Anti-taq enzyme antibody or functional fragment thereof and use thereof

Abstract

This invention relates to the field of biotechnology, specifically to anti-Taq enzyme antibodies or their functional fragments and their applications. This invention not only provides a monoclonal antibody (wild-type) with high affinity for binding to Taq enzyme antibodies, but also performs site-directed mutations on this wild-type antibody and verifies the affinity of each mutation and its related applications, providing a reference for PCR research in the field of nucleic acid detection.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to anti-Taq enzyme antibodies or their functional fragments and their applications.

[0002] This invention claims priority to application No. 202311056423.0, filed on August 22, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Polymerase chain reaction (PCR) is a method for synthesizing double-stranded DNA in vitro. PCR amplification can amplify a single DNA molecule into millions of copies of DNA molecules in just a few PCR thermal cycles, achieving exponential amplification of DNA. PCR thermal cycles usually include three different steps: denaturation, annealing, and extension. In the denaturation step (about 95°C, high temperature zone), double-stranded DNA unwinds into two single-stranded DNA molecules. Then, the temperature is lowered to perform the annealing step (about 56°C, low temperature zone), which promotes the complementary binding of primers (short DNA sequences) to single-stranded DNA. Finally, under the action of polymerase, the free nucleotides in the solution are synthesized into the second complementary strand of DNA. However, the activity of polymerase is low at the annealing temperature, so the temperature needs to be raised to about 72°C. The step at this temperature is extension. By repeating the three temperature steps, a large amount of DNA can be replicated, and the concentration of the target double-stranded DNA can be increased exponentially. This DNA replication process is called PCR[1].

[0004] At present, there are many ways to improve the specificity of PCR amplification. The simplest method is to use hot-start enzyme amplification. The optimal temperature of ordinary DNA polymerase is 72℃, at which time the enzyme activity is the best. Below this temperature, the enzyme has weak activity, and both 5'-3' polymerase activity and 3'-5' exonuclease activity can play a role. In this way, during the process of PCR amplification from low temperature to high temperature, the enzyme has weak activity, and the system is very prone to mismatch or primer dimer formation, especially when the 3' end of the designed primer is G or C, the mismatched strand is difficult to untie. To improve the specificity of PCR amplification and reduce the reaction mismatch rate, the enzyme activity can be artificially controlled. Before the hot-start temperature, the enzyme is inactive, which avoids the occurrence of strand mismatch during the heating process (or preparation of the reaction system), thereby ensuring the specificity of amplification [2].

[0005] Given the high affinity of monoclonal antibodies for antigens, Taq polymerase can be blocked using monoclonal antibodies, effectively inhibiting non-specific annealing of primers and non-specific amplification caused by primer dimers under low-temperature conditions. Taq polymerase is a fundamental tool in life science research and is widely used in universities, research institutes, and biotechnology companies. Low-cost production of blocking antibodies would significantly reduce the production cost of antibody-based hot-start polymerases. Existing anti-Taq monoclonal antibodies are expensive, used in large quantities, and have relatively poor thermal stability. Therefore, there is an urgent need to develop anti-Taq monoclonal antibodies with good blocking efficacy, low cost, and good thermal stability.

[0006] [1] Shi Bing. Research on structural and functional optimization and application of polymerase chain reaction system [J]. [2023-08-09].

[0007] [2] Yisheng Biotechnology Co., Ltd. Taq polymerase 5'-3' polymerase-blocking monoclonal antibody and its application. 2020, CN 111560073 B Summary of the Invention

[0008] The purpose of this invention is to provide a combination of anti-Taq enzyme antibodies or its functional fragments, which can achieve specific binding with Taq enzyme, block Taq enzyme activity at low temperature, and ultimately be applied to the production of Taq enzyme and PCR experiments.

[0009] This invention employs hybridoma technology to screen for specific anti-Taq enzyme antibody combinations (antibody A and antibody B), and then mutates their sequences to select mutant sequences with high stability, specificity, and affinity. The mutated anti-Taq enzyme antibody combinations (antibody A and antibody B) are expressed using recombinant ExpiCHO, which not only avoids batch-to-batch variations caused by mouse ascites production but also simplifies production and reduces costs.

[0010] Therefore, the first object of the present invention is to provide an anti-Taq enzyme antibody combination (antibody A and antibody B, wild type). The second object of the present invention is to provide a mutant form of the anti-Taq enzyme antibody combination (antibody A and antibody B) that blocks Taq enzyme, and its application. The third object of the present invention is to provide a method for preparing the mutant form of the anti-Taq enzyme antibody combination (antibody A and antibody B).

