Monoclonal antibody pair for detecting cat immunodeficiency virus p24 protein and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody pair for detecting cat immunodeficiency virus p24 protein and application of the monoclonal antibody pair. The monoclonal antibody pair is a monoclonal antibody 1D8 and a monoclonal antibody 1H10 and can specifically recognize cat immunodeficiency virus p24 protein, and sequences of complementary determining regions of variable regions of a heavy chain and a light chain of the 1D8 and the 1H10 are clear and are respectively shown as SEQ ID NO.1-12. The antibody pair can effectively avoid cross reaction, and the accuracy and sensitivity of detection are remarkably improved; the colloidal gold test strip constructed on the basis of the antibody pair is simple, convenient and rapid to operate, is suitable for early antigen detection of FIV infection, solves the problem that natural infection and vaccine immunity cannot be distinguished in existing antibody detection, and provides a reliable technical means for FIV field screening and disease prevention and control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein and application thereof. BACKGROUND

[0002] Feline immunodeficiency virus (FIV) belongs to the retrovirus of the lentivirus genus and is widely spread in the world, mainly through bite, which can cause persistent infection and has a significant impact on the health of cats. When a cat is infected with FIV, the disease process is usually divided into the following stages: the first stage, acute stage, is the primary viremia and mild symptoms, but it is easy to be ignored. The second stage is the virus carrying period, FIV enters the "dormant" state, and the cat looks completely healthy, which can last for several years or even a lifetime. The third stage is the AIDS period, the immune system of the cat is severely damaged, and clinical symptoms such as immunodeficiency, lymphoma, infection or other chronic diseases appear. Although some infected cats may have no obvious symptoms and can live to the normal expected lifespan, the potential risk of transmission can still pose a serious threat to other healthy cat populations.

[0003] At present, FIV infection cannot be cured, and the targeted treatment drugs such as zidovudine and interferon have low efficacy, high cost and potential toxic side effects. The primary treatment goal for cats infected with FIV is to protect them from other disease infections and prevent the further development of FIV infection. Early and accurate detection of FIV infection and timely separation of positive and negative cat populations for feeding are crucial for controlling the spread of FIV.

[0004] Currently, veterinarians usually perform preliminary screening diagnosis on cats by detecting FIV antibodies, but cats vaccinated with FIV vaccine may also show positive results in antibody detection, so it is impossible to distinguish between infection and vaccine immunity. In addition, antibodies can usually be detected 2-8 weeks after infection, and there is a long window period, which is not conducive to early intervention. FIV p24 is a capsid protein and is the main structural protein that constitutes the core of FIV viral particles, which can be detected 1-2 weeks after infection, which can effectively shorten the window period compared with FIV antibody detection, thereby helping to confirm early infection. Therefore, it is very meaningful to develop an antibody raw material targeting FIV antigen for the early diagnosis of FIV infection.

[0005] However, there is a lack of high-efficiency monoclonal antibody pairs and related detection tools for feline immunodeficiency virus p24 protein, which limits the application of antigen detection technology in FIV prevention and control. SUMMARY

[0006] The application provides a monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein with high specificity and sensitivity and an application thereof, and solves the technical problem of lack of high-efficiency detection tools for feline immunodeficiency virus p24 protein in the prior art.

[0007] In order to achieve the above-mentioned purpose, the main technical scheme adopted by the application comprises: In a first aspect, the application provides a monoclonal antibody pair for feline immunodeficiency virus p24 protein, wherein the monoclonal antibody pair comprises a monoclonal antibody 1D8 and a monoclonal antibody 1H10, The heavy chain variable region of the monoclonal antibody 1D8 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 1-SEQ ID NO. 3. The light chain variable region of the monoclonal antibody 1D8 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 4-SEQ ID NO. 6. The heavy chain variable region of the monoclonal antibody 1H10 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 7-SEQ ID NO. 9. The light chain variable region of the monoclonal antibody 1H10 comprises three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 10-SEQ ID NO. 12.

[0008] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO. 13; and the amino acid sequence of the light chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO. 14.

[0009] In a further embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO. 15; and the amino acid sequence of the light chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO. 16.

[0010] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO. 17; and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO. 18.

