An anti-HIV-1 P24 monoclonal antibody and its application

Abstract

The present invention belongs to the field of biological detection technology and specifically relates to an anti-HIV-1 P24 monoclonal antibody and its application. The anti-HIV-1 P24 monoclonal antibodies are designated 1H7 and 1H12. 1H7 includes a heavy chain variable region as shown in SEQ ID NO. 4 and a light chain variable region as shown in SEQ ID NO. 8; 1H12 includes a heavy chain variable region as shown in SEQ ID NO. 12 and a light chain variable region as shown in SEQ ID NO. 16. The monoclonal antibodies of the present invention can specifically bind to HIV-1 P24. Based on the combination of 1H7 and 1H12, an HIV-1 P24 enzyme-linked immunosorbent assay (ELISA) kit is constructed. The kit has a minimum detection limit of 3.9 pg / mL and can detect HIV-1 P24 levels in human serum, plasma, or tissue fluid.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to an anti-HIV-1 P24 monoclonal antibody and its application. Background Art

[0002] AIDS, caused by infection with the human immunodeficiency virus (HIV), remains a major global public health challenge. Early and accurate diagnosis is crucial for blocking viral transmission and improving patient outcomes. HIV-1 P24, a core antigen of the viral capsid, can be detected in the blood 2-3 weeks after infection and is an important biomarker for breaking through the antibody detection window. Currently, clinical practice mainly relies on a combined nucleic acid detection (NAT) and antibody detection strategy, but NAT is expensive and complex to operate, while antibody testing carries the risk of missed detection during the acute infection period. Although immunoassay technology based on the P24 antigen has been incorporated into some guidelines, its sensitivity still cannot meet the detection requirements of low viral load samples, restricting its widespread application in early diagnosis.

[0003] Existing P24 detection technologies rely on double-antibody sandwich methods constructed using monoclonal antibody pairs, but detection performance is limited by the antibody affinity and epitope recognition properties. Studies have shown that the antibody pairs used in traditional commercial kits (such as Abbott ARCHITECT®) have varying binding efficiencies for some HIV-1 subtype P24 antigens, potentially leading to false-negative results. In recent years, researchers have screened novel antibodies (such as HJ2 and 28B4) using phage display technology, which have improved epitope coverage. However, testing in clinical samples still faces challenges such as plasma matrix interference and insufficient detection limits. Developing antibodies with higher affinity and orthogonal epitope pairs has become a key breakthrough in improving the sensitivity and specificity of P24 detection.

[0004] The structural properties of monoclonal antibodies directly influence the performance of detection systems. The conformation of the complementarity-determining regions (CDRs) of the heavy chain variable region (VH) and light chain variable region (VL) determines antigen-binding ability and epitope specificity. Previous studies have used directed evolution to optimize antibody CDRs, but these modified antibodies are prone to cross-reactivity or conformational shifts in epitope recognition. Furthermore, the epitope steric hindrance effects of paired antibodies in detection kits are often overlooked, resulting in reduced sandwich complex formation efficiency. Currently, no P24 antibody pairings are commercially available that simultaneously cover HIV-1 and exhibit picomolar affinity, constituting a bottleneck in the development of high-sensitivity detection technologies. Summary of the Invention

[0005] In order to solve the above problems, the present invention first provides an anti-HIV-1 P24 monoclonal antibody combination and a detection kit thereof.

[0006] The monoclonal antibodies of the present invention include 1H7 and 1H12. The complementary determining region (CDR) sequences of the heavy and light chain variable regions are specifically designed to target and bind to different epitopes of the HIV-1 P24 protein, forming high-affinity antibody pairs and significantly improving detection sensitivity and specificity.

[0007] In certain embodiments, the complete sequence of the heavy chain variable region of the monoclonal antibody 1H7 is SEQ ID NO.4, and the complete sequence of the light chain variable region is SEQ ID NO.8; the complete sequence of the heavy chain variable region of the monoclonal antibody 1H12 is SEQ ID NO.12, and the complete sequence of the light chain variable region is SEQ ID NO.16.

[0008] The present invention provides an application, wherein the application is to use the 1H7 and 1H12 antibody combination to quantitatively detect the HIV-1 P24 protein content in human serum, plasma or tissue fluid by enzyme-linked immunosorbent assay (ELISA), which is used for early diagnosis of HIV infection, viral load monitoring and treatment effect evaluation.

[0009] In certain embodiments, the detection sensitivity of the kit reaches 3.9 pg / mL.

