Anti-hiv antibody or functional fragment thereof and use thereof

Abstract

This invention discloses an anti-HIV antibody or a functional fragment thereof and its applications. The anti-HIV antibody or functional fragment disclosed in this invention includes a heavy chain complementarity-determining region and a light chain complementarity-determining region. This antibody or functional fragment has good specificity and sensitivity for HIV and can be used to detect human immunodeficiency virus (HIV) and for the diagnosis or auxiliary diagnosis of related diseases using HIV as a biomarker.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an anti-HIV antibody or its functional fragment and its applications. Background Technology

[0002] AIDS, also known as Acquired Immunodeficiency Syndrome, is a highly dangerous infectious disease caused by the Human Immunodeficiency Virus (HIV).

[0003] The human immunodeficiency virus (HIV) first invades the body's lymphocytes, multiplies rapidly, and renders them unable to recognize foreign antigens, causing the body to lose its immune function. Therefore, the body's resistance is extremely weakened, leading to various infections such as shingles, oral candidiasis, tuberculosis, enteritis, pneumonia, and encephalitis caused by specific pathogens, and severe infections caused by Candida, Pneumocystis, and other pathogens. In later stages, malignant tumors often develop, and prolonged wasting occurs, eventually leading to systemic failure and death. The average incubation period for HIV in the human body is 7–10 years. During this incubation period, infected individuals can live and work for many years without any symptoms, but remain highly infectious. HIV testing to identify the source of infection and interrupt transmission routes is the most effective means of preventing AIDS.

[0004] Existing HIV antibodies are not well applied to the detection of HIV proteins due to their low activity, poor affinity and sensitivity. Therefore, there is a strong demand in the field for antibodies that can effectively and specifically bind to and detect HIV. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide an anti-HIV antibody or its functional fragment, which can specifically bind to HIV protein and be used for in vitro quantitative detection of HIV content in human samples (serum, plasma or whole blood), mainly for auxiliary diagnosis of human immunodeficiency virus in clinical practice.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution, as detailed below:

[0007] Anti-HIV antibodies or their functional fragments have the following complementarity-determining regions:

[0008] CDR-VH1: N-X1-GM-X2, where X1 is Y and X2 is N or S;

[0009] CDR-VH2: YIN-X1-YS-X2-APTYAD-X3-FKG, where X1 is F or T, X2 is G or A, and X3 is D or P;

[0010] CDR-VH3: RGIFT-X1-X2-GYFDV, where X1 is I or L, and X2 is V or Y;

[0011] CDR-VL1: RS-X1-QSLV-X2-I-X3-GNTYLH, where X1 is Q or S, X2 is H or M, and X3 is N or K;

[0012] CDR-VL2: T-X1-SS-X2-FS, where X1 is V or T, and X2 is R or E;

[0013] And CDR-VL3: X1-QS-X2-HV-X3-WT, where X1 is S, X2 is W or T, and X3 is P or D.

[0014] Furthermore, in the complementary determination region, X1 of CDR-VH1 is Y, X1 of CDR-VH2 is T, X2 of CDR-VH3 is V, X1 of CDR-VL1 is S, X2 of CDR-VL2 is R, and X1 of CDR-VL3 is S.

[0015] The CDR segmentation described in this invention uses the Kabat algorithm.

[0016] 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.

[0017] 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.

[0018] The inventors of this invention have discovered that when the mutation sites in each complementarity-determining region are the aforementioned amino acid residues, the antibody has a better affinity for HIV proteins.