[0011] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO. 19; and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO. 20.

[0012] In a second aspect, the application provides the monoclonal antibody pair described above for use in the preparation of a tool for detecting feline immunodeficiency virus p24 protein.

[0013] In further embodiments, the tool comprises a colloidal gold detection test strip, reagent, kit and antibody chip.

[0014] In further embodiments, the colloidal gold detection test strip uses monoclonal antibody 1D8 as a capture antibody and monoclonal antibody 1H10 as a labeled antibody.

[0015] In further embodiments, the colloidal gold detection test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad and an absorbent paper connected to a back plate.

[0016] In further embodiments, the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated with monoclonal antibody 1D8, and the quality control line comprises goat anti-mouse IgG; and the colloidal gold pad is coated with monoclonal antibody 1H10.

[0017] Beneficial effects: The application provides a monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein, which is monoclonal antibody 1D8 and monoclonal antibody 1H10, and can specifically recognize feline immunodeficiency virus p24 protein, wherein the heavy chain and light chain variable region complementarity determining region (CDR) sequences of 1D8 and 1H10 are clear (as shown in SEQ ID NO. 1-12, respectively), which ensures the high affinity and specific binding ability of the antibody pair to the target antigen; the antibody pair can effectively avoid cross-reaction, significantly improve the accuracy and sensitivity of detection, and the minimum detection limit can reach 1 ng / mL; the colloidal gold detection test strip constructed based on the antibody pair is simple and fast to operate, is suitable for early antigen detection of FIV infection, solves the problem that existing antibody detection cannot distinguish between natural infection and vaccine immunization, and provides a reliable technical means for on-site screening and disease prevention and control of FIV. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0019] Figure 1 Results of identification of proteins by SDS-PAGE; Figure 2 Results of identification of FIV / p24 recombinant protein; Figure 3Purification of monoclonal antibody identification results; Figure 4 Colloidal gold assembly schematic diagram; Figure 5 Specificity analysis results of test strip; Figure 6 Sensitivity test results of test strip; Figure 7 Antibody binding identification results. DETAILED DESCRIPTION

[0020] The embodiments of the present application will be further described in detail with reference to the drawings and examples. The following detailed description of the examples and drawings is provided for exemplarily illustrating the principles of the present application, but cannot be used to limit the scope of the present application, and the present application can be implemented in many different forms, and is not limited to the specific examples disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0021] The present application provides these examples in order to make the present application thorough and complete, and fully express the scope of the present application to those skilled in the art. It should be noted that: unless otherwise specified, the relative arrangement of components and steps, the composition of materials, numerical expressions and values set forth in these examples should be interpreted as merely exemplary, and not as a limitation.

[0022] The detection method of the present application is not used for disease diagnosis and treatment.

[0023] The present application obtains a pair of monoclonal antibodies for targeted detection of feline immunodeficiency virus p24 protein by hybridoma technology, and successfully applies it to colloidal gold detection test paper, which has been verified to have good detection sensitivity and specificity for p24 recombinant protein, and provides raw material basis for the development of FIV infection diagnostic reagents.

[0024] The feline immunodeficiency virus p24 protein includes feline immunodeficiency virus p24 recombinant protein and feline immunodeficiency virus p24 natural protein.

[0025] The feline immunodeficiency virus p24 recombinant protein is also called FIV / p24 recombinant protein.

[0026] The p24 protein is a capsid protein of feline immunodeficiency virus (FIV). When a cat is infected with FIV in the early stage, the virus replicates in the body and the p24 antigen can be detected in the blood. As the infection progresses, the cat's body will produce antibodies against p24, so antigen detection can detect infection earlier than antibody detection. After the cat is vaccinated with FIV, the virus does not undergo the process of infection and replication, and the p24 antigen cannot be detected in the body, but the cat's immune system will produce p24 antibodies, which will cause the antibody detection result to be positive, causing misjudgment, which will prevent the differentiation between antibodies produced after vaccination and antibodies produced after natural infection, and the p24 antigen detection is not affected by FIV vaccination.

