Broadly specific antibodies and uses thereof

Abstract

The present application relates to the field of biological detection, and particularly relates to broad-spectrum specific antibodies and application thereof.The present application provides a monoclonal antibody of human immunodeficiency virus P24 protein, three CDR regions of the heavy chain of which respectively have the amino acid sequences shown in SEQ ID NO:3, 4 and 5; and three CDR regions of the light chain of which respectively have the amino acid sequences shown in SEQ ID NO:7, 8 and 9.The present application uses a recombinant human immunodeficiency virus P24 protein as an immunogen, and successfully prepares a monoclonal antibody capable of simultaneously detecting HIV-1 and HIV-2 core protein P24 through a different polymorphic antigen sequential immunization strategy, and determines the epitope interval thereof through a recombinant P24 fragment, thereby laying a foundation for establishing a higher-sensitivity detection kit.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to broad-spectrum specific antibodies and their applications. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) has been widely used in the diagnosis of various clinical diseases, especially in the immunodiagnosis of infectious diseases such as hepatitis and HIV infection, as well as in blood donor screening and epidemiological surveys. However, the sensitivity of ELISA is currently not high, often resulting in missed diagnoses that affect the early diagnosis of diseases, delay treatment, or cause post-transfusion hepatitis C and HIV infection, seriously endangering people's health.

[0003] AIDS, or Acquired Immunodeficiency Syndrome, is a chronic infectious disease caused by infection with the Human Immunodeficiency Virus (HIV). HIV belongs to the Human Lentiviral Group within the Lentiviral Genus of the Family Retroviridae. It is a spherical particle approximately 100-120 nm in diameter, composed of a core and an envelope. The core includes two single-stranded RNA chains, core structural proteins, and enzymes essential for viral replication, including reverse transcriptase (RT, P51 / P66), integrase (INT, P32), and protease (PI, P10). Surrounding the core are the viral capsid proteins (P24, P17). The outermost layer of the virus is the envelope, which contains two glycoproteins: gp120 (outer membrane glycoprotein) and gp41 (transmembrane glycoprotein). HIV is highly variable, with varying degrees of variation across its genes. The env gene has the highest variation rate, while the gag and pol genes are relatively conserved. The Gag gene encodes a 55kd precursor protein, which, under the processing of the protease encoded by the pol gene, produces P17, P24, and P55 proteins. Therefore, the amino acid sequence of the P24 protein is highly conserved, which makes it show good regularity in the detection of HIV-1 infection.

[0004] Two types of human immunodeficiency virus (HIV) have been identified: HIV-1 and HIV-2. HIV-2 is less virulent and its prevalence is mainly limited to West Africa, while HIV-1 is widespread globally and is the main pathogen causing the AIDS epidemic in my country. Ten subtypes have been identified in my country: A, B (European and American B), B′ (Thailand B), C, D, F, G, H, J, and K, as well as various circulating recombinant types (CRFs). Currently, the main circulating HIV-1 subtypes are the AE recombinant type and the BC recombinant type.

[0005] After a person is infected with HIV, the HIV core protein P24 first appears in the serum of the infected person. Specific antibodies gradually develop over the next 1-2 months; this period is called the window period. Currently, widely used P24 antigen and HIV antibody combined detection kits can significantly shorten the detection window period for HIV infection. Given the low homology of the amino acid sequences of the HIV-1 and HIV-2 core protein P24, HIV-2 detection is generally achieved by using multiple P24 antibodies in combination or by detecting HIV-2 antibodies simultaneously to detect both HIV-1 and HIV-2. Currently, there are no reports in the literature of using a pair of P24 monoclonal antibodies to simultaneously detect the HIV-1 and HIV-2 core protein P24. Summary of the Invention

[0006] In view of this, the present invention provides a broad-spectrum specific antibody and its applications. Using recombinant human immunodeficiency virus P24 protein as an immunogen, the present invention successfully prepared a monoclonal antibody capable of simultaneously detecting the core protein P24 of HIV-1 and HIV-2 through a sequential immunization strategy with different polytype antigens. Furthermore, the epitope region of the recombinant P24 fragment was determined, laying the foundation for establishing a more sensitive detection kit.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a monoclonal antibody against the human immunodeficiency virus p24 protein, (I) wherein the three CDR regions of its heavy chain have amino acid sequences as shown in SEQ ID NO:3, 4, and 5, respectively; and

[0009] (II) The three CDR regions of its light chain have amino acid sequences as shown in SEQ ID NO:7, 8 and 9, respectively; or

[0010] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences described in (III), (I), or (II), and which have the same or similar function as the amino acid sequences shown in (I) or (II); or

[0011] (IV) An amino acid sequence that is at least 80% homologous to the sequence described in (I), (II) or (III).

