An elisa kit and method for detecting feline leukemia virus antibodies

Abstract

The application relates to a feline leukemia virus antibody ELISA detection kit and method, and is based on feline leukemia virus gp70 protein as a coating antigen. An indirect ELISA method of the feline leukemia virus is established through optimization of optimal reaction conditions, and the method is applied to prepare an ELISA detection kit. The application fills the blank of the domestic FeLV gp70 specific antibody ELISA detection method, and provides an effective technical means for epidemiological investigation and vaccine effectiveness evaluation of the feline leukemia virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an ELISA detection kit and detection method for feline leukemia virus antibodies. Background Technology

[0002] Feline leukemia virus (FeLV) is a common pathogenic retrovirus in felines, belonging to the genus Gammaretrovirus within the family Retroviridae. FeLV is primarily classified into three subtypes—A, B, and C—based on differences in antigenic characteristics and nucleotide sequences. FeLV-A is the most common naturally occurring subtype, causing almost all initial FeLV infections. FeLV can lead to a range of neoplastic and non-neoplastic diseases in cats, including lymphoma, leukemia, aplastic anemia, and immunosuppression, posing a serious threat to pet health and the cat industry.

[0003] FeLV is an enveloped RNA virus whose genome encodes the gp70 protein, a viral surface glycoprotein that is crucial for viral binding to host cells and possesses high antigenicity. The gp70 protein contains multiple B-cell antigenic epitopes and is one of the key targets in current vaccine development and immunodiagnostic research.

[0004] Currently, FeLV detection methods mainly include viral nucleic acid detection, antigen detection, and antibody detection. Among these, antibody detection plays an important role in evaluating vaccine efficacy and tracking infection. Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive, reproducible, and easy-to-operate immunological detection method, particularly suitable for large-scale serum sample testing and antibody level monitoring.

[0005] However, there is currently a lack of domestically developed ELISA methods for detecting specific antibodies against the FeLV gp70 protein in the Chinese market, which makes it difficult to meet the clinical needs for accurate assessment of FeLV immune status and evaluation of vaccine efficacy. Therefore, developing an indirect ELISA method based on the FeLV gp70 protein is crucial for the specific detection of antibodies in FeLV-infected cats, providing important technical support for the clinical diagnosis, evaluation of immunization efficacy, and vaccine development of FeLV. Summary of the Invention

[0006] The purpose of this invention is to provide an ELISA detection kit and method for feline leukemia virus antibodies, which can effectively detect specific antibodies in the serum of feline leukemia virus-infected and immunized cats. This kit can be used for epidemiological surveys of feline leukemia virus and for evaluating the effectiveness of vaccines, thereby improving the efficiency and accuracy of effectiveness evaluation.

[0007] Technical solution: An ELISA detection kit for feline leukemia virus antibodies, comprising: an antigen-coated enzyme-labeled plate, sample dilution buffer, washing buffer, enzyme-labeled antibody, chromogenic solution, stop solution, positive control, and negative control.

[0008] Furthermore, the antigen is recombinant feline leukemia virus gp70 protein; the sample dilution solution is 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid and 100 mM NaCl; the washing solution is PBST solution; the enzyme-labeled antibody is rabbit anti-cat enzyme-labeled antibody diluted 5000 times; the colorimetric solution is TMB single-component solution; and the stop solution is 2 M sulfuric acid.

[0009] Furthermore, the method for preparing the recombinant feline leukemia virus gp70 protein includes: cloning the feline leukemia virus gp70 gene into the prokaryotic expression vector pET28a(+) between the BamHI and XhoI restriction sites to construct the recombinant expression plasmid pET28a-FeLV-gp70; transforming the recombinant expression plasmid into Escherichia coli BL21(DE3) competent cells; inducing expression with IPTG; and purifying the His-tagged recombinant gp70 protein using a nickel ion affinity chromatography column.

[0010] Furthermore, the conditions for IPTG-induced expression are: final IPTG concentration of 0.5 mM, and expression induced at 25°C for 8 h.

[0011] An ELISA detection method for feline leukemia virus antibodies, wherein the detection method is based on the aforementioned ELISA detection kit for feline leukemia virus antibodies.