[0027] After FIV infection, viremia occurs in the early stage, and the structural protein p24 protein of the virus can be detected in the blood sample. As the disease progresses, p24 antibodies are produced, and the antibodies persist for a long time. Currently, all reagents on the market detect antibodies. The window period from infection to detection of antibodies, i.e., the early stage of infection, is prone to missed detection when only antibodies are detected. Therefore, antigen-antibody combined detection can effectively improve accuracy and avoid the influence of vaccination. The present application provides a pair of antibodies for detecting p24 protein, which can be used to detect FIV p24 antigen in blood and can be used in combination with existing antibody detection to shorten the window period and improve detection accuracy.

[0028] In summary, antigen detection can not only detect infection early and distinguish between vaccination and natural infection, but also, when used in combination with antibody detection, it can provide more comprehensive and accurate judgments of the disease progression caused by FIV infection.

[0029] Example 1 1. FIV / p24 recombinant protein expression With reference to the FIV / p24 gene sequence, the gene was synthesized by GenScript and cloned into the pET28a vector, the nucleotide sequence of which is shown as SEQ ID NO. 21 : CAGATGGGATTAGACACTAGACCGTCTACAAAAGAAGCGGGAGGAAAAGAGGAAGGCCCTCCACAGGCATATCCTATTCAAACAGTAAATGGAGCACCACAATATGTAGCACTTGACCCAAAAATGGTGTCCATTTTTATGGAAAAGGCAAGAGAGGGATTAGGAGGTGAGGAAGTTCAACTATGGTTTACAGCCTTCTCTGCAAATTTAACACCTACTGACATGGCCACATTAATAATGGCCGCACCCGGGTGCGCTGCAGATAAAGAAATATTGGATGAAAGCTTAAAGCAATTGACAGCAGAATATGATCGGACACATCCCCCTGATGGTCCTAGACCATTACCCTATTTTACTGCAGCAGAAATTATGGGTATAGGATTAACTCAAGAACAACAAGCAGAAGCAAGATTTGCACCAGCTAGGATGCAATGTAGAGCATGGTATCTTGAGGCATTAGGAAAATTAGCCGCCATAAAGGCTAAATCTCCTAGAACTGTGCAGTTAAGACAAGGAGCTAAGGAAGATTATTCATCCTTTATAGACAGATTGTTTGCCCAAATAGATCAAGAACAAAATACAGCTGAAGTTAAGTTATATCTAAAACAGTCATTAAGCATAGCTAATGCTAATGCAGAATGCAAAAAGGCAATGAGTCATCTTAAGCCAGAAAGTACCCTAGAAGAAAAGTTGAGAGCTTGTCAAGAGATAGGATCACCAGGATATAAAATGCAACTCTTGGCAGAACCTGAATAA.

[0030] The amino acid sequence is shown as SEQ ID NO. 22: QMGLDTRPSTKEAGGKEEGPPQAYPIQTVNGAPQYVALDPKMVSIFMEKAREGLGGEEVQLWFTAFSANLTPTDMATLIMAAPGCAADKEILDESLKQLTAEYDRTHPPDGPRPLPYFTAAEIMGIGLTQEQQAEARFAPARMQCRAWYLEALGKLAAIKAKSPRTVQLRQGAKEDYSSFIDRLFAQIDQEQNTAEVKLYLKQSLSIANANAECKKAMSHLKPESTLEEKLRACQEIGSPGYKMQLLAEPE.

[0031] The recombinant plasmid pET28a-FIV / p24 was transformed into BL21 (DE3) competent cells and induced for expression according to a conventional method. Specifically, the transformed bacteria were spread on LB agar plates (containing 50 μg / mL kanamycin) and incubated at 37°C overnight. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C, 220 rpm overnight. The culture was inoculated into LB medium (containing 50 μg / mL kanamycin) at a ratio of 1% of the total volume of the medium, and incubated at 37°C, 220 rpm for about 3 hours until the OD 600 was 0.6-0.9. The final concentration of IPTG was 0.1 mM, and the bacteria were induced at 30°C, 200 rpm for 4 hours, after which the bacteria were collected.