[0019] In an optional embodiment, X1 in CDR-VH1 is Y;

[0020] In an optional embodiment, X2 in CDR-VH1 is N;

[0021] In an optional embodiment, X2 in CDR-VH1 is S;

[0022] In an optional embodiment, X1 in CDR-VH2 is F;

[0023] In an optional embodiment, X1 in CDR-VH2 is T;

[0024] In an optional embodiment, X2 in CDR-VH2 is G;

[0025] In an optional embodiment, X2 in CDR-VH2 is A;

[0026] In an optional embodiment, X3 in CDR-VH2 is D;

[0027] In an optional embodiment, X3 in CDR-VH2 is P;

[0028] In an optional embodiment, X1 in CDR-VH3 is I;

[0029] In an optional embodiment, X1 in CDR-VH3 is L;

[0030] In an optional embodiment, X2 in CDR-VH3 is V;

[0031] In an optional embodiment, X2 in CDR-VH3 is Y;

[0032] In an optional embodiment, X1 in CDR-VL1 is Q;

[0033] In an optional embodiment, X1 in CDR-VL1 is S;

[0034] In an optional embodiment, X2 in the CDR-VL1 is H;

[0035] In an optional embodiment, X2 in the CDR-VL1 is M;

[0036] In an optional embodiment, X3 in CDR-VL1 is N;

[0037] In an optional embodiment, X3 in CDR-VL1 is K;

[0038] In an optional embodiment, X1 in CDR-VL2 is V;

[0039] In an optional embodiment, X1 in CDR-VL2 is T;

[0040] In an optional embodiment, X2 in CDR-VL2 is R;

[0041] In an optional embodiment, X2 in CDR-VL2 is E;

[0042] In an optional embodiment, X1 in CDR-VL3 is S;

[0043] In an optional embodiment, X2 in the CDR-VL3 is W;

[0044] In an optional embodiment, X2 in CDR-VL3 is T;

[0045] In an optional embodiment, X3 in CDR-VL3 is P;

[0046] In an optional embodiment, X3 in CDR-VL3 is D.

[0047] In an optional embodiment, each complementarity-determining region is selected from any of the following combinations of mutations:

[0048]

[0049]

[0050] Further, the antibody 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 in sequence; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO:5-8 in sequence.

[0051] Furthermore, the antibody also contains 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 VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0054] Furthermore, the antibody is a coated antibody.

[0055] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. Antibodies according to the invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, provided they retain the characteristic properties according to the invention. An "antibody fragment" comprises a portion of a full-length antibody, preferably its variable domains, or at least its antigen-binding site. Examples of antibody fragments include biantibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; biantibodies; and multispecific antibodies formed from antibody fragments.

[0056] On the other hand, the present invention provides a carrier containing a nucleic acid fragment encoding an antibody or a functional fragment thereof as described in any of the preceding claims.

[0057] On the other hand, the present invention provides a recombinant cell containing the above-mentioned carrier.

[0058] On the other hand, embodiments of the present invention provide a reagent or kit for diagnosing diseases with abnormal HIV levels, which contains antibodies or functional fragments thereof as described above.

[0059] On the other hand, the present invention discloses the application of the above-mentioned antibody or its functional fragment in the preparation of a kit for detecting human immunodeficiency virus in test samples.

[0060] Preferably, the samples tested by the kit are selected from bodily fluids such as whole blood, serum, plasma, urine, or saliva.

[0061] Beneficial Effects: The purpose of this invention is to provide an anti-HIV antibody and a method for detecting HIV. The antibody provided by this invention can specifically bind to HIV, exhibiting good binding activity and affinity, which is beneficial for improving the specificity and sensitivity of detection. It can be used for HIV detection and the diagnosis of diseases with abnormal HIV levels, providing more diverse protein options for HIV detection and the diagnosis of diseases with abnormal HIV levels. Detailed Implementation

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligo Nucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0066] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0067] Example 1: Preparation of Monoclonal Antibodies

[0068] (1) Mouse immunization and antibody detection

[0069] Five 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant was mixed with human HIV protein at a concentration of 2 mg / ml in equal volumes and emulsified. The emulsified antigen was then used to immunize the 6-8 week old SPF-grade female BALB / c mice via paw injection or subcutaneous injection in the back, with each mouse receiving 100 μg of antigen protein. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and again injected via paw injection or subcutaneous injection in the back, with each mouse receiving 100 μg of antigen protein. Two weeks after the second immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and again injected via paw injection or subcutaneous injection in the back, with each mouse receiving 100 μg of antigen protein. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was measured using ELISA. Immunization was repeated every two weeks, and serum titer was measured. After three immunizations, the serum titer, after a million-fold dilution, reached a level 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.