[0012] In some embodiments of the present invention, the above-described monoclonal antibody, (V), has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:10; and

[0013] (VI) Its light chain variable region has an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:11; or

[0014] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences described in (VII), (V), or (VI), and which have the same or similar function as the amino acid sequences shown in (V) or (VI); or

[0015] (VIII) amino acid sequences that are at least 80% homologous to the sequences described in (V), (VI) or (VII).

[0016] In some embodiments of the present invention, the plurality of monoclonal antibodies described above is 2, 3, 4 or 5.

[0017] In some embodiments of the present invention, among the above-mentioned monoclonal antibodies, those having the sequences shown in SEQ ID NO:2 and SEQ ID NO:6 are named P24-Ab1#; those having the sequences shown in SEQ ID NO:10 and SEQ ID NO:11 are named P24-Ab2#.

[0018] In some embodiments of the present invention, the antigenic epitope of P24-Ab1# in the above-mentioned monoclonal antibody is located in the range of 83-138aa.

[0019] In some embodiments of the present invention, the antigenic epitope of P24-Ab2# in the above-mentioned monoclonal antibody is located in the interval of 138-172a.

[0020] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0021] In some embodiments of the present invention, the above-described nucleic acid molecule, (IX), wherein its heavy chain has nucleotide sequences as shown in SEQ ID NO:12 and / or as shown in SEQ ID NO:13; and

[0022] (X) Its light chain has a nucleotide sequence as shown in SEQ ID NO:14 and / or as shown in SEQ ID NO:15; or

[0023] Nucleotide sequences obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequences described in (XI), (IX) or (X);

[0024] (XII) sequences that have at least 80% homology with the nucleotide sequences described in (IX), (X) or (XI);

[0025] The complementary sequence of the nucleotide sequence described in (XIII), (IX), (X), (XI) or (XII).

[0026] In some embodiments of the present invention, the sequence of SEQ ID NO:12 in the above-mentioned nucleic acid molecule is: GAGGTGCAGCTGAAGGAGTCTGGCGCTGAACTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTATAAATTCAGTGGGTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGCGATATTTTACCTGGAAGTAATACTACTGTCTACAATGAGAAGTTCAGGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATACAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGACCGTACTATGATTACGATGTTGTGGACTACT. (P24-Ab1#)

[0027] In some embodiments of the present invention, the sequence of SEQ ID NO:13 in the above-mentioned nucleic acid molecule is: CAGGTCCAGCTGCAGCAGTCTGGCGCTGAACTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGCTATAAATTCAGTGGGTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGCGATATTTTACCTGGAAGTAATACTACTGTCTACAATGAGAAGTTCAGGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATACAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGGGACCGTACTATGATTACGATGTTGTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA. (P24-Ab2#)

[0028] In some embodiments of the present invention, the sequence of SEQ ID NO:14 in the above-mentioned nucleic acid molecule is: GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCCGTCTTGGAGATCAAGCCTCCATGCCTTGCAATTCTAGTCAGAGCATTGTCTATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATTTTCAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACATTCGGAGGGGGGACCAAGCTGGAAATAAAA. (P24-Ab1#)

[0029] In some embodiments of the present invention, the sequence of SEQ ID NO:15 in the above-mentioned nucleic acid molecule is: GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCCGTCTTGGAGATCAAGCCTCCATGTCTTGCAATTCTAGTCAGAGCATTGTCTATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATTTTCAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACATTCGGAGGGGGGACCAAGCTGGAAATCAAACGT. (P24-Ab2#)

[0030] The present invention also provides an expression vector comprising the above-described nucleic acid molecule.