[0032] 2. Purification of FIV / p24 recombinant protein The expressed recombinant protein had a histidine tag, and was purified using a protein purification instrument from Suzhou Taide Biological Company, a HisTrap TM HP affinity chromatography column. The A buffer was 50 mM PB, 300 mM NaCl, pH 8.0, and the B buffer was 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The chromatography column was equilibrated with the A buffer, and then the fermented bacteria were centrifuged at 8000 rpm for 10 min, the precipitate was resuspended with the A buffer, and was ultrasonically broken in ice water for 30 min, with 5 seconds of ultrasonic treatment every 5 seconds. The supernatant was filtered with a 0.22 micron filter from Jet Bioengineering, and was then loaded onto the chromatography column. The chromatography column was washed with the A buffer, and then was eluted with the B buffer. The elution peak of the target protein was dialyzed overnight at 4°C with the A buffer, and the purified protein was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown in Figure 1 The protein concentration was determined using an ultramicro spectrophotometer, and the purified protein was stored at -20°C. The following is a photograph of the purified protein: Figure 1M: protein Marker, 1 is: flow through of the chromatography column, 2 is: 100 mM imidazole eluted purified FIV / p24 recombinant protein, 3 is: 500 mM imidazole eluted purified FIV / p24 recombinant protein, the estimated molecular weight of FIV / p24 recombinant protein is: 31.2 kDa. The purity of FIV / p24 recombinant protein is higher under the condition of 500 mM imidazole elution, the main band is located at 25-33 kDa, which is close to the expected molecular weight, indicating that the target protein is successfully expressed and effectively purified.

[0033] 3. Identification of FIV / p24 recombinant protein The purified recombinant FIV / p24 protein was used to coat the enzyme-labeled plate, and the indirect ELISA method was used to identify its reaction with FIV / p24 positive antibody. The FIV / p24 antibody is a commercial mouse clone antibody (Sant Cruz, SC-65669). First, the recombinant protein was coated in the microplate (coating buffer: carbonate buffer, sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, constant volume to 1 L of pure water, pH 9.6), the coating concentration was 1 μg / mL, 50 μL / well, 4°C overnight. The next day, the coating solution was discarded, and 3% sucrose + 2% BSA was used for blocking, 150 μL per well, 37°C incubation for 2 hours, then washed the plate once with PBST (0.05% Tween-20 in PBS, pH 7.4), and then dried. The FIV / p24 positive clone antibody was diluted with PBS at a gradient of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL, and 50 μL was added to the microplate coated with the antigen. At the same time, other mouse monoclonal antibodies were diluted at the same concentration as the negative control, 37°C for 30 min. The liquid in the well was shaken out, the plate was washed 4 times with PBST, and then dried. HRP-labeled goat anti-mouse IgG secondary antibody (Solebo, diluted 5000 times with PBS) 50 μL / well was added, 37°C for 30 min, then the plate was washed 4 times, and then dried. TMB color developing liquid 50 μL / well was added, and color development was carried out at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) was added to terminate the reaction, and the OD450nm value was measured with an enzyme-labeled instrument. The results are shown in Figure 2 , Ctrl represents CPV mouse monoclonal antibody (Hytest, CAT#3PV16), SC-65669 represents FIV / p24 commercial mouse clone antibody, FIV / p24 recombinant protein can specifically bind to mouse monoclonal antibody SC-65669, while the negative control has no significant reaction, indicating that the protein has specific recognition ability.

[0034] 4. Screening of FIV / p24 monoclonal antibody 4.1, Mouse immunization The mice were immunized with the FIV / p24 recombinant protein, and other recombinant proteins expressed by the pET28a vector were used as negative antigens for monoclonal antibody screening. Specifically, the purified FIV / p24 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and injected subcutaneously into 6-week-old female BALB / c mice at a dose of 30 μg per mouse. At weeks 2 and 4, the mice were boosted with the same dose of the FIV / p24 recombinant protein mixed with an equal volume of Freund's incomplete adjuvant. At week 6, the mice were immunized by directly injecting the spleen with insulin needles at a dose of 5 μg per mouse. Seven days after the last immunization, the serum of the mice was collected for antibody titer detection. The mice with higher titers were selected and boosted with 20 μg of the FIV / p24 recombinant protein by intraperitoneal injection. Three days later, the spleen of the mice was collected for hybridoma cell preparation.