[0010] Compared with the prior art, the present invention has at least the following beneficial effects:

[0011] The present invention is the first to construct an HIV-1 P24 enzyme-linked immunosorbent assay kit based on the combination of monoclonal antibodies 1H7 and 1H12. The minimum detection limit of the kit is 3.9 pg / mL, and it can detect the HIV-1 P24 content in human serum, plasma or tissue fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 ABCpred prediction results for CAT99392.1 p24, partial [HIV-1 M:B_06NOVAX10].

[0013] Figure 2 ABCpred prediction results for QHQ96706.1 p24, partial [Human immunodeficiency virus 1].

[0014] Figure 3 SDS-PAGE and elution peak diagram of P24-S antigen fragment.

[0015] Figure 4 Screening of anti-HIV-1 p24 monoclonal antibody strains.

[0016] Figure 5Affinity testing of 1H7 and 1H12 monoclonal antibodies.

[0017] Figure 6 Double antibody sandwich ELISA for detection of HIV-1 P24 antigen. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0019] Example 1 Preparation of HIV-1 P24 recombinant antigen fragment

[0020] The sequence of HIV-1 P24 found in the NCBI database is as follows:

[0021] >CAT99392.1 p24, partial [HIV-1 M:B_06NOVAX10]

[0022] PEVIPMFTALSEGATPQDLNTMLNTIGGHQAAMQMLKETINEEAAEWDRIHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTSNPPIPVGEIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNSNPDCKTILKALGPAATLEEMMTACQGVG (SEQ ID NO: 1);

[0023] >QHQ96706.1 p24, partial [Human immunodeficiency virus 1]

[0024] ILDIRQGPKEPFRDYVDRFYKTLRAEQATQEVKNWMTETLLVQNANPDCKSILKALGSGASLEEMMTACQGVGGPSHKARVLAEAMSHTQQANIMMMQRGNFKGQKRIKCFNCGKEGHLARL (SEQ ID NO: 2);

[0025] ABCpred is an online B cell epitope prediction tool based on artificial neural networks. It is mainly used to predict linear B cell epitope regions in antigen sequences. The prediction results of ABCpred for CAT99392.1 p24, partial [HIV-1 M:B_06NOVAX10] (partial) are shown in Figure 1ABCpred prediction results for QHQ96706.1 p24, partial [Humanimmunodeficiency virus 1], see Figure 2 The above B cell epitope predicted sequence (Score>0.8) was used as a linker (GGSGG) to construct the HIV-1 P24 enhanced antigen fragment (P24-S), whose amino acid sequence is:

[0026] HAGPIAPGQMREPRGSGGSGGPVGEIYKRWIILGLNKGGSGGLSEGATPQDLNTMLNTGGSGGGQMREPRGSDIAGTTSGGSGGCKTILKALGPAATLEEGGSGGPEVIPMFTALSEGATPGGSGGAAMQMLKETINEEAAEGGSGGLNTIGGHQAAMQMLKEGGSGGGWMTSNPPIPVGEIYKGGSG GLGPAATLEEMMTACQGGGSGGLRAEQATQEVKNWMTEGGSGGPKEPFRDYVDRFYKTLGGSGGSHTQQANIMMMQRGNFGGSGGACQGVGGPSHKARVLAGGSGGCKSILKALGSGASLEEGGSGGPKEPFRDYVDRFYKTLGGSGGALGSGASLEEMMTACQGGSGGLRAEQATQEVKNWMTE (SEQ ID NO:3).

[0027] The codon encoding the HIV-1 P24 enhancer antigen fragment (P24-S) was designed based on its preference for expression in E. coli. It was synthesized and cloned into the plasmid vector pET-28a by Wuhan Jinkairui Bioengineering Co., Ltd., and designated pET-28a-P24-S. The recombinant plasmid pET-28a-P24-S was transformed into E. coli BL21(DE3) and the culture was expanded. When the D600 of the bacterial solution reached 0.6, the bacterial solution before induction was used as a control, and IPTG inducer was added to the LB medium to a final concentration of 0.5 mmol / L. At 37°C, the expression of P24-S protein was induced. Samples of the uninduced bacterial solution and the bacterial solution 4 hours after induction were taken respectively, centrifuged at 10,000 r / min, and the supernatant was discarded. The precipitate was resuspended in PBS and analyzed by SDS-PAGE. After the bacterial cells were induced to express in large quantities, the bacterial precipitate was collected and ultrasonically disrupted, and the precipitate was resuspended in PBS. The target protein P24-S was purified according to the instructions of Ni-NTA Agarose. Impurities were removed with 25 column volumes of binding buffer, and the target protein was eluted with elution buffer. The purified target protein P24-S was then dialyzed and concentrated at 4°C, and the target protein solution in the dialysis bag was filtered with a 0.22 μm filter membrane. After filtration, it was bound to a Ni-NTA affinity column again and impurities were eluted with low concentrations of imidazole (20 mmol / L, 60 mmol / L, and 200 mmol / L) in sequence. The target protein P24-S was eluted with 500 mmol / L imidazole and the concentration was determined. The protein purification and renaturation results were analyzed by SDS-PAGE. Figure 3 .