[0070] (2) Cell fusion and screening and subcloning of positive hybridoma cells

[0071] Lymphocytes from immunized mice were isolated and fused with cultured SP2 / 0 cells via PEG1500-mediated fusion or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum for selection. After one week, the medium was changed, and after another 7 days of culture, the culture supernatant was used for positive clone selection. Human HIV protein was used for screening positive wells. Wells with a high ELISA positive value to cell number ratio were selected for multiple subcloning. ELISA plates were coated with human HIV protein. The culture supernatant of the subclones was used to screen for monoclonal clones that showed affinity under antigen-coated conditions. The monoclonal hybridoma cell line with the highest affinity was selected, ultimately yielding a hybridoma cell line with a high antibody titer that secretes HIV protein monoclonal antibodies, named 6#HIV, which exhibited good stability.

[0072] (3) Production and purification of monoclonal antibodies

[0073] Two groups of 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil to suppress the immune response. One week after injection, 0.5 ml of HIV-6 cells (approximately 1 × 10⁻⁶ cells) was injected intraperitoneally into the mice. 6Ascites fluid collection began two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification of protein A to obtain the target antibody 6#HIV.

[0074] Example 2: Validation of antibody performance

[0075] (1) ELISA Affinity Test

[0076] Dilute HIV antigen to 1 μg / mL with carbonate coating buffer (pH 9.6), add 100 μL per well to a 96-well ELISA plate, and incubate overnight at 4°C. Remove the plate, wash three times with PBS buffer containing 0.05% Tween-20, and blot dry. Add 100 μL / well of diluent (1% BSA, 0.1% PBST) (excluding A-well). Dilute antibody to 333.33 ng / mL, add to A-well, and serially dilute 3-fold to G-well; H-well serves as a blank control. Incubate at 37°C for 30 min. Remove the plate, wash three times, and blot dry. Dilute goat anti-mouse-HRP 1:3000 with diluent, add 100 μL / well, and incubate at 37°C for 30 min. Remove the plate, wash three times, and blot dry. Mix colorimetric solutions A and B at a 1:1 ratio, and immediately add 100 μL of the mixture to each well. Incubate at room temperature for 3 minutes. Stop the reaction by adding 50 μL of 0.5 M sulfuric acid to each well. Place the plate in a microplate reader and read the OD450 value. The results are shown in Table 1 below.

[0077] Table 1 Valence Measurement

[0078]

[0079] As shown in Table 1, HIV antibody #6 can recognize HIV antigens, and a signal is still present even at low antibody concentrations, indicating that HIV antibody #6 has high sensitivity and is suitable for subsequent tests.

[0080] The above-mentioned HIV antibody #6 has a heavy chain sequence of SEQ ID NO:11 and a light chain sequence of SEQ ID NO:12. The variable region of the heavy chain is shown in SEQ ID NO:9, and the amino acid sequences of the complementarity-determining regions on the variable region of the heavy chain are as follows:

[0081] CDR-VH1: NY(X1)-GMS(X2)

[0082] CDR-VH2:YINF(X1)-YSG(X2)-APTYADP(X3)-FKG

[0083] CDR-VH3: RGIFTL(X1)-V(X2)-GYFDV

[0084] Its light chain variable region is shown in SEQ ID NO:10, and the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0085] CDR-VL1: RSQ(X1)-QSLVH(X2)-IN(X3)-GNTYLH

[0086] CDR-VL2: TV(X1)-SSR(X2)-FS

[0087] CDR-VL3:S(X1)-QSW(X2)-HVP(X3)-WT

[0088] Based on antibody 6#HIV, mutations were made at sites related to antibody activity in the complementarity-determining region, where X1, X2, and X3 are mutation sites.