[0035] 4.2. Screening of hybridoma cells After the spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase, the cells were cultured in HAT medium. When the fused cells grew to 1 / 2 of the bottom of the well, positive clones that reacted with the FIV / p24 recombinant protein were screened by indirect ELISA. Since the immunogen was derived from prokaryotic expression of the pET28a vector and contained a His tag, the background components needed to be screened to obtain specific cell strains targeting the FIV / p24 protein. The positive cells were cloned into a single clone by limiting dilution, and the cell strains were expanded and cryopreserved.

[0036] Screening of positive clones by indirect ELISA: FIV / p24 recombinant protein and other recombinant proteins of the pET28a vector (pET28a-HPV16 / E7, His tag) were coated in a microplate (coating buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, and purified water to 1 L), at a concentration of 1 μg / mL, at 4°C overnight. The next day, the coating solution was discarded, and 3% sucrose + 2% BSA was added to each well (150 μL) for blocking at 37°C for 2 hours. Then, the plate was washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and dried. Fifty microliters of cell culture supernatant were added, and the reaction was carried out at 37°C for 30 minutes. The liquid in the wells was removed, and the plate was washed four times with PBST. After drying, 50 μL of HRP-labeled goat anti-mouse secondary antibody (Solebo, diluted 5000 times with PBS) was added to each well, and the reaction was carried out at 37°C for 30 minutes. The plate was washed four times, dried, and 50 μL of TMB color developing solution was added to each well for color development at room temperature for 10 minutes. Finally, 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) was added to terminate the reaction, and the OD was measured using a microplate reader. 450nm values. The selected monoclonal antibodies are shown in Table 1, and the positive cell strains which reacted with FIV / p24 recombinant protein but not with the control recombinant protein were selected for further experiments.

[0037] Table 1: Screening results of monoclonal antibodies

[0038] After the selected hybridoma cell strains were expanded, 0.2 ml (containing 2.5 x 10 6 cells) was injected into the abdominal cavity of female BALB / c mice, and about 10 days later, when the abdominal cavity of the mice was significantly enlarged, the ascites was collected using a sterile syringe needle.

[0039] 4.3 Purification of monoclonal antibodies The ascites was centrifuged at 12000 r / min for 10 minutes, 1 ml of supernatant was taken, 4 ml of acetic acid-sodium acetate buffer (0.06 M, pH 4.5) was added, mixed well, then 10 μl of n-octanoic acid was slowly added while stirring, after addition, continue to stir for 30 minutes, centrifuge at 2-8°C at 12000 r / min for 30 minutes, take the supernatant. The supernatant was filtered with defatted cotton, and saturated ammonium sulfate was added at a ratio of 50% (V / V) of the final volume, and stirred while adding, after addition, continue to stir for 30 minutes, precipitate overnight at 2-8°C, centrifuge at 12000 r / min for 30 minutes at 2-8°C, take the precipitate. The precipitate was completely dissolved with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filtered through a 0.22 μm filter, and the filtered sample was pumped into a Protein L purification column equilibrated with binding buffer at low speed by peristaltic pump, connected to a protein purification instrument, washed with 5-10 column volumes of binding buffer until the ultraviolet absorption peak was washed flat, then eluted with elution buffer (0.1 M glycine, pH 2.7), collected the elution peak, the collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, and was loaded into a dialysis bag (MW: 8000-14000), and was dialyzed in 20 mM PBS pH 7.4 solution at 2-8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min for 5 minutes, and the supernatant was the purified monoclonal antibody. Figure 3 For the identification results of the purified monoclonal antibodies, Figure 3 see Table 2 for specific data.

[0040] Table 2: Data of monoclonal antibody identification results

[0041] 5. Preparation of test strips coated with different monoclonal antibodies of FIV / p24 The selected FIV / p24 monoclonal antibodies were respectively drawn on the nitrocellulose membrane with different specifications of 20 mm x 300 mm, using a membrane drawing instrument to spray the diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS at pH 7.4) in a horizontal line, with a spraying amount of 0.8 uL / cm, to form the test line (T line). After 6 mm interval, the goat anti-mouse IgG antibody was sprayed in a horizontal line with the same spraying amount, with a dilution concentration of 1 mg / mL in 0.01 M PBS at pH 7.4, coated on the nitrocellulose membrane at an amount of 0.8 uL / cm, to form the control line (C line).