[0031] The present invention also provides a host for transforming or transfecting the above-mentioned expression vector.

[0032] The present invention also provides the use of the above-mentioned monoclonal antibody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector and / or the above-mentioned host in the preparation of products for detecting human immunodeficiency virus.

[0033] In some embodiments of the present invention, in the above applications, the human immunodeficiency virus includes: Type and / or type.

[0034] The present invention also provides a kit comprising: the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described expression vector and / or the above-described host, and acceptable adjuvants, vectors and / or devices.

[0035] This invention uses recombinant human immunodeficiency virus (HIV) P24 protein expressed in HEK293F cells as an antigen to prepare a novel mouse monoclonal antibody specifically targeting HIV P24 protein. Using the mouse monoclonal antibody prepared in this invention, an ELISA method for detecting HIV P24 protein using a double-antibody sandwich assay is established. Compared to existing technologies, this method requires less antibody and offers higher sensitivity and specificity, and exhibits higher correlation with the Roche HIV P24 protein detection kit. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 The results of SDS-PAGE analysis of recombinant P24 protein of human immunodeficiency virus are shown below. Lane A shows the electrophoresis pattern of recombinant P24 protein from HIV mutant strains of types A / B / C / D / E / F (lane 1: P24-A-UG273; lane 2: P24-B-WMJ22; lane 3: P24-C-ETH2220; lane 4: P24-D-SE365; lane 5: P24-E-CM235; lane 6: P24-F-BZ163; lane 8: Marker); Lane B shows the electrophoresis pattern of recombinant P24 protein from HIV mutant strains of types G / H / O (lane 1: P24-G-BCF-Dioum; lane 2: P24-H-BCF-KITA; lane 3: P24-O-BCF11; lane 4: Marker).

[0038] Figure 2The results of SDS-PAGE analysis of human immunodeficiency virus P24 recombinant protein fragment antigen are shown below; where: A shows P24-WMJ22 (1-172aa) recombinant protein fragment antigen (lane 1: P24-WMJ22 (1-172aa; lane 2: Marker); B shows P24-WMJ22 (138-231aa) recombinant protein fragment antigen (lane 1: P24-WMJ22 (138-231aa; lane 2: Marker); C shows P24-WMJ22 (1-83aa) recombinant protein fragment antigen (lane 1: P24-WMJ22 (1-83aa; lane 2: Marker);

[0039] Figure 3 SDS-PAGE analysis results of mouse monoclonal antibody P24-Ab1#; where: lane 1: P24-Ab1#; lane 2: marker;

[0040] Figure 4 SDS-PAGE analysis results of mouse monoclonal antibody P24-Ab2#; where: lane 1: P24-Ab2#; lane 2: marker. Detailed Implementation

[0041] This invention discloses a broad-spectrum specific antibody and its applications.

[0042] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0043] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0044] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0045] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0046] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0047] The mouse monoclonal antibodies against human immunodeficiency virus P24 protein described in this invention are named P24-Ab1# and P24-Ab2#, respectively. The mouse monoclonal antibody P24-Ab1# has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:2 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:6; the mouse monoclonal antibody P24-Ab2# has a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:10 and a light chain variable region of the amino acid sequence shown in SEQ ID NO:11.

[0048] Specifically, the amino acid sequence of SEQ ID NO:2 is as follows: EVQLKESGAELMKPGASVKISCKASGYKFSGYWIEWIKQRPGHGLEWIGDILPGSNTTVYNEKFRGKATFTADTSSNTAYIQLSSLTSEDSAVYYCARGPYYDYDVVDYWGQGTTVTVSS.

[0049] The corresponding CDR sequences are as follows:

[0050] CDR1: SEQ ID NO:3: GYKFSGYW;

[0051] CDR2: SEQ ID NO:4: ILPSNTT;

[0052] CDR3: SEQ ID NO:5: ARGPYYDYDVVDY.

[0053] The amino acid sequence of SEQ ID NO:6 is as follows: DAVMTQTPLSLPVRLGDQASMPCNSSQSIVYSNGNTYLEWYLQKPGQSPKLLIFKVSNRFSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKR.