[0012] An ELISA method for detecting feline leukemia virus antibodies includes the following steps: S1. Using prokaryotic expression of recombinant feline leukemia virus gp70 protein as the coating antigen, the coating antigen was diluted to a concentration of 0.5-2.0 μg / mL with coating buffer, added to the ELISA plate for coating, and incubated overnight at 4°C. S2. Block the coated ELISA plate with blocking solution. The blocking conditions are 37°C incubation for 1.5 hours. S3. Add the cat serum to be tested as the primary antibody and incubate. Dilute the serum to be tested with diluent at a ratio of 1:(100-400). Incubate at 37°C for 2 hours. S4. Add enzyme-labeled secondary antibody and incubate at a dilution ratio of 1:5000. S5. Develop color with a colorimetric solution, and then add a stop solution to terminate the reaction; S6. Measure the OD450 value of the liquid in the wells of the enzyme-labeled plate, and determine the negative or positive result of the serum to be tested based on the OD450 critical value.

[0013] Furthermore, the coating solution in step S1 includes 20mM borate-borax buffer, 0.5%-1.5% Ficoll 400, 0.01%-0.05% Y-30 (polyethylene glycol derivative), and 0.1% trehalose.

[0014] Furthermore, the blocking solution in step S2 contains 5% skim milk powder, 20mM boric acid-borax buffer, and 0.3% southern turbot antifreeze protein.

[0015] Furthermore, in step S6, the critical value of OD450 is 0.234. If OD450 ≥ 0.234, the serum to be tested is determined to be positive.

[0016] Furthermore, the method for determining the OD450 critical value in step S6 is as follows: detect the OD450 values ​​of at least 35 negative serum samples, calculate the mean X and standard deviation SD, and use X+3SD as the critical value.

[0017] The above-described ELISA detection kit and method for feline leukemia virus antibodies are used in the evaluation of the immunization efficacy of feline leukemia virus vaccines.

[0018] Beneficial effects: This invention selects the FeLV gp70 recombinant protein as the coating antigen. The gp70 protein is a surface antigen unique to FeLV, containing specific epitopes, which significantly improves the specificity of the detection method. The recombinant protein is expressed by genetic engineering methods, which does not involve live virus operations, making the production process safe, without biosafety risks, and the product has good uniformity, which is convenient for quality control.

[0019] The indirect ELISA method and kit established in this invention are the first detection technology in China for FeLV gp70 specific antibodies, filling a market gap in this field, eliminating dependence on imported reagents and reducing costs. This method can not only be used for clinical auxiliary diagnosis and epidemiological monitoring of FeLV infection, but is also particularly suitable for screening non-viremia infections. More importantly, it can provide a key tool for evaluating the immunogenicity of FeLV subunit vaccines, serving the pet medical and biopharmaceutical industries.

[0020] The method of this invention exhibits good sensitivity, specificity, and repeatability, providing objective and reliable detection results. Sensitivity testing results show that the detection limit for positive cat serum is greater than 6400-fold dilution. Repeatability testing results show that the inter-batch and intra-batch variability of this detection method is less than 10%. This invention establishes a positive judgment criterion of OD450 ≥ 0.234 by testing 35 healthy non-immunized cat serum samples, using the mean (X) + 3 standard deviations (SD), thus ensuring the accuracy and reliability of the detection results.

[0021] The detection method showed a 100% positive result for antibodies in cat serum after feline leukemia virus infection; the antibody detection results after immunization with recombinant feline leukemia virus gp70 showed a 100% positive rate of serum antibodies 14 days after immunization, which can effectively evaluate the immunization effect of the vaccine. Attached Figure Description

[0022] Figure 1 This diagram illustrates the identification of purified recombinant feline leukemia virus (FLLV) gp70 protein. A shows the Coomassie Brilliant Blue staining results after SDS-PAGE, indicating a single, clear band at approximately 45.23 kDa for the purified gp70 protein. Note: M is the Protein Marker, 1 is the pET28a empty vector control, and 2 is the pET28a-gp70 protein. B shows the Western blot identification results of the purified pET28a-gp70 protein, demonstrating a specific reaction with feline anti-FeLV positive serum. Note: M is the Protein Marker, 1 is the pET28a-gp70 protein, and 2 is the pET28a empty vector control. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Example 1

[0024] Preparation and identification of recombinant feline leukemia virus gp70 protein: S1. Gene cloning and expression vector construction: Based on the FeLV gp70 gene sequence published in GenBank, the gene fragment was synthesized by a biotechnology company and cloned into the BamHI and XhoI restriction sites of the prokaryotic expression vector pET28a(+) to construct the recombinant expression plasmid pET28a-FeLV-gp70. After sequencing verification, it was transformed into Escherichia coli BL21(DE3) competent cells for protein expression and purification. S2. Protein Identification: The purified recombinant gp70 protein was subjected to SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue. The results are as follows: Figure 1 As shown in Figure A, a clear protein band appears at approximately 45.23 kDa, consistent with the expected molecular weight. S3. Immunological identification: The protein was transferred to a PVDF membrane. Western blot analysis was performed using feline anti-FeLV positive serum (1:500 dilution) as the primary antibody and HRP-labeled rabbit anti-feline IgG (1:5000 dilution) as the secondary antibody. The results are as follows: Figure 1 As shown in Figure B, a specific reaction band appears at approximately 45.23 kDa, indicating that the purified recombinant protein has good immunoreactivity. Example 2