[0042] 6. Colloidal gold pairing of FIV / p24 monoclonal antibodies Preparation of antibody-colloidal gold labeling complex: Antibody labeling: The colloidal gold solution was prepared by the trisodium citrate reduction method. Specifically, 100 mL of 0.01% chloroauric acid solution was heated and boiled, then 1 mL of 1% trisodium citrate solution was quickly added until the solution color was wine red. Boiling was continued for 5 minutes, and after the colloidal gold particles were stable, the solution was cooled to room temperature for standby. To optimize the coupling efficiency of the antibody and colloidal gold, 1 mL of colloidal gold solution was taken in a centrifuge tube, and 1 uL, 2 uL, 3 uL, 4 uL, 5 uL, and 6 uL of 0.2 M potassium carbonate solution were added respectively to adjust the pH environment of the colloidal gold solution in a gradient. Finally, 5 uL was selected when the coupling effect was optimal. After mixing, 5 ug of the FIV / p24 monoclonal antibody to be labeled was added, and after quick mixing, it was incubated at room temperature for 10 min. Then, 10 uL of 10% (w / v) bovine serum albumin (BSA) was added to block the non-specific binding sites, and the incubation was continued at room temperature for 10 min. Then, 10 uL of 10% (w / v) polyethylene glycol 20000 (PEG20000) was added to enhance the labeling stability, and after mixing, it was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The lower precipitate was resuspended with 1 / 10 volume of resuspension solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4), to obtain the antibody-colloidal gold labeling complex, which was stored at 4°C in the dark for standby.

[0043] 7. Screening of paired monoclonal antibodies The FIV / p24 recombinant protein was diluted to 20 ng / mL for detection, and the HPV16 / E7 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection, and the combination that showed a deep color development for FIV / p24 protein and did not react with the control protein was screened. The screening process is shown in Table 3. Therefore, the 1D8 membrane and 1H10 gold-labeled antibody pair is the best pair for detecting FIV / p24 recombinant protein.

[0044] Table 3: Results of screening paired monoclonal antibodies with FIV / p24 recombinant protein

[0045] - indicates negative, i.e. no color development; + / ++ / +++ indicates positive, i.e. color development, the more "+" signs, the deeper the color development, i.e. the stronger the positive reaction.

[0046] 8. Preparation and assembly of colloidal gold test paper Preparation of gold-labeled pad: 6mm X 300mm glass fiber membrane was treated with PBS containing 1% BSA and 1% Tween-20, pH 7.4, and the prepared colloidal gold-labeled antibody was added to the glass fiber at 1200ul / strip, naturally dried, and then placed in a 37°C oven for 2h for standby.

[0047] See Figure 4 , Figure 4 The schematic diagram of colloidal gold assembly is shown in Figure 1. A 60mm X 300mm PVC backboard was used as support, and sample pad, gold-labeled pad, nitrocellulose membrane and absorbent paper were attached to it, respectively. The nitrocellulose membrane was coated with two lines, and was dried at 37°C for 12h for standby. The nitrocellulose membrane was coated with a detection line (monoclonal antibody 1D8 membrane) and a quality control line (goat anti-mouse IgG), and the colloidal gold pad was coated with monoclonal antibody 1H10. The assembled large plate was cut into 4mm naked strips with a cutting machine, and was wrapped with a colloidal gold plastic shell to expose the sample pad to the sample hole position of the plastic shell. The quality control line and the detection line were exposed to the result observation hole position, and the colloidal gold test paper card was assembled.

[0048] 9. Specificity test of test strip Recombinant protein samples: FIV / p24 recombinant protein, FeLV / p27 recombinant protein (E. Coli recombinant expression FeLV / p27 271-519 aa, gene derived from NC_001940.1), FPV / VP2 recombinant protein (E. Coli recombinant expression FPV / VP2 full-length protein, amino acid sequence: GenBank # UVI41039.1), FCV / VP1 recombinant protein (E. Coli recombinant expression FCV / VP1 full-length protein, amino acid sequence: GenBank # QZA82901.1), HPV16 / E7 recombinant protein sample dilution was diluted to 1 ug / mL for detection. Take 80 uL of the above diluted sample and add it to the sample well of the test strip card, and the preparation of HPV16 / E7 recombinant protein is described in 202510831708.X. Determine the results within 20 min. If clear red bands appear on both T and C lines, it is positive; only C line is colored negative; if C line is not colored, it is invalid.