[0089] Table 2 Mutation sites related to antibody activity

[0090]

[0091] Table 3 Antibody activity analysis data

[0092] 333.33 1.8519 1.4605 2.1012 0.8675 1.9843 1.1570 111.11 1.3944 0.8468 1.7832 0.6849 1.5983 0.7864 37.04 0.7483 0.6014 1.2892 0.5143 0.9725 0.5711 12.35 0.5477 0.4132 0.8606 0.3347 0.7701 0.4367 4.12 0.3500 0.2934 0.5498 0.2716 0.4782 0.3143 1.37 0.2478 0.2244 0.3419 0.1751 0.3100 0.2568 0.46 0.1902 0.1716 0.1982 0.1567 0.1764 0.1661 0 0.1767 0.1522 0.1546 0.1553 0.1620 0.1579

[0093] As can be seen from the table above, mutation 2 exhibits the best activity. Therefore, using mutation 2 as the backbone sequence, other mutation sites with good affinity were screened, and some results are shown below:

[0094] Table 4 Mutation sites related to antibody affinity

[0095]

[0096]

[0097] HIV protein concentration of 1000 ng / ml was measured, and the absorbance at OD450 for each mutation was measured. The results are as follows:

[0098] Table 5. Affinity test for antibody mutations

[0099] Mutation 2-2 1.8764 Mutation 2-12 2.5000 Mutation 2-22 2.1285 Mutation 2-3 2.1788 Mutation 2-13 2.1750 Mutation 2-23 2.3406 Mutations 2-4 2.1619 Mutation 2-14 2.3634 Mutation 2-24 2.3450 Mutations 2-5 1.9950 Mutation 2-15 2.0629 Mutation 2-25 1.9767 Mutations 2-6 2.3406 Mutation 2-16 2.3475 Mutation 2-26 2.4483 Mutations 2-7 2.3124 Mutation 2-17 2.4087 Mutation 2-27 2.0057 Mutations 2-8 2.2253 Mutation 2-18 2.2131 Mutation 2-28 2.3661 Mutations 2-9 2.0201 Mutation 2-19 1.8594 Mutation 2-29 2.2257 Mutation 2-10 2.4185 Mutation 2-20 2.2319 Mutation 2-30 2.3444

[0100] The test results in Table 5 show that the above-mentioned mutant antibodies all have high affinity.

[0101] (2) Stability determination

[0102] The above-mentioned antibodies were subjected to accelerated thermal treatment at 37°C for 7 and 14 days in a predetermined buffer (PBS, 0.05% ProClin™ 300). The accelerated antibodies were evaluated using an indirect ELISA method, with a control at 4°C, to assess their long-term stability. Additionally, the antibodies were subjected to five freeze-thaw cycles at -20°C, and the results are shown as the deviation between the values ​​at 4°C and the accelerated values. The measurement results are as follows:

[0103] Table 6 Stability Study

[0104]

[0105]

[0106]

[0107] As can be seen from the results in Table 6 above, all of the above-mentioned mutant antibodies can be stably stored at 4℃, and their properties remain stable even after accelerated storage at 37℃ for 7 and 14 days (the slight decrease in reactivity did not affect the performance of the reagents), ensuring the performance of the reagents after opening the bottle, thereby ensuring the accuracy and stability of the test results.

[0108] Example 3: Antibody Application Research

[0109] Because the performance of HIV antibody mutations 1-30 is comparable, the examples cannot exhaust the effects of all antibodies. In this example, antibodies with mutations 2-12 were selected for detection by magnetic microparticle chemiluminescence method.

[0110] (1) Coating with biotinylate-labeled antibody

[0111] a. Turn on the high-speed refrigerated centrifuge, set the speed to 6500 rpm / min, the time to 20 minutes, and the temperature to 4℃;

[0112] b. Antibody desalting: Take a PD-10 desalting column, 0.3 mg of antibody, A0 = 0.3 mg, and volume V0 (mL) = mass A0 / antibody concentration. Add V0 to the PD-10 desalting column. Then add biotinylated buffer 1, V1 = 2.5 mL - V0. When no more liquid flows out from the bottom of the PD-10 desalting column, place a liquid collection tube at the bottom and add 3 mL of biotinylated buffer 1 to elute the antibody and collect the antibody eluent.

