ELISA DETECTION KIT AND DETECTION METHOD FOR IGA ANTIBODY AGAINST PORCINE ROTAVIRUS GROUP A

Abstract

This invention provides an ELISA detection kit and method for IgA antibody against Porcine rotavirus group A, belonging to the field of animal husbandry detection technology. The ELISA detection method of this invention utilizes popular PoRV variant strains as parental strains to successfully express recombinant VP6 fusion protein in vitro. Subsequently, the VP6 fusion protein was used as an antigen to be coated onto enzyme-labeled plate to establish a novel ELISA detection method. The method provided by this invention possesses strong sensitivity, high specificity, robust stability, good reproducibility, and ease of preparation, and holds important clinical practical significance for the diagnosis, prevention and control of PoRV.

Claims

1. An ELISA detection kit for IgA antibody against porcine rotavirus group A, comprising an enzyme-labeled plate coated with a VP6 fusion recombinant protein, a sample diluent, a wash solution, a substrate chromogenic solution, and a stop solution.

2. The detection kit according to claim 1, wherein the VP6 fusion recombinant protein being synthesized use against Porcine rotavirus group A as a template.

3. The detection kit according to claim 1, wherein an encoding sequence of the VP6 fusion recombinant protein is as shown in SEQ ID NO. 1.

4. The detection kit according to claim 1, wherein a coating concentration of the VP6 fusion recombinant protein is 3.5 ?g/mL.

5. The detection kit according to claim 1, wherein a coating time of the VP6 fusion recombinant protein is 10-14 h.

6. An ELISA detection method for IgA antibody against Porcine rotavirus group A, comprising the following steps of: coating: diluting a VP6 fusion recombinant protein to 3.5 ?g/mL using a 0.05 mol/L Na.sub.2CO.sub.3NaHCO.sub.3 buffer solution with a pH of 9.6 to obtained a diluted protein solution, and adding 100 ?L of the diluted protein solution to each well such that the VP6 fusion recombinant protein is adsorbed at 4? C. for 14 h; after the coating, discarding the diluted protein solution in the well, followed by adding 250 L of PBS wash solution containing 0.05% Tween-20 to each of the well, and washing the well for three times, with the final wash being tapped dry; blocking: adding 200 ?L of PBS blocking solution containing 5% BSA and 0.05% Tween-20 to each of the well and blocked at 37? C. for 1 h; after the blocking, discarding the PBS blocking solution in the well, followed by adding 250 ?L of the PBS wash solution to each of the well, and washing the well for three times, with the final wash being tapped dry; primary antibody incubation: diluting a milk sample to be tested with a sample diluent at a ratio of 1:100 or diluting a serum sample to be tested with the sample diluent at a ratio of 1:200 to obtain a primary antibody solution, adding 100 ?L of the primary antibody solution to each of the well and incubated at 37? C. for 60 min; discarding the primary antibody solution in the well, followed by adding 250 ?L of the PBS wash solution to each of the well, and washing the well for five times, with the final wash being tapped dry; secondary antibody incubation: diluting an enzyme-labeled secondary antibody with an antibody diluent at a ratio of 1:10000 to obtain a secondary antibody solution, followed by adding 100 ?L of the secondary antibody solution to each of the well and incubated at 37? C. for 60 min; discarding the secondary antibody solution in the well, followed by adding 250 ?L of the PBS wash solution to each of the well, and washing the well for five times, with the final wash being tapped dry; chromogenic time and reading: adding 100 ?L of a single-component TMB substrate chromogenic solution to each of the well for a chromogenic reaction, and allowing the chromogenic reaction to proceed in dark at 37? C. for 15 minutes, subsequently, adding 100 ?L of a stop solution to each of the well, and reading an OD value at 450 nm using an enzyme-labeled instrument; wherein the method is used for non-disease diagnostic purposes.

7. The method according to claim 6, wherein the VP6 fusion recombinant protein being synthesized use against Porcine rotavirus group A as a template.