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] An anti-Taq enzyme antibody or a functional fragment thereof, said antibody or functional fragment comprising the following complementarity-determining regions:

[0013] CDR-VH1: D-X1-YI-X2, where X1 is Y or D, and X2 is N;

[0014] CDR-VH2: W-X1-YPG-X2-GHTQ-X3-NERFKG, where X1 is I, X2 is S or T, and X3 is D or Y;

[0015] CDR-VH3: S-X1-FYAM-X2-Y, where X1 is R or A, and X2 is D or N; CDR-VL1: R-X1-SQSIG-X2-SIH, where X1 is A or N, and X2 is T or S;

[0016] CDR-VL2: Y-X1-SES-X2-S, where X1 is A or R, and X2 is I or P;

[0017] And CDR-VL3: Q-X1-TNS-X2-PLT, where X1 is Q and X2 is W or P;

[0018] Named antibody A;

[0019] or

[0020] The antibody or its functional fragment includes the following complementarity-determining regions:

[0021] CDR-VH1: S-X1-W-X2-N, where X1 is F or E, and X2 is I;

[0022] CDR-VH2: D-X1-YPG-X2-GSTHYN-X3-KFKN, where X1 is I or N, X2 is S or T, and X3 is E or T;

[0023] CDR-VH3: S-X1-DGYLF-X2-Y, where X1 is G or P, and X2 is A or R;

[0024] CDR-VL1: R-X1-SQSVSTS-X2-YSFIH, where X1 is A and X2 is T or S; CDR-VL2: Y-X1-SN-X2-DS, where X1 is A or R and X2 is L;

[0025] And CDR-VL3: Q-X1-SWEIP-X2-T, where X1 is H and X2 is W or P;

[0026] It was named antibody B.

[0027] Furthermore, in the complementarity-determining region of antibody A, X2 of CDR-VH1 is N, X1 of CDR-VH2 is I, X2 of CDR-VH3 is D, X1 of CDR-VL1 is A, X2 of CDR-VL2 is I, and X1 of CDR-VL3 is Q.

[0028] or

[0029] The complementarity-determining region of antibody B has X2 of I for CDR-VH1, X1 of I for CDR-VH2, X2 of A for CDR-VH3, X1 of A for CDR-VL1, X2 of L for CDR-VL2, and X1 of H for CDR-VL3.

[0030] The CDR segmentation described in this invention uses the Kabat algorithm.

[0031] In this article, "CDR" refers to the "complementarity-determining region" within the variable sequence of the antibody. Each of the heavy and light chains has three CDRs, starting from the N-terminus of either the heavy or light chain.

[0032] Antigen binding sites may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 in this invention). A polypeptide containing a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, or CDR-VL3) can be termed a "molecular recognition unit." Crystallographic analysis of antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may primarily be responsible for the specificity of the antigen binding site. Generally, CDR residues directly and substantially participate in influencing antigen binding.

[0033] Furthermore, in the CDR-VH1 of antibody A, X1 is Y;

[0034] Or, in the CDR-VH1 of antibody A, X1 is D;

[0035] Or, in the CDR-VH1 of antibody A, X2 is N;

[0036] Or, in the CDR-VH2 of antibody A, X1 is I;

[0037] Or, in the CDR-VH2 of antibody A, X2 is S;

[0038] Or, in the CDR-VH2 of antibody A, X2 is T;

[0039] Or, in the CDR-VH2 of antibody A, X3 is D;

[0040] Or, in the CDR-VH2 of antibody A, X3 is Y;

[0041] Or, in the CDR-VH3 of antibody A, X1 is R;

[0042] Or, in the CDR-VH3 of antibody A, X1 is A;

[0043] Or X2 in the CDR-VH3 of antibody A is D; or X2 in the CDR-VH3 of antibody A is N; or X1 in the CDR-VL1 of antibody A is A; or X1 in the CDR-VL1 of antibody A is N; or X2 in the CDR-VL1 of antibody A is T; or X2 in the CDR-VL1 of antibody A is S; or X1 in the CDR-VL2 of antibody A is A; or X1 in the CDR-VL2 of antibody A is R; or X2 in the CDR-VL2 of antibody A is... X2 is I; or X2 in the CDR-VL2 of antibody A is P; or X1 in the CDR-VL3 of antibody A is Q; or X2 in the CDR-VL3 of antibody A is W; or X2 in the CDR-VL3 of antibody A is P; or X1 in the CDR-VH1 of antibody B is F; or X1 in the CDR-VH1 of antibody B is E; or X2 in the CDR-VH1 of antibody B is I; or X1 in the CDR-VH2 of antibody B is I; or X2 in the CDR-VH2 of antibody B is I; or X1 in the CDR-VH2 of antibody B is P; or X1 in the CDR-VH1 of antibody B is P; or X2 in the CDR-VH1 of antibody B is P; or X1 in the CDR-VH2 of antibody B is P; or X2 in the CDR-VH1 of antibody B is P; or X1 in the CDR-VH1 ... In CDR-VH2, X1 is N; or in CDR-VH2 of antibody B, X2 is S; or in CDR-VH2 of antibody B, X2 is T; or in CDR-VH2 of antibody B, X3 is E; or in CDR-VH2 of antibody B, X3 is T; or in CDR-VH3 of antibody B, X1 is G; or in CDR-VH3 of antibody B, X1 is P; or in CDR-VH3 of antibody B, X2 is A; or in CDR-VH3 of antibody B, X2 is R; or in CDR-VH3 of antibody B, X1 is G; or in CDR-VH3 of antibody B, X1 is P; or in CDR-VH3 of antibody B, X2 is A; or in CDR-VH3 of antibody B, X2 is R; or in CDR-VH3 of antibody B, X1 is G; or in CDR-VH3 of antibody B, X1 is P; or in CDR-VH3 of antibody B, X2 is A; or in CDR-VH3 of antibody B, X2 is R; or in CDR-VH2 ... X1 in CDR-VL1 of antibody B is A; or X2 in CDR-VL1 of antibody B is T; or X2 in CDR-VL1 of antibody B is S; or X1 in CDR-VL2 of antibody B is A; or X1 in CDR-VL2 of antibody B is R; or X2 in CDR-VL2 of antibody B is L; or X1 in CDR-VL3 of antibody B is H; or X2 in CDR-VL3 of antibody B is W; or X2 in CDR-VL3 of antibody B is P.