[0113] c. Antibody ultrafiltration concentration after desalting: Transfer the antibody eluent to a 15ml 30KD ultrafiltration tube. After centrifugation, transfer the supernatant from the ultrafiltration tube to an EP tube. Vortex to mix the liquid after the transfer. Determine the concentration and concentrate to 2-3mg / mL. Measure the antibody volume V2 and calculate the antibody mass and yield 1. (Mass A1 = volume * concentration, yield 1 = A1 / A0 * 100%);

[0114] d. Preparation of biotin solution: Weigh out biotin by mass D (≥1 mg). Dissolve the weighed biotin in DMSO to a concentration of 2.8385 mg / ml. The required volume of DMSO is E: E (ml) = D (mg) ÷ 2.8385 (mg / ml). Add DMSO to the biotin and mix well.

[0115] e. Antibody-Biotin Conjugation: Calculate the required volume of biotin solution to be added to the antibody solution, based on the requirement of 6.6 μL of biotin solution for 1 mg of antibody. Mix immediately, and then allow the mixed antibody and biotin solution to react in the dark at (25±2)℃ for 4 hours.

[0116] f. Purification after antibody-biotin conjugation: Take a PD-10 desalting column, add V2 mL of antibody, then add (2.5 mL - V2) mL of biotin-labeled buffer 2; wait until no liquid flows out from the bottom of the PD-10 desalting column, place a liquid collection tube at the bottom, add 3 mL of biotin-labeled buffer 2 to elute and collect the antibody-conjugate eluent. Vortex to mix after collection;

[0117] g. Antibody-biotin conjugate concentration determination: The concentration of antibody-biotin conjugate was determined by ultraviolet spectrophotometer.

[0118] (2) Labeling with antibody alkaline phosphatase

[0119] a. Turn on the high-speed refrigerated centrifuge, set the speed to 12000 rpm / min, the time to 6 minutes, and the temperature to 4℃;

[0120] b. Antibody desalting: Take a 0.5 ml 30KD ultrafiltration tube, 0.3 mg of antibody, B0 = 0.3 mg, and volume V0 (mL) = mass B0 / antibody concentration. Add TSE (pH 8.5) solution to the ultrafiltration tube, volume V2 = 0.5 mL - V0. Then add the mixed antibody volume V0; repeat centrifugation 4 times. After desalting, transfer the antibody to a 1.5 ml EP tube. After the liquid transfer, place it on a vortex mixer to mix. Determine the concentration by measuring the antibody volume with a pipette and calculating the antibody mass B1. (Mass B1 = volume * concentration). Adjust the antibody concentration to 3-4 mg / mL;

[0121] c. ALP Desalting: Take one 0.5ml 30KD ultrafiltration tube and label it "ALP". Calculate the volume of ALP used: ALP mass C0 = B0 × 1.1mg, volume V4 (mL), = mass C0 / ALP concentration (mg / ml). Add ALP dialysis buffer (pH 7.6) solution V5 = 0.5mL - V4 to the "ALP" ultrafiltration tube. Repeat centrifugation 4 times. After desalting, transfer the ALP to a 1.5ml EP tube and vortex to mix. Determine the concentration using a Nano-300 microscope and measure the antibody volume using a pipette. Calculate the antibody mass C1. (Mass C1 = volume * concentration);

[0122] d. Activator Preparation: Remove the activators Traut's Reagent 2-Iminothiolane·HCl and Sulfo-SMCC from the refrigerator and allow them to warm to room temperature. Weigh ≥1 mg of 2-IT and dissolve it in TSE (pH 8.5) solution to a concentration of 13.76 mg / ml. The required volume of TSE (pH 8.5) solution, E: E(ml) = D(mg) ÷ 13.76 (mg / ml). After adding the TSE (pH 8.5) solution to the 2-IT, label it "2-IT Solution" and mix well. Weigh ≥1 mg of Sulfo-SMCC and dissolve it in purified water to a concentration of 3.7 mg / ml. The required volume of purified water, E: E(ml) = D(mg) ÷ 3.7 (mg / ml). After adding the purified water to the Sulfo-SMCC, label it "SMCC Solution" and mix well on a vortex mixer.