[0049] By Figure 5 It can be seen that the test strip card can well detect FIV / p24 recombinant protein, and has no cross reaction with other recombinant proteins, indicating that the test strip card has good specificity.

[0050] 10. Sensitivity test of test strip FIV / P24 recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL for detection. Figure 6 The results show that the colloidal gold test strip still has weak coloration at a recombinant protein concentration of 1 ng / mL, while the blank diluent, i.e. sample diluent (0.01M Tris + 1% BSA + 0.9% NaCl + 0.1% Tween20, pH 8.0) (0 ng / mL) does not color, indicating that the minimum detection limit of the test strip card for FIV / P24 recombinant protein is 1 ng / mL.

[0051] The present application obtains a pair of monoclonal antibodies that specifically recognize FIV / P24 protein by hybridoma technology. The pair of antibodies can efficiently recognize FIV / P24 recombinant protein and has good specificity and sensitivity. The present application applies the pair of monoclonal antibodies to an immunodetection platform and constructs a rapid detection test strip or test strip card based on colloidal gold immunochromatography technology. The test strip has high sensitivity for FIV / P24 recombinant protein and has no cross reaction with other proteins.

[0052] 11. Monoclonal antibody binding activity detection Based on the screening of potential paired antibodies by colloidal gold, the paired monoclonal antibodies screened and other irrelevant mouse antibodies were gradiently diluted (concentrations were 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL and 0.1 ng / mL, respectively) to evaluate their binding activity with FIV / p24 recombinant protein by referring to the foregoing indirect ELISA method. In the experiment, CPV mouse monoclonal antibody (Hytest, CAT#3PV16) was used as a negative control to exclude the influence of non-specific binding. The results are shown in Figure 7 . Figure 7 In the above table, "Ctrl" represents the negative control CPV mouse monoclonal antibody, indicating that the monoclonal antibody 1D8 and the monoclonal antibody 1H10 have strong binding activity.

[0053] 12. Gene sequence of the monoclonal antibody Total RNA of the hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription with Random Primers. Mouse antibody variable region universal primers were designed, and VHand VLgenes were amplified by 2 rounds of PCR, and an Age1 and Bsiw1 enzyme cutting site was introduced in the primer of the 3rd round of PCR. The PCR product was cut and purified, and then connected to the pUC19 vector, and the TOP10 strain was transformed and cultured at 37°C for 14 h. Single colonies were picked for sequencing to obtain the gene sequence of the light and heavy chains of the monoclonal antibody.

[0054] Monoclonal antibody 1D8 sequence: Nucleotide sequence of the light chain variable region: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D8 is shown as SEQ ID NO. 18: GAGATCCAGATGACCCAGAGCCCCCTGAGCCTGCCCGTGAGCCTGGGCGACCAGGCCAGCATCAGCTGCAGGAGCAGCCAGACCCTGGTGTACAACAACGGCAACACCTACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGAGCAGGTGGGGCACCGAGGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTTCTGCCACGTGGACAGGACCTTCTACCAGCTGACCTTCGGCAGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0055] Light chain variable region amino acid sequence: The amino acid sequence of the light chain variable region of monoclonal antibody 1D8 is shown in SEQ ID NO. 14: EIQMTQSPLSLPVSLGDQASISCRSSQTLVYNNGNTYLHWYLQKPGQSPKLLIYKSRWGTEGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCHVDRTFYQLTFGSGTKLELKRTV.

[0056] Light chain CDR region annotation: The amino acid sequence of the complementarity determining region CDR-L1 of the light chain variable region of monoclonal antibody 1D8 is shown in SEQ ID NO. 4: CDR-L1: RSSQTLVYNNGNTYLH; The amino acid sequence of the complementarity determining region CDR-L2 of the light chain variable region of monoclonal antibody 1D8 is shown in SEQ ID NO. 5: CDR-L2: KSRWGTE; The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of monoclonal antibody 1D8 is shown in SEQ ID NO. 6: CDR-L3: HVDRTFYQLT.