8. The method according to claim 6, wherein an encoding sequence of the VP6 fusion recombinant protein is shown in SEQ ID NO.1.

9. The method according to claim 6, wherein a cut-off value of the milk sample to be tested is 0.262; when the OD value is ?0.262, a positive test result is obtained, and when the OD value is <0.262, a negative test result is obtained.

10. The method according to claim 6, wherein a cut-off value of the serum sample to be tested is 0.319; when the OD value is ?0.319, a positive test result is obtained, and when the OD value is <0.319, a negative test result is obtained.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is an electropherogram of the enzyme digestion product of the pColdII-VP6 recombinant expression vector constructed by this invention;

[0034] FIG. 2 is a result of the VP6 fusion recombinant protein purified by nickel column affinity chromatography.

[0035] FIG. 3 is the specific detection result of the detection method of this invention;

[0036] FIG. 4 is the sensitivity test result of the detection method of this invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] A variety of exemplary embodiments of this invention were described in detail, and such detailed description shall not be regarded as a limitation of this invention, but shall be understood as a more detailed description of certain aspects, characteristics and embodiments of this invention.

[0038] It should be understood that the terms described in the invention were only intended to describe special embodiments and were not intended to limit the invention. In addition, with respect to the numerical range in this invention, it should be understood that each intermediate value between the upper and lower limits of the range was also specifically disclosed. The intermediate value in any stated value or range of representations, and each smaller range between any other stated value or intermediate value in said range was also included in this invention. The upper and lower limits of these smaller ranges can be included or excluded independently.

Example 1: Construction of pColdII-VP6 Recombinant Expression Vector

[0039] (1) Referring to the VP6 sequence (SEQ ID NO.1) of the CHN/SCCD/A/2017 strain in GenBank, the sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. [0040] (2) After enzymatic digestion and purification, the synthetic product was inserted into the BamHI/HindIII multiple cloning site of the pColdII-VP6 vector. The expected result was verified through sequencing, thus construct the recombinant expression vector pColdII-VP6.

[0041] As shown in FIG. 1 of enzymatic digestion result, the target gene was correctly inserted into the pColdII-VP6 vector.

Example 2: Expression and Purification of Recombinant Protein

[0042] (1) The constructed recombinant plasmid pColdII-VP6 was transformed into BL21 competent cells to obtain the recombinant expression strain BL21-pColdII-VP6. [0043] (2) The cells were cultured at 37? C. with 220 rpm oscillation for 2-3 hours until the OD value at 600 nm reached 0.6-0.8. Subsequently, IPTG with a final concentration of 0.1 mmol/L was added, and the cells were induced for expression at 16? C. with 220 rpm oscillation culture. [0044] (3) The bacteria were collected by centrifugation and disrupted and lysed by ultrasonic treatment. [0045] (4) The cell lysate was centrifuged to separate the supernatant and precipitate. Both fractions were analyzed by SDS-PAGE to determine the expression form of the VP6 recombinant protein. Samples containing the target protein were collected, and the pColdII-VP6 recombinant protein was purified by nickel column affinity chromatography according to the Ni-NTA Superflow Cartridge manual. The purified protein samples were analyzed by SDS-PAGE.

[0046] As shown in FIG. 2, the soluble protein was purified using nickel column affinity chromatography, resulting in a purity of pColdII-VP6 recombinant protein above 95%.

Example 3: Determination of Optimal Antigen Coating Concentration and Optimal Secondary Antibody Working Concentration

[0047] (1) A matrix assay was used to determine the optimal antigen coating concentration and optimal secondary antibody working concentration. The VP6 fusion recombinant protein was diluted to six concentrations by coating buffer: 7 ?g/mL, 3.5 ?g/mL, 1.75 ?g/mL, 0.625 ?g/mL, 0.3125 ?g/mL and 0.156 ?g/mL. Each concentration of the VP6 fusion recombinant protein was coated on one row of the ELISA plate, with 100 ?L per well. [0048] (2) The HRP-labeled 3 against porcine IgA antibody was sequentially diluted at ratios of 1:5000, 1:10000 and 1:20000 by antibody diluent. Two columns of wells were added for each dilution, with 100 ?L per well, forming a matrix pattern. [0049] (3) The antigen concentration and secondary antibody dilution that produced the highest ratio of positive milk OD value at 450 nm to negative milk OD value at 450 nm (P/N value) were selected as the optimal antigen coating concentration and optimal secondary antibody working concentration. The results are presented in Table 1.