[0044] Furthermore, the complementarity-determining regions of antibody A are selected from any one of the following mutation combinations:

[0045]

[0046]

[0047] Alternatively, the complementary determinant regions of antibody B may be selected from any of the following mutation combinations:

[0048]

[0049]

[0050] Furthermore, the anti-Taq enzyme antibody or its functional fragment further includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H are selected sequentially from SEQ ID NO:1-4 or from SEQ ID NO:5-8; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected sequentially from SEQ ID NO:9-12 or SEQ ID NO:13-16.

[0051] Furthermore, the anti-Taq enzyme antibody or its functional fragment further includes a constant region;

[0052] Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. Preferably, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0053] Preferably, the functional fragment is selected from any one of the antibody's F(ab')2, Fab', Fab, Fv, and scFv.

[0054] Furthermore, the antibody is a labeled antibody;

[0055] Preferably, the labeling agents for the labeled antibodies include, but are not limited to, latex microspheres, biotin, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labeling agents.

[0056] On the other hand, the present invention discloses the use of the antibody or its functional fragment in the preparation of PCR detection kits or in the preparation of blocking agents.

[0057] On the other hand, the present invention discloses a blocking agent comprising the antibody or a functional fragment thereof;

[0058] Optionally, the concentration of the antibody in the blocking agent is 1-5 mg / mL.

[0059] The "blocking agent" mentioned in this invention refers to a reagent capable of blocking Taq enzyme activity.

[0060] On the other hand, the present invention discloses a detection reagent or kit, the reagent or kit comprising the antibody or its functional fragment or the labeled antibody.

[0061] On the other hand, the present invention discloses a method for blocking Taq enzyme activity, which involves adding the antibody or its functional fragment or the antibody conjugate to an immunoassay system.

[0062] On the other hand, the present invention discloses a method for detecting Taq enzyme, the method comprising:

[0063] A) Under conditions sufficient for a binding reaction to occur, the antibody or its functional fragment, the antibody-drug conjugate, the blocking agent, or the reagent or kit described herein is contacted with a sample from the subject to initiate a binding reaction; and

[0064] B) Detect the immune complexes produced by the binding reaction.

[0065] Beneficial effects:

[0066] This invention employs a combination of cell biology and bioinformatics methods to prepare anti-Taq enzyme monoclonal antibodies expressed in hybridoma cell lines, and obtains antibodies with the highest affinity and specificity through sequence mutation. Expression in CHO cells is not only cost-effective but also exhibits good thermal stability, effectively avoiding the inhibition of non-specific primer annealing and non-specific amplification caused by primer dimers under low-temperature conditions. Attached Figure Description

[0067] Figure 1 PCR spectrum of the blocking effect of anti-Taq antibody combination;

[0068] Figure 2 PCR spectrum for Chlamydia pneumoniae detection;

[0069] Figure 3 PCR spectrum for Mycoplasma pneumoniae detection. Detailed Implementation

[0070] To make the objectives, 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0072] As used herein, the terms “comprising,” “including,” “having,” “may,” and their variations are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0073] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); and *Handbook of Experimental Immunology* (D.M. Weir and CC. Blackwyn). The references cited are: J.M. Miller and M.C. Salos (eds., 1987); F.M. Usubel et al. (eds., 1987); PCR: The Polymerase Chain Reaction (eds., Mullis et al., 1994); and J.E. C. Olgan et al. (eds., 1991), each of which is explicitly incorporated herein by reference.

[0074] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0075] Example 1: Preparation of Monoclonal Antibodies

[0076] 1. Mouse immunization and antibody detection

[0077] Five 6-8 week old SPF-grade female BALB / c mice were selected. Taq polymerase protein (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.) was chosen as the antigen protein and mixed with Freund's complete adjuvant in equal volumes and emulsified. The emulsified antigen was used to immunize the 6-8 week old SPF-grade female BALB / c mice, with each mouse receiving 50 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks after the initial immunization, Taq polymerase was mixed with Freund's incomplete adjuvant and emulsified, and each mouse was again injected with 50 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was detected by ELISA. Immunization was repeated every two weeks, and serum titer was measured. After two immunizations, the serum titer, after a million-fold dilution, was higher than 2.0. A serum titer of 10 was selected for screening. 6 Lymphocytes were isolated from the mice mentioned above for cell fusion.

[0078] 2. Cell fusion and screening of positive hybridoma cells and subcloning

[0079] The hybridoma cell groups obtained were screened through three steps: 1) confirming the ability to bind to Taq polymerase; 2) screening for specific antibody pairs.

[0080] 1) To screen for high-affinity anti-Taq polymerase antibodies, Taq polymerase-bound wells were used for screening. Wells with high ELISA positive values ​​to cell count ratios were selected for multiple subclonings to identify monoclonal hybridoma cells with the highest affinity.

