating: Dilute the T4-T4 antibody complex (a conjugate of T4 and anti-T4 monoclonal antibody (6E2C8) hereinafter the same) to 2 μg / mL with carbonate buffer, add 100 μL to each well of a 96-well microplate, and coat overnight at 4°C.

[0108] Blocking: Discard the coating solution, add 200 μL of 3% BSA to each well, and block at 37°C for 2 hours.

[0109] Primary antibody incubation: The purified T4M21 antibody or its mutant protein was serially diluted 3-fold with PBS (8 dilutions in total). 100 μL of the diluted antibody was added to each well and incubated at 37°C for 1 hour.

[0110] ④ Washing: Wash the microplate with PBST 3 times, soaking for 1 minute each time and patting it dry on thick absorbent paper.

[0111] ⑤ Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-human IgG antibody diluted 1:5000 with PBS to each well and incubate at 37°C for 1 hour.

[0112] ⑥ Washing: Same as step 4, but wash 4 times.

[0113] ⑦ Color development and termination: Add 100 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 10 minutes. Then add 50 μL of 2 M H2SO4 to each well to terminate the reaction.

[0114] ⑧ Detection and Analysis: Immediately read the OD450nm value on the microplate reader. Use software for data analysis and graphing, fit the EC50, and derive its K0. D .

[0115] Table 1. Activity and affinity of anti-T4-T4 antibody complex wild-type and mutant antibodies.

[0116]

[0117]

[0118]

[0119] Based on the data in Table 1, mutations targeting position 93 of the light chain of the wild-type anti-complex antibody showed significant changes in affinity. Most mutants exhibited varying degrees of decreased affinity, with the mutant ΔY93 (deleting position 93) showing a nearly 10-fold decrease in affinity. Only the mutant Y93A showed a slight increase in affinity. Overall, these mutants... D All have an E-9 value, indicating high affinity, which meets the requirements for T4 detection using the double-antibody sandwich method.

[0120] Example 5: Mutant Cross-Reactivity Test (Chemiluminescent Immunoassay)

[0121] A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Anti-T4 monoclonal antibody was immobilized with magnetic beads as a capture antibody. An anti-T4-T4 antibody complex monoclonal antibody (a mutant monoclonal antibody suitable for T4 detection) was conjugated to ALP. The T4-containing sample, ALP substrate, and corresponding buffer components were placed in a fully automated chemiluminescence immunoassay analyzer from Wondfo Biotech, and the instrument program was set for detection. The relative light intensity (RLU) measured by a photomultiplier tube showed a specific proportional relationship with the T4 concentration in the sample.

[0122] (1) Preparation of capture antibodies

[0123] ① Magnetic bead pretreatment: Take 80 mg of magnetic beads (Magnosphere™, catalog number: MS160), wash 3 times with 0.1 M MES buffer (pH 6.0), and resuspend in the same buffer for later use.

[0124] ② Antibody activation: Dilute the anti-T4 monoclonal antibody to 2 mg / mL with 0.1 M PBS (pH 7.4). Add 4 μg of cross-linking agent BS(PEG)5 (polyethylene glycol-modified bis(sulfosuccinimide) octanoic acid) and incubate at room temperature in the dark for 30 minutes (e.g., 25°C, 30 minutes) to activate the amino groups on the antibody surface.

[0125] ③ Coupling reaction: Add the activated antibody solution to the pretreated magnetic beads and react at room temperature for 1 hour under gentle mixing conditions.

[0126] ④ Blocking: After the reaction is complete, magnetically separate and discard the supernatant. Wash the magnetic beads three times with washing buffer (0.05 M Tris-HCl, pH 7.4). Then add 0.05 M Tris buffer (pH 7.4) containing 0.5% BSA (bovine serum albumin) and block at 37°C for 1 hour (e.g., 37°C, 1 hour) to block unreacted active sites on the magnetic beads.

[0127] ⑤ Preservation of final product: After blocking, the blocking solution is discarded by magnetic separation. The magnetic beads are washed and resuspended with preservation buffer (Tris buffer containing 0.1% BSA and 0.05% sodium azide) to achieve a final concentration of 1 mg / mL. The final product of immunomagnetic microparticles is obtained by storage at 4℃.

[0128] (2) Preparation of detection antibodies

[0129] ① Antibody activation (introduction of thiol reactive groups): Replace the anti-T4-T4 antibody complex monoclonal antibody (the monoclonal antibody and its mutant) with conjugation buffer (e.g., 0.1 M PBS, pH 7.4) to an antibody concentration of 5 mg / mL. Add 2 μL of 5 mg / mL SPDP (succinimide 3-(2-pyridyldithio)-propionate) and incubate at room temperature in the dark for 1 hour.