TABLE-US-00001 TABLE 1 Determination of the optimal antigen coating concentration and the optimal secondary antibody working concentration Antigen Enzyme-labeled secondary antibody dilution coating 1:5000 1:10000 1:20000 concentration P N P N P N (?g/mL) OD.sub.450 nm OD.sub.450 nm P/N OD.sub.450 nm OD.sub.450 nm P/N OD.sub.450 nm OD.sub.450 nm P/N 7.000 1.336 0.219 6.100 1.056 0.152 6.947 0.717 0.097 7.392 3.500 0.972 0.154 6.312 0.962 0.111 8.667 0.522 0.089 5.865 1.250 0.855 0.135 6.333 0.685 0.121 5.661 0.430 0.083 5.181 0.625 0.614 0.128 4.797 0.474 0.093 5.097 0.332 0.073 4.548 0.313 0.496 0.109 4.550 0.353 0.082 4.305 0.225 0.071 3.169 0.156 0.391 0.093 4.204 0.278 0.076 3.658 0.195 0.064 3.047

[0050] As shown in Table 1, the concentration of VP6 fusion recombinant protein was diluted to 1.25?7 ?g/mL, and the dilution of enzyme-labeled secondary antibody was in the range of 1:5000?20000, among which the highest P/N value was obtained when the concentration of VP6 fusion recombinant protein was 3.5 ?g/mL and the dilution of enzyme-labeled secondary antibody was 1:10000, indicating that the dose combination was the optimal combination.

Example 4: Determination of Optimal Antigens Coating Conditions

[0051] The optimal antigen coating concentration and the optimal secondary antibody working concentration obtained in example 2 were used as test conditions. Incubation were set into three test groups at 37? C. for 1 h, 37? C. for 2 h and 4? C. overnight (15?16 h) for ELISA detection, and the P/N value (P/N value=OD.sub.450 nm mean value of positive samples/OD.sub.450 nm mean value of negative samples) is calculated, selecting a maximum P/N value as an optimal antigen coating condition. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Determination of optimal antigen coating conditions Antigen coating conditions Groups 37? C. 1 h 37? C. 2 h 4? C. overnight Positive milk OD.sub.450 nm 0.732 0.824 0.965 0.724 0.867 0.972 0.735 0.888 0.983 Positive milk OD.sub.450 nm mean 0.730 0.860 0.973 value Positive milk OD.sub.450 nm 0.098 0.102 0.111 0.093 0.107 0.112 0.092 0.103 0.114 Negative milk OD.sub.450 nm mean 0.094 0.104 0.112 value P/N value 7.742 8.266 8.665

[0052] As shown in Table 2, the P/N value of the overnight coating at 4? C. and the 1?2 h coating at 37? C. have obvious advantages, so the method for the VP6 fusion recombinant protein coated with overnight coating at 4? C. was optimal.

Example 5: Determination of Optimal Blocking Conditions

[0053] The Enzyme-labeled plate is coated with the optimal antigen coating concentration and the optimal coating conditions. Three test groups were set up for 37? C. blocked for 1 h, 37? C. blocked for 2 h and 4? C. overnight. The OD 450 nm value of positive and negative milk in each group is measured separately, and the P/N value is calculated to determine the optimal blocking time, and the results are shown in Table 3.