[0081] 2) Evaluation of antibody pair screening: The specificity of the 10 antibodies generated from the clones selected in 1) was evaluated by pairwise pairing, and the hybridoma cells with the highest antibody reactivity were named anti-Taq-A and anti-Taq-B, respectively.

[0082] 3. Production and purification of monoclonal antibodies

[0083] Two groups of 6-8 week old BALB / c mice were selected, and 500 μL of paraffin oil was injected intraperitoneally to suppress the immune response. One week after injection, 0.5 ml of anti-Taq-A hybridoma cells (approximately 1 × 10⁶ cells) was injected intraperitoneally into one group of mice; 0.5 ml of anti-Taq-B hybridoma cells were injected intraperitoneally into the other group. Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification of protein A to obtain two target antibodies, named antibody A (primary antibody) and antibody B (secondary antibody).

[0084] 4. Determination of the titers of antibody A and antibody B against immobilized Taq enzyme.

[0085] Taq enzyme protein was diluted 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate buffer), and 100 μL / well was added to the microplate and incubated overnight at 4°C. For detection, each antibody was diluted to 100 ng / mL and incubated with the antigen at 37°C. HRP-labeled goat anti-mouse polyclonal antibody was used for color development, and the absorbance of the peak values ​​was measured. The results are shown in Table 1 below.

[0086] Table 1 Valence Measurement

[0087]

[0088] The presence of a signal at a low antibody concentration, specifically at 0.1 ng / mL, indicates that both antibody strains have high sensitivity and are suitable for subsequent experiments.

[0089] E. Identification of monoclonal antibody subtypes and cloning of gene sequences

[0090] The heavy and light chain isotypes of monoclonal antibodies were identified using the Southern Biothech SBA Clonotyping System-HRP kit, following the manufacturer's instructions. The specific procedures were as follows:

[0091] 1. Dilute the capture antibody to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer).

[0092] 2. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lambda-HRP) 1:3000 with diluent.

[0093] 3. After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the chromogenic buffer and incubate for approximately 5 minutes (depending on the reaction strength). Then, add 2M sulfuric acid to stop the reaction and read the OD450 absorbance. Identification showed that the anti-Taq-A heavy chain isotype was IgG1, and the light chain was Kappa; the anti-Taq-B heavy chain isotype was IgG2b, and the light chain was Kappa.

[0094] Based on the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instruction manual, and the full-length sequence of the target antibody was amplified.

[0095] Example 2: Validation of antibody performance

[0096] 1. ELISA Affinity Test

[0097] Goat anti-mouse antibody was diluted to 1 μg / mL using coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), and 100 μL was added to each well of a 96-well microplate. The plates were incubated overnight at 4°C. The plates were washed three times with PBS buffer containing 0.05% Tween-20. The antibody was then diluted to 1 μg / mL using dilution buffer (1% BSA, 0.1% PBST), and 100 μL was added to each well of a 96-well microplate. The plates were incubated at 37°C for 30 minutes. Dilute the DNA polymerase to 100 ng / mL with diluent. Remove the ELISA plate from its 37°C incubation and wash it three times. Add 150 μL of the prepared canine C-reactive protein antibody dilution to the 96-well plate in row A. Add 100 μL of diluent to each well in rows B-H. Take 50 μL of the antibody dilution from row A and perform a 3-fold serial dilution from row B onwards. Incubate at 37°C for 30 minutes. Dilute the horseradish peroxidase-labeled avidin 1:5000 with diluent. Remove the ELISA plate and wash it three times. Add 100 μL of the diluted avidin to each well in a 96-well plate and incubate at 37°C for 30 minutes. Remove the ELISA plate and wash it three times. Add the chromogenic buffer and incubate at room temperature for 3 minutes. Stop the reaction by adding 0.5 M sulfuric acid and read the OD450 value using a plate reader.

[0098] 2. The antibody anti-Taq-A in Example 1 has a heavy chain variable region as shown in SEQ ID NO:17, wherein the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0099] CDR-VH1: DY(X1)-YIN(X2);

[0100] CDR-VH2:WI(X1)-YPGT(X2)-GHTQD(X3)-NERFKG;

[0101] CDR-VH3: SR(X1)-FYAMN(X2)-Y;

[0102] Its light chain variable region is shown in SEQ ID NO:18, wherein the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0103] CDR-VL1: RN(X1)-SQSIGT(X2)-SIH;

[0104] CDR-VL2: YA(X1)-SESP(X2)-S;

[0105] CDR-VL3: QQ(X1)-TNSP(X2)-PLT.

[0106] Based on the antibody anti-Taq-A, mutations were performed on sites related to antibody activity in the complementarity-determining region. The results are shown in Tables 2 and 3, where X1, X2, and X3 are all mutation sites.