[0025] Establishment of an indirect ELISA detection method: S1. Coating: The purified pET28a-FeLV-gp70 recombinant protein was coated at a rate of 100 ng per ELISA well (i.e., a coating concentration of 1.0 μg / mL). The coating solution consisted of 20 mM borate-borax buffer, 0.5%-1.5% Ficoll 400, 0.01%-0.05% Y-30 (polyethylene glycol derivative), and 0.1% trehalose. The coating was incubated overnight at 4°C. S2. Washing: Discard the coating solution, pat dry on absorbent paper, add 100 μL of PBST washing solution to each reaction well, place in a horizontal shaker, shake at 520 rpm / min for 3 min each time, discard the washing solution, pat dry the residual liquid on absorbent paper, repeat this step four times. S3. Blocking: Prepare 5% skim milk with skim milk powder, then prepare 20mM borate-borax buffer and 0.3% turbot antifreeze protein for blocking. Add 100μL to each reaction well and incubate at 37℃ for 1.5h. Discard the blocking solution and pat dry on absorbent paper. Add 150μL of PBST washing solution to each well using a pipette. Place the well in a horizontal shaker and shake at 520rpm / min for 3min each time. Discard the washing solution and pat dry the remaining liquid on absorbent paper. Repeat this step four times. S4. Serum sample incubation: Take positive serum and dilute it at 1:200. Add 100 μL to each reaction well and incubate at 37°C for 2 h. Discard the sample and pat it dry on absorbent paper. Add 100 μL of PBST washing buffer to each reaction well and place it on a horizontal shaker. Shake at 520 rpm for 3 min each time. Discard the washing buffer and pat the remaining liquid dry on absorbent paper. Repeat this step four times. S5. Dilution and incubation of secondary antibody: Dilute the commercial secondary antibody at a ratio of 1:5000, add 100 μL to each reaction well, incubate at 37°C for 30 min, discard the secondary antibody, pat dry on absorbent paper, add 100 μL of PBST washing buffer to each reaction well, place on a horizontal shaker, and shake at 520 rpm / min for 3 min each time. Discard the washing buffer, pat dry the remaining liquid on absorbent paper, and repeat this step four times. S6. Color development: Add 100 μL of Beyotime TMB color development solution to each reaction well and incubate at room temperature in the dark for 15 min. To stop the color development, add 50 μL of 2M H2SO4 stop solution to each reaction well and incubate at room temperature for 5 min. Then, take the OD450nm reading using a microplate reader. Example 3

[0026] Optimization of ELISA reaction conditions 1. Optimization of coating protein concentration Protein solutions of different concentrations were added to each well of the ELISA plate: 0.25 μg / mL, 0.50 μg / mL, 1.00 μg / mL, 2.00 μg / mL, 5.00 μg / mL, and 10.00 μg / mL. After incubation, data were read at OD450 nm using a microplate reader, and the P / N value for each sample was calculated. The protein concentration corresponding to the highest P / N value was selected as the optimal concentration.

[0027] Table 1 shows that when the pET28a-FeLV-gp70 recombinant protein was coated at a concentration of 1.0 μg / mL, the OD450nm value of positive serum was 0.773, and the OD450nm value of negative serum was 0.057, with the highest corresponding P / N value of 13.561. Therefore, the protein coating concentration was determined to be 1.0 μg / mL.

[0028] Table 1 Optimization results of optimal coating concentration

[0029] Coating temperature and time optimization The optimal protein concentration was used for coating experiments, and incubation was performed under the following conditions: 37°C for 1 h, 4°C for 1 h, 37°C for 2 h, 4°C for 2 h, and 4°C overnight. After incubation, data were read at OD450 nm using a microplate reader, and the P / N ratio was calculated. The coating temperature and time conditions corresponding to the highest P / N ratio were selected as the optimization conditions.

[0030] Table 2 shows that when adsorption and coating were performed overnight at 4℃, the OD450nm value of positive serum was 0.673, and the OD450nm value of negative serum was 0.074, corresponding to the highest P / N value of 9.094. Therefore, the optimal adsorption and coating condition was determined to be overnight at 4℃.