[0054] The corresponding CDR sequences are as follows:

[0055] CDR1: SEQ ID NO:7: QSIVYSNGNTY;

[0056] CDR2: SEQ ID NO:8: KVS;

[0057] CDR3: SEQ ID NO:9: FQGSHVPYT.

[0058] The amino acid sequence of SEQ ID NO:10 is as follows: QVQLQQSGAELMKPGASVKISCKASGYKFSGYWIEWIKQRPGHGLEWIGDILPGSNTTVYNEKFRGKATFTADTSSNTAYIQLSSLTSEDSAVYYCARGPYYDYDVVDYWGQGTSVTVSS.

[0059] The corresponding CDR sequences are as follows:

[0060] CDR1: SEQ ID NO:3: GYKFSGYW;

[0061] CDR2: SEQ ID NO:4: ILPSNTT;

[0062] CDR3: SEQ ID NO:5: ARGPYYDYDVVDY.

[0063] The amino acid sequence of SEQ ID NO:11 is as follows: DAVMTQTPLSLPVRLGDQASMSCNSSQSIVYSNGNTYLEWYLQKPGQSPKLLIFKVSNRFSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKR.

[0064] The corresponding CDR sequences are as follows:

[0065] CDR1: SEQ ID NO:7: QSIVYSNGNTY;

[0066] CDR2: SEQ ID NO:8: KVS;

[0067] CDR3: SEQ ID NO:9: FQGSHVPYT.

[0068] Furthermore, the mouse monoclonal antibody also includes a heavy chain constant region and a light chain constant region.

[0069] Furthermore, the heavy chain constant region and the light chain constant region are derived from mouse IgG.

[0070] The mouse monoclonal antibody against human immunodeficiency virus (HIV) P24 protein described in this invention is prepared by immunizing Balb / c mice with recombinant HIV P24 protein expressed in HEK293F cells, followed by fusion of the spleen with myeloma cells NS1, and subsequent screening and purification. The prepared mouse monoclonal antibody against HIV P24 protein exhibits an affinity greater than 1.0 × 10⁻⁶ for the recombinant HIV P24 antigen expressed in HEK293 cells. -10 M.

[0071] Given the numerous mutant strains of human immunodeficiency virus (HIV) and the varying distribution of new and prevalent strains globally, the ability to detect all HIV mutant strains using a single detection method is crucial for curbing HIV transmission and improving the success rate of AIDS treatment. Current methods for detecting HIV P24 protein typically employ a combination of multiple antibodies to ensure the detection of all mutant strains. This increases the cost per test and hinders the widespread application of HIV screening. This invention utilizes recombinant HIV P24 proteins of different subtypes as immunogens. Balb / c mice are immunized using a sequential immunization strategy, and different subtypes of recombinant HIV P24 proteins are used as detection sources to screen for antibodies that simultaneously recognize all mutant strains of HIV P24 protein. This allows for the detection of all known HIV mutant strains using only one antibody pair.

[0072] By using recombinant fragment antigens of the human immunodeficiency virus P24 protein, namely P24-(1-83aa), P24-(1-172aa), and P24-(138-231aa), the antibody epitope regions of the present invention were identified. The mouse monoclonal antibody P24-Ab1# epitope is located in the 83-138aa region, and the mouse monoclonal antibody P24-Ab2# epitope is located in the 138-172a region.

[0073] Using the mouse monoclonal antibody against human immunodeficiency virus (HIV) P24 protein prepared in this invention, a double-antibody sandwich ELISA detection kit was established. The mouse monoclonal antibody P24-Ab1# against HIV P24 protein was coated on magnetic beads as a capture antibody, and the mouse monoclonal antibody P24-Ab2# against HIV P24 protein was labeled with biotin as a detection antibody. The double-antibody sandwich method detects HIV P24 protein in patient serum and has extremely high sensitivity and specificity, which can greatly shorten the detection window period for HIV infection.

[0074] In Examples 1 to 3 of this invention, all raw materials and reagents used can be purchased from the market.