[0107] Table 2 Mutation sites related to antibody activity

[0108]

[0109]

[0110] Table 3 Antibody activity analysis data

[0111]

[0112] As can be seen from the table above, mutation 3 exhibits the best activity. Therefore, mutation 3 was used as the backbone sequence to screen for other mutation sites with good affinity. Some results are shown in Table 4:

[0113] Table 4 Mutation sites related to antibody affinity

[0114]

[0115]

[0116] Taq enzyme protein 100 ng / ml, absorbance at OD450 was measured for each mutant, and the results are shown in Table 5:

[0117] Table 5. Affinity test for antibody mutations

[0118] Mutation 3-2 2.3095 Mutation 3-12 2.4408 Mutation 3-22 2.1591 Mutation 3-3 2.2268 Mutation 3-13 2.0563 Mutation 3-23 2.4127 Mutation 3-4 2.1243 Mutation 3-14 2.1253 Mutation 3-24 2.5061 Mutations 3-5 2.5709 Mutation 3-15 2.2476 Mutation 3-25 2.1232 Mutations 3-6 2.1988 Mutation 3-16 2.2083 Mutation 3-26 2.0377 Mutations 3-7 2.3155 Mutation 3-17 2.3500 Mutation 3-27 2.4009 Mutations 3-8 2.3666 Mutation 3-18 2.0038 Mutation 3-28 2.3146 Mutations 3-9 2.2470 Mutation 3-19 2.2504 Mutation 3-29 2.0133 Mutation 3-10 2.3696 Mutation 3-20 2.1352 Mutation 3-30 2.2379

[0119] It can be seen that the affinity of each mutation is high.

[0120] 3. Anti-Taq-A mutation stability assay

[0121] The concentration of the antibody in this invention is 1 μg / mL, and the concentration of the Taq enzyme protein is 100 ng / mL.

[0122] The antibody was prepared in a predetermined buffer (PBS, 0.05% ProClin). TM The antibody was accelerated at 37°C for 7 days (300). The accelerated antibody was evaluated by SDS-PAGE and ELISA indirect methods, with a control at 4°C, to determine the long-term stability of the antibody. In addition, the antibody was subjected to repeated freeze-thaw cycles at -20°C 5 times. The test results are shown as the deviation between the values ​​at 4°C and the values ​​after acceleration. The measurement results are shown in Table 6.

[0123] Table 6 Stability Study

[0124]

[0125]

[0126] As can be seen from the above, mutations 3 to 3-30 can be stably stored at 4°C, and their properties remain stable even after being accelerated at 37°C for 0.5 months.

[0127] 4. The antibody anti-Taq-B in Example 1 has a heavy chain variable region as shown in SEQ ID NO:19, wherein the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0128] CDR-VH1: SE(X1)-WI(X2)-N;

[0129] CDR-VH2: DN(X1)-YPGT(X2)-GSTHYNF(X3)-KFKN;

[0130] CDR-VH3: SG(X1)-DGYLFR(X2)-Y;

[0131] Its light chain variable region is shown in SEQ ID NO:20, wherein the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0132] CDR-VL1:RA(X1)-SQSVSTSS(X2)-YSFIH;

[0133] CDR-VL2: YA(X1)-SNL(X2)-DS;

[0134] and CDR-VL3:QH(X1)-SWEIPP(X2)-T.

[0135] Based on the anti-Taq-B antibody, mutations were performed on sites related to antibody activity in the complementarity-determining region. The results are shown in Tables 7 and 8, where X1, X2, and X3 are all mutation sites.

[0136] Table 7 Mutation sites related to antibody activity

[0137]

[0138] Table 8 Antibody Activity Analysis Data

[0139]

[0140] As can be seen from the table above, mutation 5 exhibits the best activity. Therefore, mutation 5 was used as the backbone sequence to screen for other mutation sites with good affinity. Some results are shown in Table 9:

[0141] Table 9 Mutation sites related to antibody affinity

[0142]

[0143]

[0144] Taq enzyme protein 100 ng / ml, absorbance at OD450 was measured for each mutant, and the results are shown in Table 10:

[0145] Table 10 Affinity test for antibody mutations

[0146] Mutation 5-1 2.0234 Mutation 5-12 2.3317 Mutation 5-22 2.4107 Mutation 5-2 2.5070 Mutation 5-13 2.4427 Mutation 5-23 2.4417 Mutation 5-3 2.4207 Mutation 5-14 2.2127 Mutation 5-24 2.0567 Mutation 5-4 2.1327 Mutation 5-15 2.5200 Mutation 5-25 2.1327 Mutation 5-5 2.4227 Mutation 5-16 2.6378 Mutation 5-26 2.5252 Mutations 5-6 2.5157 Mutation 5-17 2.007 Mutation 5-27 2.0050 Mutations 5-8 2.5427 Mutation 5-18 2.0257 Mutation 5-28 2.4457 Mutations 5-9 2.3527 Mutation 5-19 2.4207 Mutation 5-29 2.3357 Mutations 5-10 2.5125 Mutation 5-20 2.1237 Mutation 5-30 2.5017

[0147] It can be seen that all mutations have high affinity.

[0148] 5. Anti-Taq-B mutation stability assay

[0149] The concentration of the antibody in this invention is 1 μg / mL, and the concentration of the Taq enzyme is 100 ng / mL.

[0150] The antibody was prepared in a predetermined buffer (PBS, 0.05% ProClin). TM The antibody was subjected to accelerated thermal treatment at 37°C for 7 days (300), and the accelerated antibody was evaluated by indirect ELISA. A control was used at 4°C to assess the long-term stability of the antibody. Additionally, the antibody underwent five freeze-thaw cycles at -20°C. The results are shown as the deviation between the values ​​at 4°C and the accelerated values. The measurement results are shown in Table 11.