[0123] e. Antibody activation and desalting: Calculate the volume of 2-IT solution required for the desalted antibody in step (2) based on the requirement of 5 μL of 2-IT solution per 1 mg of antibody. Add the 2-IT solution to the desalted antibody and vortex immediately after addition. After mixing, place the antibody at (25±2)℃ for 20 min to react. This operation is called antibody activation. After activation, replace the activated antibody with TSE (pH 7.3) solution. Follow the same procedure as in step (2) for ultrafiltration 4 times. During ultrafiltration, add TSE (pH 7.3) solution. After desalting, transfer the antibody to a 1.5 ml EP tube and vortex after transfer. Measure the concentration using Nano-300. Measure the antibody volume with a pipette and calculate the antibody mass B1. (Mass B1 = volume * concentration);

[0124] f. ALP activation and desalting: Calculate the volume of SMCC solution required for ALP after desalting in step (3) based on the requirement of 10 μL of SMCC solution per 1 mg of ALP. Add the SMCC solution to the desalted ALP and vortex immediately after addition. After mixing, place the antibody at (25±2)℃ for 20 min to react. This operation is called antibody activation. After activation, replace the activated ALP with TSMZ (pH 7.3) solution. Follow the procedure in step (3) to ultrafilter 4 times. During ultrafiltration, add TSMZ (pH 7.3) solution. After desalting, transfer the ALP to a 1.5 ml EP tube using a pipette. Vortex after liquid transfer. Measure the concentration using Nano-300. Measure the volume of ALP using a pipette and calculate the antibody mass C2 (mass C2 = volume * concentration).

[0125] g. Antibody-ALP ligation reaction: Dilute the desalted antibody to 0.3 mg / ml with TSE (pH 7.3) solution, and dilute the desalted ALP to 0.4 mg / ml with TSMZ (pH 7.3) solution. Calculate the antibody and ALP volumes to be used for ligation according to a mass ratio of antibody to ALP of 1:0.91. Mix the calculated antibody and ALP volumes, and vortex to mix thoroughly. After mixing, allow to stand at 2–8°C for 12–20 h.

[0126] h. Preparation of the stop solution: Weigh out a mass D (≥1 mg) of (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide sodium salt). Dissolve the weighed (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide sodium salt) in DMSO to a concentration of 9.7 mg / ml. The required DMSO volume E is: E(ml) = D(mg)) ÷ 9.7 (mg / ml). Add DMSO to (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide sodium salt) and vortex to mix thoroughly. After mixing, dilute 10-fold with TSMZ (pH 7.3) to obtain the stop solution.

[0127] i. Termination of the reaction: Allow the antibody-ALP conjugate, which has been reacted at 2–8°C, to return to room temperature. Calculate the required volumes of stop solution for both the control and test antibody-ALP conjugates, based on a requirement of 20 μL per 1 ml of antibody-ALP conjugate. Add the stop solution to the antibody-ALP conjugate and immediately vortex to mix.

[0128] (3) Reagent preparation

[0129] a. R1 reagent: Dilute the biotinylated antibody to 1.2 ug / ml with HIV anti-reagent 1 buffer, prepare 7 mL, label it, place it on a vortex mixer, mix for no less than 60 s, and set aside.

[0130] b. R2 reagent: Dilute the biotin-labeled antibody to 1.2 ug / ml with HIV anti-reagent 2 buffer, prepare 7 mL, label it, place it on a vortex mixer, mix for at least 60 seconds, and set aside.

[0131] (4) On-machine test

[0132] a. Set the instrument according to the "EXI1800 Fully Automated Chemiluminescence Immunoassay Analyzer Operation and Maintenance Manual" and HIV project parameters, and load R1, R2, and magnetic bead reagents.

[0133] b. Testing the enterprise reference and clinical samples: Place the HIV enterprise reference and clinical samples into the sample tray of the EXI1800 instrument and apply for testing according to the "EXI1800 Fully Automated Chemiluminescence Immunoassay Analyzer Operation and Maintenance Manual".

[0134] Following the above process, reagents were prepared and tested on the instrument. The deviations between the 2-12 antibody and the Abcam antibody were compared, and the results are shown in Table 7 below.