[0075] The present invention will be further illustrated below with reference to the embodiments:

[0076] Example 1: Preparation of recombinant antigen of human immunodeficiency virus p24

[0077] ①Full genome synthesis of the P24 protein coding sequence of HIV mutant strain WMJ22

[0078] The mRNA sequence (GeneID:155030) of the P24 protein of the HIV M group B mutant strain (WMJ22) was retrieved from NCBI (National Center for Biotechnology Information). Its CDS region sequence is (SEQ ID NO:1):

[0079] The entire genome was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0080] ②Construction and identification of P24 recombinant plasmid

[0081] The full-length P24 coding region gene sequence and the pCMV3 vector were double-digested with Xbal I and Hind III restriction endonucleases for 4 h each. The digestion products were subjected to 1% agarose gel electrophoresis (110 V, 35 min), and the target fragment was recovered using a gel extraction kit. The P24 target fragment was ligated to the linear vector using T4 DNA ligase overnight at 4 °C. The ligation product was transformed into E. coli Top10 competent cells and plated on Luria-Bertani (LB) solid medium containing 100 μg / mL ampicillin, and incubated overnight at 37 °C. Positive clones were picked and the plasmid was extracted by shaking. The plasmid was identified by digestion with Xbal I and Hind III. The digestion products were identified by 1% agarose gel electrophoresis. A specific band was visible at approximately 1200 bp, indicating that a fragment of approximately 1200 bp had been inserted into the pCMV3 vector.

[0082] The correctly identified positive plasmid was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing identification. The sequencing results showed that it was completely consistent with the P24 gene sequence reported in NCBI.

[0083] ③Preparation of P24 recombinant protein

[0084] HEK293F cells were cultured in 500mL cell culture flasks using SMM293-TII medium, with a culture volume of 200mL. On the day of transfection, the HEK293F cell density was adjusted to 3×10⁶ cells / year. 6 Cells / mL, viability not less than 90%. Mix 200 μg plasmid and 1 mL transfection reagent, incubate at room temperature for 10 min, then slowly add to a shake flask and incubate at 37°C with 5% CO2. Sample and count cell viability every 24 h after transfection. When cell viability is below 40%, centrifuge at 2000 rpm to collect the culture supernatant.

[0085] Cell culture supernatant was dialyzed overnight in PBS buffer (pH 7.4, 20 mmol / mL). After equilibrating the Ni column with PBS buffer (pH 7.4, 20 mmol / mL), the dialyzed cell supernatant was slowly loaded. After eluting away contaminating proteins with PBS buffer (pH 7.4, 20 mmol / mL) containing 200 mmol / mL imidazole, the supernatant was dissociated with PBS buffer (pH 7.4, 20 mmol / mL) containing 500 mmol / mL imidazole. The collected dissociated solution was concentrated to a protein concentration of 1 mg / mL and stored at 4°C. Figure 2 The highly purified P24 recombinant protein collected was named P24-B-WMJ22.

[0086] Using the same method, P24 recombinant proteins corresponding to different HIV isolates were prepared using the core protein P24 gene of different HIV isolates as templates, and were named as shown in Table 1.

[0087] Table 1

[0088]

[0089] Electrophoresis diagram of the corresponding recombinant protein is as follows Figure 1 and Figure 2 As shown, the P24 fragment antigen was expressed using the core protein P24 gene of HIV isolate WMJ22 as a template in HEK293F cells and named P24-WMJ22 (1-83aa), P24-WMJ22 (1-172aa), and P24-WMJ22 (138-231aa).

[0090] Example 2: Preparation of mouse monoclonal antibody against human immunodeficiency virus p24 protein

[0091] ① Mouse immunization

[0092] To prepare monoclonal antibodies that can simultaneously recognize the P24 protein of different HIV mutant strains, mice were sequentially immunized with recombinant P24 proteins from different mutant strains.

[0093] Five-week-old female Balb / c mice were intraperitoneally immunized with the human immunodeficiency virus (HIV) P24-B-WMJ22 recombinant antigen, which was fully emulsified with Freund's complete adjuvant at a primary dose of 40 μg / mouse. A second immunization was administered 21 days after the first, using HIV P24-O-BCF11 recombinant antigen, which was fully emulsified with Freund's incomplete adjuvant at a secondary dose of 20 μg / mouse. A third immunization was administered 21 days after the second, using HIV P24-C-ETH2220 recombinant antigen, which was fully emulsified with Freund's incomplete adjuvant at a secondary dose of 10 μg / mouse. Maintenance immunizations were then performed 21 days later, using a cycle of P24-B-WMJ22 recombinant antigen, P24-O-BCF11 recombinant antigen, and P24-C-ETH2220 recombinant antigen, with a maintenance dose of 10 μg / mouse.