[0151] Table 11 Stability Study

[0152] WT -9％ 2.5％ 6.4％ 6.5％ Mutation 5 7％ 0.9％ -4.2％ -2.3％ Mutation 5-1 -4％ -7.2％ -8.2％ -3.5％ Mutation 5-2 -4％ 1.7％ -3.0％ -6.3％ Mutation 5-3 6％ 0.0％ -4.0％ -10.2％ Mutation 5-4 11％ 3.4％ -11.4％ -8.7％ Mutation 5-5 8％ 2.8％ 2.1％ 10.5％ Mutations 5-6 1％ 2.9％ 2.1％ -12.3％ Mutations 5-7 -7％ -9.1％ 2.5％ 4％ Mutations 5-8 8％ 4.6％ 0.9％ -2％ Mutations 5-9 -2％ -5.1％ -7.2％ 2％ Mutations 5-10 -14％ 0.9％ 1.7％ 3％ Mutation 5-11 5％ 1.6％ 0.0％ 3％ Mutation 5-12 5％ -0.7％ 3.4％ -3％ Mutation 5-13 4％ 11.4％ 2.8％ -14％ Mutation 5-14 6％ 9.1％ 2.9％ -7％ Mutation 5-15 -5％ -8.0％ 2％ -4％ Mutation 5-16 0.5％ 3.0％ 1％ -4％ Mutation 5-17 3％ -0.5％ -4％ -7％ Mutation 5-18 -7％ 2.3％ 5％ -8％ Mutation 5-19 12％ -2.3％ 14％ -4％ Mutation 5-20 12％ -6.4％ 11％ -5％ Mutation 5-21 1％ -0.5％ 1％ -8％ Mutation 5-22 -3％ -6.3％ 9％ -6％ Mutation 5-23 -4％ -3％ 1％ -3％ Mutation 5-24 9％ 1％ 7％ 4％ Mutation 5-25 -6％ -3％ -4％ -1％ Mutation 5-26 11％ 9％ 1％ -1％ Mutation 5-27 0％ -4％ 10％ 2％ Mutation 5-28 -2％ -1％ 1％ -10％ Mutation 5-29 -5％ 5％ 2％ -7％ Mutation 5-30 5％ -1％ -5％ -2％

[0153] As can be seen from the above, mutations 5 to 5-30 can be stably stored at 4°C, and their properties remain stable even after being accelerated at 37°C for 0.5 months.

[0154] Example 3: Production and Purification of Recombinant Antibodies

[0155] To study the application performance of antibodies, it is necessary to first produce and purify recombinant antibodies. From the performance experiments of the antibodies themselves, it can be seen that antibody A, mutation 3-30, antibody B, and mutation 5-30 all have good affinity and stability. It can be inferred that the application performance of antibody A and mutation 3-30, and antibody B and mutation 5-30 are comparable. Due to the limitations of the examples themselves, it is impossible to exhaustively demonstrate the effects. In Examples 3-4, mutation 3-5 and mutation 5-16 were randomly selected for subsequent experiments.

[0156] Recombinant antibody production, especially using the Chinese hamster ovary cell (CHO) system, results in lower host protein levels, higher expression levels, and is commercially mature, widely used in antibody drug preparation, with production capacities ranging from gram to tens of grams. Compared to mouse ascites antibodies, it offers not only more stable production and smaller batch-to-batch variation but also significantly lower costs. The specific implementation plan is as follows:

[0157] 1. Plasmid extraction: Extract plasmids according to the instructions of Thermo Fisher Scientific's plasmid extraction kit "PureLink Rapid Low Endotoxicity Plasmid Purification Kit".

[0158] 2. Cell transfection: When the cell density of CHO-K1Q cells (cells and their accompanying culture medium were purchased from Lonza) reached 3*E6 cells / mL, electroporation was performed according to the Bio-RAD cell electroporator instruction manual. The electroporation parameters used were square wave, 300V, and 950Ω. 48 hours after transfection, positive cells were sorted by flow cytometry and seeded as single clones. The cells with the highest production capacity were selected as the final cell line for fermentation, with an expression capacity of 5g / L.

[0159] 3. Cell Fermentation: Before fermentation, cells were passaged every 72 hours. Before passage, samples were taken to test the viable cell density and cell viability. Cell slurry and preheated culture medium were added according to the theoretical seeding density (0.3–0.6) × 10⁶ cells / ml and the culture volume. The cells were then cultured on a shaker at 37°C, 8% CO₂, 120 rpm, and 25 mm amplitude. The cell passages were at P4–P5. 14 It is available for use during this period. The reactor (Applikon) is a 15L unit (10L liquid volume) for fermentation. The theoretical inoculation density is (1.0±0.2)×10⁻⁶. 6Calculate the cell slurry and culture medium addition volume (15L fermenter culture volume: 9000mL) using cells / mL, and transfer the cell slurry and culture medium to the injection bottle in the ultra-clean workbench according to the calculated volume, and then add them to the fermenter.

[0160] 4. Purification: Protein G column and conventional cation purification were used to obtain antibody mutations 3-5 and 5-16, which were named anti-Taq A3-5 antibody and anti-Taq B5-16 antibody, respectively.