[0028] Figure 3 The results showed that the molecular weight band of the P24-S antigen fragment was equivalent to 36.91 kDa, and the molecular weight and purity were in line with expectations.

[0029] Example 2 Preparation of anti-HIV-1 P24 monoclonal antibody

[0030] Six- to eight-week-old BALB / c mice were immunized with p24-S protein as the immunogen according to a conventional immunization schedule. Multiple subcutaneous injections were administered into the abdomen and back of the neck at a dose of 100 μg per mouse. Matrix titration was used to optimize the coating concentration and serum dilution of HIV-1 p24 protein (MedChemExpress, p24 protein, HIV-1, Catalog No. AAB50258) for an indirect ELISA assay. An indirect ELISA assay was established. Seven days after the second immunization, orbital blood was collected from each mouse to measure the anti-HIV-1 p24 antibody titer in the serum. Mice with high titers and good performance were selected for a third booster immunization. Three days later, the mice were sacrificed for preparation of spleen cell suspensions and fused with myeloma cells via PEGylation. HIV-1 P24 protein was used as the coating antigen, cell culture supernatant was used as the primary antibody, and goat anti-mouse IgG-HRP diluted to an appropriate working concentration was used as the secondary antibody. After 2 to 3 rounds of cell subclone screening, a strain that continuously secreted anti-HIV-1 P24 monoclonal antibodies was identified. Figure 4 .

[0031] Figure 4 The results showed that the strains 1H7 and 1H12, which could continuously secrete anti-HIV-1 P24 monoclonal antibodies, had the highest binding activity.

[0032] The subclasses of monoclonal antibodies 1H7 and 1H12 were identified using a mouse antibody subclass identification kit.

[0033] Table 1 Identification of subtypes of monoclonal antibodies 1H7 and 1H12

[0034]

[0035] The results in Table 1 show that the heavy chains of 1H7 and 1H12 are both IgG3 subtype, and the light chains are both Lambda chains.

[0036] Monoclonal antibodies were produced using an in vivo induction method: 0.5 mL of pristane was injected intraperitoneally into each mouse to induce ascites formation. The mice were housed in an SPF environment for 1 week and no abnormal reactions were observed. 1H7 and 1H12 hybridoma cells were inoculated in DMEM medium containing 10% FBS and 1% double-antibody and cultured at 37°C and 5% CO2 until the logarithmic growth phase (cell density approximately 1×10 6 The cells were collected by centrifugation (1000 rpm, 5 min), washed three times with sterile PBS, and resuspended in PBS to adjust the concentration to 1×10 7cells / mL; continue to feed the mice, observe the abdominal distension every day, and provide sufficient drinking water and feed; 7-14 days after inoculation, start collecting when the mouse abdomen is significantly swollen, puncture the mouse abdominal cavity with an 18G needle, tilt the needle to allow the ascites to flow out naturally, and gently press the abdomen to help collection. 3-5 mL of ascites can be collected from each mouse each time, and repeat the collection at intervals of 2-3 days until the mouse is moribund. Centrifuge the ascites (3000 rpm, 10 min) to remove cell debris, take the supernatant, filter the supernatant with a 0.45μm filter membrane, aliquot and store at -80℃, mix the ascites with binding buffer (pH 7.4 PBS) and load it onto the chromatography column, elute the target antibody with elution buffer, immediately adjust the pH to neutral with neutralization buffer, place the eluate into a dialysis bag, dialyze in PBS at 4℃ overnight, and change the buffer 3 times. Antibodies were concentrated to 1-5 mg / mL using centrifugal concentrators and stored at -80°C. ELISA was used to verify the binding ability of antibodies 1H7 and 1H12 to HIV-1 p24 antigen. The antigen concentration of HIV-1 p24 protein (MedChemExpress, p24 protein, HIV-1, catalog number: AAB50258) in the ELISA experiment was 0.1 μg / mL. Figure 5 .