[0094] Approximately 10 days after the twelfth immunization, blood was collected from the tail, and serum titer was detected using an indirect method in a 96-well plate coated with human immunodeficiency virus P24-O-BCF11 recombinant antigen.

[0095] ② Hybridoma cell preparation

[0096] Mice with serum titers greater than 1 / 256K as detected by the indirect method were selected for intrasplenic booster immunization at a dose of 200 μg per mouse. Three days after booster immunization, mouse spleens were harvested and fused with mouse myeloma cells NS1 at a ratio of 10:1 using PEG. The fused cells were then cultured on DMEM medium (Gibco) containing HAT.

[0097] Approximately 6-7 days after fusion, the content of specific antibodies in the cell culture supernatant was detected by indirect method using a 96-well plate coated with nine recombinant antigens of human immunodeficiency virus (HIV) P24-B-WMJ22, P24-C-ETH2220, P24-H-BCF-KITA, P24-G-BCF-Dioum, P24-A-UG273, P24-D-SE365, P24-F-BZ163, P24-E-CM235, and P24-O-BCF11. Positive wells with OD values ​​not lower than 0.5 were selected for three rounds of subcloning using limiting dilution to finally obtain a hybridoma cell line that stably secretes anti-HIV P24 protein.

[0098] ③ Purification of mouse monoclonal antibody against human immunodeficiency virus p24 protein

[0099] The mouse hybridoma cells that can stably secrete anti-human immunodeficiency virus P24 protein were injected into the peritoneal cavity of mice, and the ascites fluid was collected and purified by SPA to obtain mouse monoclonal antibodies against human immunodeficiency virus P24 protein with a purity of over 90%.

[0100] ④ The ability of mutant strains of anti-human immunodeficiency virus (HIV) P24 protein monoclonal antibodies to recognize the P24 protein.

[0101] The recombinant human immunodeficiency virus (HIV) P24 protein obtained in Example 1 was diluted to 0.5 μg / mL with 0.05 mmol / L, pH 9.6 CB buffer. 50 μL was added to each well of a 96-well ELISA plate (Corning) and incubated overnight at 4°C. The plate was washed three times with PBST the next day, and then blocked with 1% Casein (100 μL / well) at 37°C for 2 hours. The purified monoclonal antibodies (all at 5 mg / mL) were serially diluted 1:1000 with 0.05 mmol / L, pH 9.6 CB buffer. The diluted antibodies were added to the ELISA plates coated with the recombinant HIV P24 protein. 50 μL of 0.05 mmol / L, pH 9.6 CB buffer was added to each negative control well. After incubating at 37℃ for 30 minutes, the plate was washed 5 times with PBST, dried, and 100 μL of HRP-goat anti-mouse IgG (SIGMA) diluted 1:4000 was added to each well. The plate was then incubated at 37℃ for 30 minutes. After washing 5 times with PBST and drying, 100 μL of standard enzyme immunoassay substrate was added to each well. The plate was incubated at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μL of 0.1 mol / L sulfuric acid, and the absorbance at 450 nm was measured. The results are shown in Table 2.

[0102] Table 2. Reactivity results of the antibodies prepared in this invention with recombinant proteins of different subtypes of human immunodeficiency virus p24.

[0103]

[0104] As shown in Table 2, the antibodies prepared by this invention have strong recognition ability for P24 protein of different HIV mutant strains. Among them, Ab1# and Ab2# antibodies can recognize almost all P24 proteins including HIV type 1 and HIV 2A.