[0161] Example 4: Antibody Application Research

[0162] A. Application of anti-Taq antibody combination and identification of its blocking effect on Taq DNA polymerase

[0163] 1. To investigate the combined application of anti-Taq A3-5 and anti-Taq B5-16 antibodies, this invention studied the combined concentrations of the two antibodies. The concentrations of anti-Taq A3-5 and anti-Taq B5-16 antibodies were adjusted to 2 mg / mL, and then mixed with 25 U of Taq DNA polymerase at volume ratios of 1:0, 0:1, 1:1, 1:2, and 2:1, respectively. The PRC program was set according to Table 12.

[0164] Table 12 PCR Program Settings

[0165]

[0166] Enter the "Sample Information" interface of the SLAN 96P PCR instrument analysis system to edit the well plate; then enter the "Experimental Analysis" interface, click the table option, select Ct, average Ct, CV and unique identifier, click OK, and finally click Analyze to obtain the final results.

[0167] Note: (1) Definition of anti-Taq antibody blocking power: The amount of Taq enzyme monoclonal antibody that can inhibit 100% of the activity of 1U of Taq DNA polymerase after mixing Taq enzyme monoclonal antibody with Taq DNA polymerase and incubating at 25℃ for 10 min, and then at 55℃ for 10 min, is defined as 1U (refer to the Takara anti-Taq enzyme activity definition). If the slope k of the sample reaction curve is not different from that of anti-Taq (judgment criterion: Δslope k(sample-anti-Taq control)<10), then it is considered that the Taq enzyme monoclonal antibody has blocked 100% of the Taq DNA polymerase activity.

[0168] (2) Reaction Curves: Export the original reaction curves to Excel to obtain the fluorescence intensity data for each cycle in program segment 2. Plot a fitted straight line with the cycle number as the x-axis and the fluorescence value corresponding to the cycle number as the y-axis. This is called the reaction curve for each reaction. Record the slope k of each reaction curve. Note: Generally, data with cycle numbers of 5-24 are used for the reaction curves. Because the fluorescence value detected by the instrument is unstable at the beginning of the reaction, the first 3 data are not used.

[0169] Different ratios of Taq enzyme monoclonal antibodies were mixed with 25 U Taq DNA polymerase and reacted. The slopes of the reaction curves are shown in Table 13. The slope K of the antibody group with a 1:1 anti-Taq ratio after being mixed with 25 U Taq was not significantly different from that of the blank control (anti-Taq, containing only anti-Taq, without Taq enzyme), indicating that the antibody combination with a 1:1 anti-Taq ratio can best block 25 U Taq.

[0170] Table 13 Slope of Taq enzyme response to anti-Taq antibody combinations

[0171]

[0172]

[0173] 2. To further clarify the blocking effect of the anti-Taq enzyme monoclonal antibody combination on Taq enzyme, the test samples were placed at room temperature for 2 hours and 0 hours, and the fluorescence values ​​were measured respectively. If the deviation was less than 5%, it indicates that the anti-Taq antibody combination can fully block the active region of Taq DNA polymerase. The specific operation is as follows:

[0174] Add the reagents to each sample according to Table 14, vortex to mix, centrifuge briefly for 5 seconds, and place on ice for later use. (Prepare fresh for each sample, and calculate based on the number of samples + 2, where 2 represents the sample loss). Mix 25 μL of 5 U / μL Taq DNA polymerase with 25 μL of anti-Taq antibody, and incubate at room temperature for 10 minutes. Then, add 5 μL of the mixture to the working reaction solution.

[0175] Table 14 Working Solution Preparation Table

[0176]

[0177] The centrifuged sample from the previous step was quickly placed into a real-time PCR instrument to begin detection.

[0178] The procedure is as follows:

[0179] The reaction system was 50 μL, and the temperature was controlled by the module.

[0180] Instrument program settings:

[0181] Program segment 1 (37℃ 20s), loop count: 1;

[0182] Program segment 2 (60℃ 25s), number of cycles: 24; fluorescence is detected in this stage.

[0183] like Figure 1 As shown in Table 15, the fluorescence values ​​at 2h and 0h of sample placement were both less than 5%, indicating that the anti-Taq antibody combination can fully block the active region of Taq DNA polymerase and effectively reduce non-specific amplification.

[0184] Table 15 Results of Taq DNA polymerase activity blocking by anti-Taq antibody combination

[0185] Ct mean 2.2315 2.2530 -2.2％

[0186] 3. To investigate the blocking ability of different concentrations of anti-Taq antibody combinations, the PRC program was set up according to Table 12. Enter the "Sample Information" interface of the SLAN 96P PCR instrument analysis system to edit the well plate; then enter the "Experimental Analysis" interface, click the table option, select Ct, average Ct, CV, and unique identifier, click OK, and finally click Analyze to obtain the final results.

[0187] Taq enzyme monoclonal antibody (25 U / μL) was serially diluted; mixed with 25 U Taq DNA polymerase, and the reaction was carried out; the slope of the reaction curve is shown in Table 16. After the 12.5-25 U anti-Taq was mixed with 25 U Taq and reacted, the slope K was not significantly different from that of the blank control (anti-Taq buffer), indicating that 12.5-25 U anti-Taq can block 25 U Taq.