[0130] ② Enzyme activation (introduction of free sulfhydryl groups): Dilute alkaline phosphatase (ALP) to 20 mg / mL with DTT activation buffer (e.g., 0.1 MPBS, pH 8.0). Then add 2 μL of 5 mg / mL DTT to 50 μL of alkaline phosphatase and incubate at room temperature for 1 hour.

[0131] ③ Cross-linking reaction: Mix the SPDP-activated antibody with the DTT-activated alkaline phosphatase and cross-link at room temperature in the dark for 1 hour.

[0132] ④ Desalting: After the reaction, the desalted and purified conjugate was recovered to 0.1 mg / mL using 0.01 M PBS buffer at pH 7.4.

[0133] ⑤ Preparation of final product: Dilute the purified antibody-alkaline phosphatase cross-linked product to the working concentration with enzyme preservation buffer (Tris buffer containing 1% BSA, 1 mM MgCl2, 0.1 mM ZnCl2, and 0.05% sodium azide, pH 8.0), aliquot and store at -20℃ to obtain the detection antibody.

[0134] The instrument selected was a fully automated chemiluminescence immunoassay analyzer (Guangzhou Wondfo Biotech Co., Ltd., model Shine i2900).

[0135] 1) The detection includes the following steps:

[0136] a) The sample to be tested (containing T4 or not containing T4) and magnetic microspheres coated with anti-T4 monoclonal antibody are placed in the reaction system and incubated, so that the T4-T4 antibody complex formed is captured on the surface of the microspheres.

[0137] b) Perform magnetic separation and washing to remove unbound substances;

[0138] c) Add ALP-labeled wild-type monoclonal antibody or its mutant antibody of the present invention and incubate to allow the labeled antibody to bind to the captured T4-T4 antibody complex;

[0139] d) Perform magnetic separation and washing again to remove unbound labeled antibodies;

[0140] e) Add a chemiluminescent substrate (acridinium ester) and measure the resulting chemiluminescent signal, wherein the relative light unit (RLU) value of the signal is directly proportional to the concentration of T4.

[0141] 2) Cross-reactivity and signal-to-noise ratio test

[0142] Using the above detection method, the cross-reactivity and signal-to-noise ratio of wild-type and mutant antibodies were detected at the same antibody concentration. The luminescence value of each antibody was detected under the conditions of presence and absence of T4 (PBS buffer). The results are shown in Figure 2.

[0143] Table 2. Cross-reactivity and signal-to-noise ratio of wild-type and mutant antibodies against the anti-T4-T4 antibody complex.

[0144]

[0145]

[0146]

[0147] Using T4-free PBS buffer for testing can reflect the cross-reactivity between anti-T4 antibody and anti-T4-T4 antibody complex antibody. Table 2 shows that the wild-type antibody T4M21 monoclonal antibody exhibits a certain degree of cross-reactivity with the anti-T4 antibody, resulting in a low signal-to-noise ratio. However, some mutants, such as ΔY93, Y93A, and Y93G, show significantly reduced cross-reactivity and better signal-to-noise ratios. Although the detection signal of ΔY93 is low, the cross-reactivity value is reduced by more than 13 times, indicating that ΔY93 has extremely low background signal during detection, avoiding false positives and fully meeting the requirements of the double-antibody sandwich method for chemiluminescence detection. Therefore, it is determined to be the optimal mutant for this invention. Its light chain variable region sequence is shown in SEQ ID NO: 3, and its heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0148] SEQ ID NO: 3:

[0149] DLDMTQTPSSVEAAVGGTVTIKCQASQNIYSNLAWYQQKPGQPPKLLISRASNLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQSYYASGSTYANAFGGGTELEIL.

[0150] SEQ ID NO: 2:

[0151] QSLEESGGRLVTPGTPLTLTCTAS GFSLSSYPLNWVRQAPGKGLEWIGF INRDGNA YYATWVKGRFTISRTSTTVDLKITSPTTEDTATYFC ARGDDNYGDLLFNL WGQGTLVTISS.