[0105] ⑤ Site identification of mouse monoclonal antibodies against human immunodeficiency virus p24 protein

[0106] The recombinant human immunodeficiency virus (HIV) P24 fragment protein obtained in Example 1 was diluted to 0.5 μg / mL with 0.05 mmol / L, pH 9.6 CB buffer. 50 μL was added to each well of a 96-well ELISA plate (Corning) and incubated overnight at 4°C. The plate was washed three times with PBST the following day, and then blocked with 1% Casein (100 μL / well) at 37°C for 2 hours. The purified monoclonal antibodies (all at 5 mg / mL) were serially diluted 1:1000 with 0.05 mmol / L, pH 9.6 CB buffer. The diluted antibodies were added to the ELISA plates coated with the HIV P24 recombinant protein. 50 μL of 0.05 mmol / L, pH 9.6 CB buffer was added to each negative control well. After incubating at 37℃ for 30 minutes, the plate was washed 5 times with PBST, dried, and 100 μL of HRP-goat anti-mouse IgG (SIGMA) diluted 1:4000 was added to each well. The plate was then incubated at 37℃ for 30 minutes. After washing 5 times with PBST and drying, 100 μL of standard enzyme immunoassay substrate was added to each well. The plate was incubated at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μL of 0.1 mol / L sulfuric acid, and the absorbance at 450 nm was measured. The results are shown in Table 3.

[0107] Table 3. Site identification results of the mouse monoclonal antibody of the present invention

[0108]

[0109] Using recombinant proteins P24-WMJ22 (1-83aa), P24-WMJ22 (1-172aa), and P24-WMJ22 (138-231aa), the antibody epitope regions of the present invention were identified. The mouse monoclonal antibody P24-Ab1# epitope is located in the 83-138aa region, and the mouse monoclonal antibody P24-Ab2# epitope is located in the 138-172a region.

[0110] ⑥ Affinity assay of mouse monoclonal antibody against human immunodeficiency virus p24 protein

[0111] The affinity between the prepared anti-human immunodeficiency virus (HIV) P24 antibody and the anti-HIV P24-WMJ22 recombinant protein was detected using a Biocare T200 microarray. The P24 antibody was chemically immobilized on the microarray, and the P24-WMJ22 recombinant protein was used as the mobile phase. The affinity test results are shown in Table 4. Both antibodies showed pmol-level affinity, indicating extremely high affinity.

[0112] Table 4. Affinity assay results of the mouse monoclonal antibody against human immunodeficiency virus p24 protein of the present invention.

[0113]

[0114] ⑦ Monoclonal antibody sequencing

[0115] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0116] 4C-LF 5′-GACATTGTGATGACCCAGTCTCCT-3′; (as shown in SEQ ID NO:16)

[0117] 4C-LR 5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′ (as shown in SEQ ID NO:17)

[0118] 4C-HF 5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′ (as shown in SEQ ID NO:18)

[0119] 4C-HR 5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′ (as shown in SEQ ID NO:19).

[0120] Extracted with Trizol Reagent reagent at a concentration of 3 × 10⁻⁶. 6 Total RNA from hybridoma cells Ab1# and Ab2# was reverse transcribed into cDNA. The heavy chain variable regions of monoclonal antibodies Ab1# and Ab2# were amplified by PCR using primers 4C-HF and 4C-HR, while the light chain variable regions of monoclonal antibodies against Ab1# and Ab2# were amplified by PCR using primers 4C-LF and 4C-LR. All PCR reactions were hot-started, with the following conditions: 95℃ for 5 minutes; 95℃ for 15 seconds, 55℃ for 45 seconds, 72℃ for 30 seconds, 30 cycles; 72℃ for 7 minutes.

[0121] The PCR products were separated by 1% agarose gel electrophoresis and the target fragment was recovered and purified. It was cloned into the PM18-T vector, transformed into *E. coli* DH5α cells, and screened on LB agar plates. White colonies were inoculated into LB liquid medium containing ampicillin for amplification. Positive clones were screened, and plasmids were extracted using a QIAGEN plasmid extraction kit and sequenced to determine the heavy and light chain variable region sequences of monoclonal antibodies Ab1# and Ab2#.

[0122] Monoclonal antibody P24-Ab1# has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:2 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:6; mouse monoclonal antibody P24-Ab2# has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:10 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:11.

[0123] Example 3: Establishment of an ELISA method for detecting human immunodeficiency virus p24 protein using a double-antibody sandwich assay.