[0135] Table 7.2-12 Results of Antibody and Abcam Antibody Function Tests

[0136]

[0137] In summary, the chemiluminescence functional detection method of the 2-12 antibody magnetic microparticles of this invention has comparable performance to that of the Abcam antibody.

[0138] 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. Other modifications can be easily made by those skilled in the art. 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 examples shown and described herein.

Claims

1. An anti-HIV antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment includes the following complementarity-determining regions: CDR-VH1: N-X1-GM-X2, where X1 is Y and X2 is N or S; CDR-VH2: YIN-X1-YS-X2-APTYAD-X3-FKG, where X1 is F or T, X2 is G or A, and X3 is D or P; CDR-VH3: RGIFT-X1-X2-GYFDV, where X1 is I or L, and X2 is V or Y; CDR-VL1: RS-X1-QSLV-X2-I-X3-GNTYLH, where X1 is Q or S, X2 is H or M, and X3 is N or K; CDR-VL2: T-X1-SS-X2-FS, where X1 is V or T, and X2 is R or E; And CDR-VL3: X1-QS-X2-HV-X3-WT, where X1 is S, X2 is W or T, and X3 is P or D.

2. The anti-HIV antibody or its functional fragment as described in claim 1, characterized in that, The complementary determination region has X1 as Y for CDR-VH1, X1 as T for CDR-VH2, X2 as V for CDR-VH3, X1 as S for CDR-VL1, X2 as R for CDR-VL2, and X1 as S for CDR-VL3.

3. The anti-HIV antibody or its functional fragment as described in claim 1, characterized in that, In the CDR-VH1, X2 is N; Preferably, X2 in the CDR-VH1 is S; Preferably, in the CDR-VH2, X1 is F; Preferably, in the CDR-VH2, X1 is T; Preferably, in the CDR-VH2, X2 is G; Preferably, in the CDR-VH2, X2 is A; Preferably, in the CDR-VH2, X3 is D; Preferably, in the CDR-VH2, X3 is P; Preferably, in the CDR-VH3, X1 is I; Preferably, in the CDR-VH3, X1 is L; Preferably, in the CDR-VH3, X2 is V; Preferably, in the CDR-VH3, X2 is Y; Preferably, X1 in the CDR-VL1 is Q; Preferably, in the CDR-VL1, X1 is S; Preferably, X2 in the CDR-VL1 is H; Preferably, X2 in the CDR-VL1 is M; Preferably, in the CDR-VL1, X3 is N; Preferably, X3 in the CDR-VL1 is K; Preferably, in the CDR-VL2, X1 is V; Preferably, in the CDR-VL2, X1 is T; Preferably, in the CDR-VL2, X2 is R; Preferably, X2 in CDR-VL2 is E; Preferably, in the CDR-VL3, X1 is S; Preferably, in the CDR-VL3, X2 is W; Preferably, in the CDR-VL3, X2 is T; Preferably, in the CDR-VL3, X3 is P; Preferably, X3 in the CDR-VL3 is D.

4. The anti-HIV antibody or its functional fragment as described in any one of claims 1-3, characterized in that, Each complementarity-determining region is selected from any of the following mutation combinations: 。 5. The anti-HIV antibody or its functional fragment as described in any one of claims 1-3, characterized in that, The antibody comprises 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 in sequence; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO:5-8 in sequence.

6. The anti-HIV antibody or its functional fragment as described in any one of claims 1-4, characterized in that, The antibody also includes a constant region; 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.

7. The anti-HIV antibody or its functional fragment as described in claim 6, characterized in that, The antibody is a coated antibody.

8. A carrier, characterized in that, It contains a nucleic acid fragment encoding an antibody or a functional fragment thereof as described in any one of claims 1-7.

9. A recombinant cell, characterized in that, It contains a carrier, which contains the carrier according to claim 8.

10. A reagent for detecting human immunodeficiency virus, characterized in that, Includes the antibody or its functional fragment as described in any one of claims 1-7.

11. The reagent as described in claim 10, characterized in that, The reagent test sample can be selected from whole blood, serum, plasma, urine or saliva.

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