[0152] Example 6: Performance Evaluation of the T4 Double Antibody Sandwich Chemiluminescent Immunoassay Kit

[0153] Using wild-type and the optimal mutant △Y93, 155 clinical samples were collected, including samples from different age groups and sexes. Sample concentrations were detected using the immunoluminescence assay kits and methods described in Examples 4 and 5 of this invention. A fully automated chemiluminescence immunoassay analyzer (Guangzhou Wondfo Biotech Co., Ltd., model Shine i2900) was used as the detection tool. The methodology was a double-antibody sandwich method: 10 μL of clinical sample was added to the instrument, followed by 50 μL of capture antibody (magnetic microparticles coated with anti-T4 monoclonal antibody (6E2C8)) and 50 μL of detection antibody (alkaline phosphatase-labeled anti-T4-T4 antibody complex monoclonal antibody and its mutant). After reacting for 10 min, magnetic separation was performed. Then, 200 μL of substrate solution was added, mixed, and the instrument sent the reaction mixture into a dark chamber. Finally, the luminescence value was recorded, and the concentration was calculated. Simultaneously, the concentration of the above clinical samples was determined using Roche's T4 detection kit.

[0154] The detection concentrations of wild-type monoclonal antibody and mutant ΔY93 were analyzed and compared with the concentration determination results of Roche's T4 detection kit. The clinical relevance of wild-type T4M21 antibody was R. 2 =0.9731, the clinical relevance of the mutant antibody ΔY93 is R. 2 =0.9902, indicating that the mutant provided by this invention has better clinical relevance. Combined with the detection results of cross-signal in Example 5, the mutant greatly reduces the cross-reactivity between antibody pairs, avoiding the risk of false positives caused by high background signal from the source. In summary, the mutant provides higher accuracy and specificity for the detection of T4.

[0155] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A monoclonal antibody mutant that can be used for T4 detection, characterized in that, The monoclonal antibody mutant includes a light chain variable region and a heavy chain variable region. The heavy chain variable region includes complementarity-determining regions CDR-VH1, CDR-VH2, and CDR-VH3, and the light chain variable region includes complementarity-determining regions CDR-VL1, CDR-VL2, and CDR-VL3. CDR-VL1 is shown as QNIYSN, CDR-VL2 is shown as RAS, and CDR-VL3 is shown as QSYYYASGSTYANA, where the fifth amino acid Y is deleted or replaced by a non-Y amino acid residue. CDR-VH1 is shown as GFSLSSYP, CDR-VH2 is shown as INRDGNA, and CDR-VH3 is shown as ARGDDNYGDLLFNL.

2. The monoclonal antibody mutant according to claim 1, characterized in that, The CDR-VL3 is as shown in QSYYASGSTYANA, or QSYYAASGSTYANA, or QSYYGASGSTYANA.

3. The monoclonal antibody mutant according to claim 1, characterized in that, The light chain variable region is based on the sequence shown in SEQ ID NO:1, in which the amino acid at position 93 is deleted or replaced by a non-Y residue.

4. The monoclonal antibody mutant according to claim 3, characterized in that, The light chain variable region is based on the sequence shown in SEQ ID NO: 1, with the 93rd amino acid deleted, or with a Y93A mutation, or with a Y93G mutation.

5. The monoclonal antibody mutant according to claim 1, characterized in that, The heavy chain variable region is shown in SEQ ID NO:

2.

6. The monoclonal antibody mutant according to any one of claims 1-5, characterized in that, The light chain variable region of the monoclonal antibody mutant is shown in SEQ ID NO:

3.

7. A biological substance characterized by, It is selected from any of the following: A. A nucleic acid molecule, said nucleic acid molecule encoding a monoclonal antibody mutant as described in any one of claims 1-6; B. A recombinant vector, wherein the recombinant vector is infused with the aforementioned nucleic acid molecules; C. Host cell, wherein the host cell is converted with the above-mentioned recombinant vector.

8. A method for preparing the monoclonal antibody mutant according to any one of claims 1-6, the method comprising: Under conditions suitable for the expression of the monoclonal antibody, the host cell as described in claim 7 is made to express the monoclonal antibody mutant, and the expressed monoclonal antibody mutant is recovered from the culture of the host cell.

9. The use of the monoclonal antibody mutant according to any one of claims 1-6 in the preparation of a detection reagent or kit for T4 detection.

10. A reagent or kit for detecting T4, characterized in that, It includes the monoclonal antibody mutant as described in any one of claims 1-6.

11. The reagent or kit according to claim 10, characterized in that, The reagent or kit employs a double-antibody sandwich immunoassay, comprising: a) binding an anti-T4 monoclonal antibody to T4 in the test sample to form a T4-T4 antibody complex under conditions sufficient to induce antigen-antibody binding; and b) binding a monoclonal antibody mutant according to any one of claims 1-6 to the T4-T4 antibody complex of step a) to form an immune sandwich complex; and c) determining the amount of T4 by detecting the amount of the immune sandwich complex.

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