[0124] ①Magnetic microparticle coating

[0125] Take 30 μL of the well-mixed magnetic microparticle stock solution and wash five times with 300 μL of PBS buffer. Then, activate the magnetic microparticles with 10% glutaraldehyde for 1 hour, followed by washing twice with pH 7-8 PBS buffer. Add the mouse monoclonal antibody Ab1# against human immunodeficiency virus p24 protein to the magnetic beads at a rate of 0.3 μg / human dose and coat at 4°C for 2 hours. Finally, block with blocking buffer containing BSA for 2 hours.

[0126] ② Biotin-labeled mouse monoclonal antibody against human immunodeficiency virus p24 protein

[0127] A mouse monoclonal antibody against human immunodeficiency virus (HIV) p24 protein (Invitrogen: PA1-73095) was labeled with biotin at a molar ratio of 20:1. The biotin-labeled mouse monoclonal antibody Ab2# was diluted to 1:500 with dilution buffer. Horseradish peroxidase (HRP)-labeled avidin was used to catalyze the substrate luminescence.

[0128] ③ P24 detection sensitivity verification

[0129] The HIV P24 antigen sensitivity internal control disc was prepared by serially diluting WHO international standard (NIBSC code: 90 / 636) with negative plasma (Rubai Biotechnology: Human Plasma). An ELISA method for detecting human immunodeficiency virus P24 protein using a double-antibody sandwich assay established with the broad-spectrum specific P24 monoclonal antibody prepared in this invention, and the Roche Diagnostics fourth-generation HIV test kit (Elecsys® HIV combi PT), were used in parallel to test the HIV P24 antigen sensitivity internal control disc and compare the sensitivity of the self-produced antibody.

[0130] Table 5. Comparison of sensitivity between the ELISA method established using the antibody prepared in this invention and commercially available kits.

[0131]

[0132] As can be seen from the test results in Table 5, the ELISA method for detecting human immunodeficiency virus (HIV) P24 protein using the broad-spectrum specific P24 monoclonal antibody prepared in this invention has reached the international first-class level in sensitivity. It is particularly noteworthy that this invention uses only one coated antibody to detect HIV M1 and O mutant strains, which is significantly superior to detection methods using multiple monoclonal antibodies on the market.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the human immunodeficiency virus p24 protein, characterized in that, (I) The amino acid sequences of the CDR1, CDR2, and CDR3 regions of its heavy chain variable region are as shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; and (II) The amino acid sequences of the CDR1, CDR2 and CDR3 regions of its light chain variable region are as shown in SEQ ID NO:7, KVS and SEQ ID NO:9, respectively.

2. The monoclonal antibody as described in claim 1, characterized in that, ( Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:2 and / or as shown in SEQ ID NO:10; and ( Its light chain variable region has an amino acid sequence as shown in SEQ ID NO:6 and / or as shown in SEQ ID NO:11; or (V), ( )or( The amino acid sequence described herein is obtained by substitution, deletion, or addition of one or more amino acids, and is related to ( )or( The amino acid sequences shown are functionally identical or similar to the amino acid sequences shown; or (VI), and ( ), ( The amino acid sequence described in (V) has at least 80% homology.

3. The monoclonal antibody as described in claim 2, characterized in that, The "multiple" can be 2, 3, 4, or 5.

4. A nucleic acid molecule encoding a monoclonal antibody as described in any one of claims 1 to 3.

5. The nucleic acid molecule as described in claim 4, characterized in that, ( Its heavy chain has a nucleotide sequence as shown in SEQ ID NO:12 and / or SEQ ID NO:13; and ( Its light chain sequence is SEQ ID NO:14 and / or the nucleotide sequence shown in SEQ ID NO:

15.

6. An expression vector, comprising the nucleic acid molecule as described in claim 4 or 5.

7. The host, characterized in that, Transformation or transfection with the expression vector as described in claim 6.

8. The use of the monoclonal antibody as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4 or 5, the expression vector as described in claim 6, and / or the host as described in claim 7 in the preparation of products for detecting human immunodeficiency virus.

9. The application as described in claim 8, characterized in that, The human immunodeficiency virus includes: Type and / or type.

10. A reagent kit, characterized in that, include: The monoclonal antibody as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4 or 5, the expression vector as described in claim 6, and / or the host as described in claim 7, as well as acceptable adjuvants, vectors, and / or devices.

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