[0188] Table 16 Slope of Taq Enzyme Response to Anti-Taq Antibody Combinations

[0189] 24U anti-Taq + 25U Taq 3.1953 15U anti-Taq + 25U Taq 4.0807 23U anti-Taq + 25U Taq 3.3791 14.5U anti-Taq + 25U Taq 4.5119 22U anti-Taq + 25U Taq 3.7643 14U anti-Taq + 25U Taq 3.87 21U anti-Taq + 25U Taq 3.9411 13.5U anti-Taq + 25U Taq 4.3007 20U anti-Taq + 25U Taq 4.1484 13U anti-Taq + 25U Taq 4.1781 19U anti-Taq + 25U Taq 4.1101 12.5U anti-Taq + 25U Taq 4.7009 18U anti-Taq + 25U Taq 3.6811 12U anti-Taq + 25U Taq 69.101 17U anti-Taq + 25U Taq 3.9293 anti-Taq buffer 3.6011 TAQ-25U 3152.5

[0190] B. Identification of the functional effects of anti-Taq antibody combinations on Taq DNA polymerase

[0191] 1. Obtain diluted influenza A virus clinical samples: Take influenza A clinical samples, dilute them with virus preservation solution (inactivated type), and extract the virus. The Ct value should be within 30±1. (Note: When clinical samples are first obtained, they need to be diluted 20 times for nucleic acid extraction to determine the appropriate concentration and dilution factor.)

[0192] 2. Semi-automated extraction of influenza A nucleic acid: Refer to the instructions for the Zhongyuan Huiji "Nucleic Acid Extraction Kit (Magnetic Bead Method)". Preparation of Working Solution 1: Mix RNA reaction solution, enzyme solution (stored at -20℃ / enzyme solution accelerated at 37℃), and influenza A primers / probes in a 7:2:1 ratio as needed. Vortex to mix and centrifuge for 5-10 seconds in a handheld centrifuge. Preparation of Working Solution 2: Mix COVID-19 reaction solution and enzyme solution (stored at -20℃) in an 8:2 ratio as needed. Vortex to mix and centrifuge for 5-10 seconds in a handheld centrifuge.

[0193] 3. Add 10 μL of each of the above working solutions to an 8-tube PCR array, with at least four replicates for each working solution. Vortex the extracted influenza A nucleic acid and SARS-CoV-2 internal standard, then add 10 μL of influenza A nucleic acid to the 8-tube PCR array containing working solution 1, and add 10 μL of SARS-CoV-2 internal standard to the 8-tube PCR array containing working solution 2. Tightly cap the 8-tube arrays (to prevent evaporation and abnormal results), mix well, and centrifuge. Amplification and detection: Place the 8-tube array containing working solution 1 and influenza A nucleic acid in a PCR instrument and set the following program:

[0194] Table 17 PCR Procedure for Influenza A Nucleic Acid Detection

[0195]

[0196] Place the above working solution 2 + COVID-19 internal standard eight-tube strip into the PCR instrument and set the following program:

[0197] Table 18 PCR Procedure for COVID-19 Nucleic Acid Testing

[0198]

[0199] 4. Influenza A functional tests: as shown in Table 19. Figure 2 and Figure 3 As shown, comparing the enzyme solution before and after acceleration at 37℃ for 3 days with the control stored at -20℃, ΔCt≤1.0 and the amplification curve is a typical S-shaped curve, indicating that the functional test and stability of the test sample are both good. With increasing temperature, the polymerase active site is gradually exposed. Under optimal annealing and extension conditions, it can completely separate from Taq DNA polymerase, maximizing enzyme activity. This gradual release process effectively protects Taq DNA polymerase activity, effectively reducing non-specific amplification and gradually releasing Taq DNA polymerase during denaturation, thus minimizing the impact of high temperature on enzyme activity and ensuring the enzyme's thermal stability. Even with higher amplification cycle numbers (up to 45 cycles), the enzyme activity can still be effectively maintained.

[0200] Table 19 Precision and stability of anti-Taq antibody combinations

[0201]

[0202]

[0203] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An anti-Taq enzyme antibody, characterized in that, The antibody includes the following complementarity-determining regions: CDR-VH1: DYYIN; CDR-VH2:WIYPGSGHTQDNERFKG; CDR-VH3: SRFYAMDY; CDR-VL1: RASQSIGTSIH; CDR-VL2: YASESIS; CDR-VL3: QQTNSWPLT.

2. The anti-Taq enzyme antibody as described in claim 1, characterized in that, The anti-Taq enzyme antibody further includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NO:1-4 or SEQ ID NO:5-8 in sequence; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO:9-12 or SEQ ID NO:13-16 in sequence.

3. The anti-Taq enzyme antibody according to claim 1, characterized in that, The anti-Taq enzyme antibody also contains a constant region.

4. The anti-Taq enzyme antibody as described in claim 3, characterized in that, The constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

5. The anti-Taq enzyme antibody as described in claim 4, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, or humans.

6. The anti-Taq enzyme antibody according to any one of claims 1 to 5, characterized in that, The antibody is a labeled antibody, and the label for the labeled antibody is selected from biotin, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labels.

7. The use of the antibody according to any one of claims 1 to 5 in the preparation of a PCR detection kit or in the preparation of a blocking agent.

8. A detection reagent or kit, characterized in that, The reagent or kit includes the antibody as described in any one of claims 1 to 5.

9. A method for blocking Taq enzyme activity, characterized in that, Add the antibody according to any one of claims 1 to 5 to the immune detection system.

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