[0037] Figure 5 The results showed that the KD values of 1H7 and 1H12 antibodies were both 3×10 -11 mol / L, which indicates that it has extremely high affinity.

[0038] LC-MS / MS scanning was performed on antibodies 1H7 and 1H12 to obtain mass-to-charge ratio (m / z) data, and fragment ions of peptides after trypsin digestion were analyzed. Peptide sequence alignment and protein identification were performed using Mascot database software to match the amino acid sequences of the heavy or light chain. The amino acid sequences of the heavy or light chain were compared with the IMGT antibody database to identify the FR and CDR regions of the VH and VL. Their CDR sequences were defined according to the IMGT rules (see Table 2).

[0039] Table 2 Amino acid sequence identification of monoclonal antibodies 1H7 and 1H12

[0040]

[0041] Example 3 Detection of HIV-1 P24 antigen based on double antibody sandwich ELISA

[0042] Add 100 µL of 1H7 antibody (2 µg / mL) to each well of a 96-well ELISA plate and incubate overnight at 4°C. After incubation, wash the plate three times with washing buffer. Add 200 µL of blocking buffer (5% BSA / PBS) and incubate at room temperature for 1 hour to prevent nonspecific binding. Wash the plate three times with washing buffer and add various concentrations of HIV-1 p24 standard (3.9, 7.8, 15.6, 31.25, 62.5, 125, and 250 pg / mL) to each well. Perform at least three replicates for each group and incubate at room temperature for 1 hour. Add 100 µL of HRP-conjugated 1H12 antibody (2 µg / mL) to each well and incubate at room temperature for 1 hour. Wash the plate three times with washing buffer to remove unbound antibody. Add TMB substrate solution and incubate for 10-20 minutes until a distinct blue reaction develops. The reaction was terminated with a stop solution (1M H2SO4) and the substrate turned yellow. The absorbance (OD value) of each well was measured at a wavelength of 450 nm. Figure 6 .

[0043] Figure 6 The results showed that the minimum detection limit of the double-antibody sandwich ELISA method for detecting HIV-1 P24 antigen was 3.9 pg / mL.

[0044] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An anti-HIV-1 P24 monoclonal antibody 1H7, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-H1 is shown in SEQ ID NO.5, the amino acid sequence of its CDR-H2 is shown in SEQ ID NO.6, and the amino acid sequence of its CDR-H3 is shown in SEQ ID NO.7; the light chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-L1 is shown in SEQ ID NO.9, the amino acid sequence of its CDR-L2 is shown in SEQ ID NO.10, and the amino acid sequence of its CDR-L3 is shown in SEQ ID NO.

11.

2. A monoclonal antibody 1H12 against HIV-1 p24, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-H1 is shown in SEQ ID NO.13, the amino acid sequence of its CDR-H2 is shown in SEQ ID NO.14, and the amino acid sequence of its CDR-H3 is shown in SEQ ID NO.15; the light chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-L1 is shown in SEQ ID NO.17, the amino acid sequence of its CDR-L2 is shown in SEQ ID NO.18, and the amino acid sequence of its CDR-L3 is shown in SEQ ID NO.

19.

3. Use of a composition comprising the anti-HIV-1 P24 monoclonal antibody according to claims 1 and 2 in the preparation of an HIV-1 P24 protein detection reagent or kit.

4. An HIV-1 P24 enzyme-linked immunosorbent assay kit, characterized in that: The kit comprises the anti-HIV-1 P24 monoclonal antibody 1H7 according to claim 1 and the anti-HIV-1 P24 monoclonal antibody 1H12 according to claim 2.

5. The HIV-1 P24 ELISA kit according to claim 4, characterized in that: The amino acid sequence of the 1H7 heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the 1H7 light chain variable region is shown in SEQ ID NO.

8.

6. The HIV-1 P24 ELISA kit according to claim 4, characterized in that: The amino acid sequence of the 1H12 heavy chain variable region is shown in SEQ ID NO.12, and the amino acid sequence of the 1H12 light chain variable region is shown in SEQ ID NO.

16.

7. Use of the HIV-1 P24 enzyme-linked immunosorbent assay kit according to any one of claims 4 to 6 in the preparation of a product for detecting HIV-1 P24 content in serum, plasma or tissue fluid.

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