[0031] Table 2 Results of optimization of antigen coating conditions

[0032] Optimization of Closure Conditions The sealing process was divided into the following groups of conditions: incubation at 4℃ for 1 hour, incubation at 4℃ for 1.5 hours, incubation at 4℃ for 2 hours, overnight at 4℃, incubation at 37℃ for 1 hour, incubation at 37℃ for 1.5 hours, incubation at 37℃ for 2 hours, and overnight at 37℃. After incubation, data were read at OD450nm using a microplate reader, and the P / N value for each group of conditions was calculated.

[0033] Table 3 shows that when the blocking condition was 37℃ for 1.5 hours, the OD450nm value of positive serum was 0.875, and the OD450nm value of negative serum was 0.081. At this time, the P / N ratio was the highest at 10.802. Therefore, the blocking condition was determined to be 37℃ for 1.5 hours.

[0034] Table 3 Optimization results of the closure condition

[0035] Optimization of serum incubation conditions Positive serum was added to an ELISA plate and incubated under the following conditions: 1 h at 4°C, 1.5 h at 4°C, 2 h at 4°C, overnight at 4°C, 1 h at 37°C, 1.5 h at 37°C, 2 h at 37°C, and overnight at 37°C. After incubation, the OD at 450 nm was measured using a microplate reader, and the P / N ratio was calculated.

[0036] Table 4 shows that when serum was incubated at 37°C for 2 hours, the OD450nm value of positive serum was 0.731, and the OD450nm value of negative serum was 0.087. At this point, the maximum P / N ratio was 8.402. Therefore, the optimal serum incubation condition was determined to be 37°C for 2 hours.

[0037] Table 4 Results of optimization of serum incubation conditions

[0038] Serum dilution ratio optimization Positive serum was diluted with 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid and 100 mM NaCl at different concentrations: 1:100, 1:200, 1:400, 1:800, 1:1000, and 1:1600. After dilution, the absorbance was measured at OD450 nm using an ELISA reader, and the P / N ratio was calculated.

[0039] Table 5 shows that when the serum was diluted at a ratio of 1:200, the OD450nm value of positive serum was 1.416, and the OD450nm value of negative serum was 0.112. At this ratio, the maximum P / N value was 12.643. Therefore, the optimal serum dilution ratio was determined to be 1:200.

[0040] Table 5 Results of Optimization of Serum Dilution Ratio

[0041] Example 4 Establishment of critical values The optimized ELISA detection method described above was used to test the OD450 values ​​of 35 healthy non-immunized cat serum samples from corresponding wells. The calculated values ​​were X = 0.183, SD = 0.017, and X+3SD = 0.234. A serum sample with an OD450 value greater than 0.234 was considered positive; a sample with an average OD450 value (X+2SD) less than 0.217 was considered negative; samples falling between these values ​​were considered suspicious. Example 5

[0042] Specificity test In the specific detection test, cat positive serum for FPV, FHV-1, FIV, FCoV, and FCV, as well as FeLV negative serum, were selected for detection.

[0043] Table 6 shows that the OD450nm values ​​of cat positive sera for FPV, FHV-1, FIV, FCoV, and FCV, as well as FeLV negative sera, were all below 0.234, while the OD450nm value of FeLV positive sera was 2.104. This indicates that the test kit has good specificity and no cross-reactivity with common feline pathogens.

[0044] Table 6 Specific detection results

[0045] Example 6 Sensitivity test One positive serum sample of feline leukemia virus was selected and serially diluted (1:400-1:25600) as the test sample. The ELISA method established in Example 3 was used for detection. Each serum sample was measured in 3 wells, and the OD450 was read and the average value was calculated.

[0046] The sensitivity test results are shown in Table 7. Positive serum can still be detected even at a dilution ratio of 1:6400 after serial dilution, indicating that it has good sensitivity.

[0047] Table 7 Sensitivity test results

[0048] Example 7 Repeatability test Six positive and six negative serum samples of feline leukemia virus were randomly selected. Following the ELISA detection method established in Example 3, intra-batch repeatability tests were performed using ELISA plates coated in the same batch, and inter-batch repeatability tests were performed using ELISA plates coated in different batches. Three replicates were set up for each serum sample. OD450 was measured, and the intra-batch and inter-batch mean values ​​and coefficient of variation were calculated.

[0049] Serum samples were tested using ELISA kits developed in the same batch. The results are shown in Table 8. The intra-batch coefficient of variation (CV) for different positive sera within the same batch of kits ranged from a maximum of 4.4% to a minimum of 2.6%, both less than 5%, indicating good intra-batch reproducibility. The inter-batch coefficient of variation for different batches of kits showed a maximum of 4.2% and a minimum of 2.5%, both less than 10%, indicating good inter-batch reproducibility.