[0057] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D8 is shown in SEQ ID NO. 17: GAGTTCCAGCTGCAGCAGTCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTCCTGGATGCACTGGGCGAAGCAGAGACCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTAGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAACCTACTATGTTAACTCCCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA.

[0058] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of monoclonal antibody 1D8 is shown as SEQ ID NO. 13: EFQLQQSGSVLVRPGASVKLSCKASGYTFTNSWMHWAKQRPGQGLEWIGEIHPSSGNTNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARTYYVNSPFAYWGQGTLVTVSS.

[0059] Heavy chain CDR region annotation: The CDR-H1 amino acid sequence of the complementarity determining region of the heavy chain variable region of monoclonal antibody 1D8 is shown as SEQ ID NO. 1: CDR-H1: NSWMH; The CDR-H2 amino acid sequence of the complementarity determining region of the heavy chain variable region of monoclonal antibody 1D8 is shown as SEQ ID NO. 2: CDR-H2: EIHPSSGNTNYNEKFKG; The CDR-H3 amino acid sequence of the complementarity determining region of the heavy chain variable region of monoclonal antibody 1D8 is shown as SEQ ID NO. 3: CDR-H3: TYYVNSPFAY.

[0060] Monoclonal antibody 1H10 sequence: Light chain variable region nucleotide sequence: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 20: GAAATCCAGATGACCCAGTCTCCATCCTCCATATCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTAGCAATAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATGTATCATGGAACCAAATTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAGATTATTCGCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATTCTCAGTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0061] Light chain variable region amino acid sequence: The amino acid sequence of the light chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 16: EIQMTQSPSSISVSLGDTVSITCHASQGISNNIGWLQQKPGKSFKGLMYHGTKLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYSQYPYTFGGGTKLEIKRTV.

[0062] Light chain CDR region annotation: The amino acid sequence of the light chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 16: CDR-L1: HASQGISNNIG; The amino acid sequence of the light chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 16: CDR-L2: HGTKLED; The amino acid sequence of the light chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 16: CDR-L3: VQYSQYPYT.

[0063] Heavy chain variable region nucleotide sequence: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 19: GACGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTACTATTCAGACACTGTGAAGGGCCGATTCACCATTTCCAGAGACAATGCCAAGTGCGCCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGATAGTTTGGGCTACTTCTGGGGCCAAGGCACCACTCTCACCGTCTCCTCA.

[0064] Heavy chain variable region amino acid sequence: The amino acid sequence of the heavy chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 15: DVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGSYTYYSDTVKGRFTISRDNAKCALYLQMSSLKSEDTAMYYCTRDSLGYFWGQGTTLTVSS.

[0065] Heavy chain CDR region annotation: The amino acid sequence of the complementarity determining region CDR-H1 of the heavy chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 7: CDR-H1: SYTMS; The amino acid sequence of the complementarity determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 8: CDR-H2: TISSGGSYTYYSDTVKG; The amino acid sequence of the complementarity determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 1H10 is shown as SEQ ID NO. 9: CDR-H3: DSLGYF.

Claims

1. A monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein, characterized in that, The monoclonal antibody pair includes monoclonal antibody 1D8 and monoclonal antibody 1H10. The heavy chain variable region of the monoclonal antibody 1D8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 1D8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively. The heavy chain variable region of the monoclonal antibody 1H10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively. The light chain variable region of the monoclonal antibody 1H10 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

2. The monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO.

14.

3. The monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO.

16.

4. The monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1D8 is shown in SEQ ID NO.

18.

5. The monoclonal antibody pair for detecting feline immunodeficiency virus p24 protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1H10 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody pair according to claim 1 in the preparation of a tool for detecting feline immunodeficiency virus p24 protein.

7. The application according to claim 6, characterized in that, The tools include colloidal gold test strips, reagents, kits, and antibody chips.

8. The application according to claim 7, characterized in that, The colloidal gold test strip uses monoclonal antibody 1D8 as the capture antibody and monoclonal antibody 1H10 as the labeling antibody.

9. The application according to claim 8, characterized in that, The colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

10. The application according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 1D8, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 1H10.

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