[0050] Table 8 Repeatability Test Results

[0051] Example 8 Clinical sample testing Antibody levels were detected in cat serum samples collected 14, 28, 42, 56, and 70 days after immunization with the recombinant subunit vaccine using the ELISA method established in Example 1, and the number of positive samples was counted.

[0052] A total of 30 serum samples from recombinant subunit vaccine recipients were tested using an optimized ELISA method. With an OD450 ≥ 0.234 as the standard, the results (Table 9) showed that the positive rate was 100% (6 / 6) at 14 days, 28 days, 42 days, 56 days, and 70 days post-immunization. This indicates that the established ELISA method can effectively detect antibody levels in serum from recombinant subunit vaccine recipients and can be used for evaluating vaccine efficacy.

[0053] Table 9. Antibody detection results in serum samples from cats immunized with recombinant subunit vaccine.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. An ELISA detection kit for feline leukemia virus antibodies, characterized in that, The test kit includes: an ELISA plate coated with recombinant feline leukemia virus gp70 protein antigen, sample diluent, washing buffer, enzyme-labeled antibody, chromogenic solution, stop solution, positive control, and negative control; wherein the sample diluent is 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid and 100 mM NaCl.

2. The ELISA detection kit for feline leukemia virus antibodies according to claim 1, characterized in that, The washing solution is PBST solution; the enzyme-labeled antibody is rabbit anti-cat enzyme-labeled antibody diluted 5000 times; the colorimetric solution is TMB single-component solution; and the stop solution is 2M sulfuric acid.

3. The ELISA detection kit for feline leukemia virus antibodies according to claim 1, characterized in that, The method for preparing the recombinant feline leukemia virus gp70 protein includes: cloning the feline leukemia virus gp70 gene into the prokaryotic expression vector pET28a(+) between the BamHI and XhoI restriction sites to construct the recombinant expression plasmid pET28a-FeLV-gp70; transforming the recombinant expression plasmid into Escherichia coli BL21(DE3) competent cells; inducing expression with 0.5 mM IPTG at 25°C for 8 h; and purifying the His-tagged recombinant gp70 protein using a nickel affinity chromatography column.

4. An ELISA method for detecting feline leukemia virus antibodies, characterized in that, The detection method is based on an ELISA detection kit for feline leukemia virus antibodies according to any one of claims 1-3.

5. The ELISA detection method for feline leukemia virus antibodies according to claim 4, characterized in that, Includes the following steps: S1. Using prokaryotic expression of recombinant feline leukemia virus gp70 protein as the coating antigen, the coating antigen was diluted to a concentration of 0.5-2.0 μg / mL with coating buffer, added to the ELISA plate for coating, and incubated overnight at 4°C. S2. Block the coated ELISA plate with blocking solution. The blocking conditions are 37°C incubation for 1.5 hours. S3. Add the cat serum to be tested as the primary antibody and incubate. Dilute the serum to be tested with diluent at a ratio of 1:(100-400). Incubate at 37°C for 2 hours. S4. Add enzyme-labeled secondary antibody and incubate at a dilution ratio of 1:5000. S5. Develop color using a colorimetric solution, and terminate the reaction by adding a stop solution; S6. Measure the OD450 value of the liquid in the wells of the enzyme-labeled plate, and determine the negative or positive result of the serum to be tested based on the OD450 critical value.

6. The ELISA detection method for feline leukemia virus antibodies according to claim 5, characterized in that: The coating solution in step S1 includes 20mM boric acid-borax buffer, 0.5%-1.5% Ficoll 400, 0.01%-0.05% Y-30 (polyethylene glycol derivative), and 0.1% trehalose.

7. The ELISA detection method for feline leukemia virus antibodies according to claim 5, characterized in that: The blocking solution in step S2 contains 5% skim milk powder, 20mM boric acid-borax buffer, and 0.3% southern turbot antifreeze protein.

8. The ELISA detection method for feline leukemia virus antibodies according to claim 5, characterized in that: In step S6, the critical value of OD450 is 0.

234. If OD450 ≥ 0.234, the serum to be tested is determined to be positive.

9. The ELISA detection method for feline leukemia virus antibodies according to claim 5, characterized in that: The method for determining the OD450 critical value in step S6 is as follows: detect the OD450 values ​​of at least 35 negative serum samples, calculate the mean X and standard deviation SD, and use X+3SD as the critical value.

10. The application of the ELISA detection kit for feline leukemia virus antibodies according to claims 1-3 in the evaluation of the immunization efficacy of feline leukemia virus vaccine.

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