TABLE-US-00003 TABLE 3 Determination of optimal blocking conditions Blocking conditions Groups 37? C. 1 h 37? C. 2 h 4? C. overnight Positive milk OD.sub.450 nm 0.975 0.825 0.735 0.982 0.863 0.721 0.983 0.889 0.724 Positive milk OD.sub.450 nm mean 0.980 0.859 0.727 value Negative milk OD.sub.450 nm 0.113 0.101 0.096 0.111 0.103 0.094 0.114 0.105 0.097 Negative milk OD.sub.450 nm mean 0.113 0.103 0.096 value P/N value 8.698 8.340 7.596

[0054] As shown in Table 3, the P/N value of the blocking solution at 37? C. for 1?2 h has obvious advantages over at 4? C. overnight, and the enzyme-labeled plate of this invention was treated with the at 37? C. for 1 h blocking method.

Example 6: Determination of Optimal Milk Dilution and Incubated Time

[0055] (1) ELISA test is carried out under the determined optimal conditions, and the male and negative milk are diluted at ratios of 1:50, 1:100, 1:200, and 1:400 times respectively, with 100 L/well. [0056] (2) After adding the diluted milk, three test groups were setup, the first group is incubated at 37? C. for 30 min, the second group is incubated at 37? C. for 45 min, and the third group is incubated at 37? C. for 60 min. [0057] (3) The OD 450 nm value of positive and negative milk in each group is measured separately, and the P/N value is calculated to determine the optimal milk dilution and incubated time, and the results are shown in Table 4.

TABLE-US-00004 TABLE 4 Optimization of milk dilution and incubated time Milk incubated time 30 min 45 min 60 min Milk OD.sub.450 nm mean OD.sub.450 nm mean OD.sub.450 nm mean dilution value value value factor P N P/N P N P/N P N P/N 1:50 1.112 0.203 5.478 1.119 0.256 4.371 1.268 0.295 4.298 1:100 0.856 0.112 7.643 0.912 0.117 7.795 0.973 0.121 8.041 1:200 0.763 0.102 7.480 0.792 0.106 7.472 0.837 0.109 7.679 1:400 0.435 0.089 4.888 0.487 0.094 5.181 0.526 0.096 5.479

[0058] As shown in Table 4, the obtained P/N value has obvious advantages in the range of milk dilution of 1:50?200, therefore, the milk samples of this invention was detected by a method with a dilution of 1:100 and an incubated time of 1 h.

Example 7: Determination of Optimal Serum Dilution and Incubated Time

[0059] The ELISA test was performed under the determined optimal conditions, and the negative and negative serum are diluted at ratios of 1:50, 1:100, 1:200, and 1:400 respectively, with 100 ?L/well. After adding the diluted serum, three test groups were setup, the first group is incubated at 37? C. for 30 min, the second group is incubated at 37? C. for 45 min, and the third group is incubated at 37? C. for 60 min. The positive and negative serum OD 450 nm values of each group are measured separately, and the P/N values are calculated to determine the optimal serum dilution and incubated time, and the results are shown in Table 5.

TABLE-US-00005 TABLE 5 Optimization of serum dilution and incubated time Serum incubated time 30 min 45 min 60 min Serum OD.sub.450 nm mean OD.sub.450 nm mean OD.sub.450 nm mean dilution value value value factor P N P/N P N P/N P N P/N 1:50 1.728 0.375 4.608 1.862 0.395 4.714 1.879 0.398 4.721 1:100 1.546 0.311 4.971 1.632 0.327 4.991 1.657 0.324 5.114 1:200 1.347 0.258 5.221 1.457 0.269 5.416 1.465 0.268 5.466 1:400 0.766 0.198 3.869 0.943 0.211 4.469 0.965 0.214 4.509

[0060] As shown in Table 5 that in the range of serum dilution of 1:100?400, the obtained P/N value has obvious advantages, therefore, the serum samples of this invention were detected by a method with a dilution of 1:200 and an incubated time of 1 h.

Example 8: Determination of the Optimal Secondary Antibody Incubated Time

[0061] (1) Perform the ELISA test under the determined optimal conditions, and add enzyme-labeled secondary antibody (100 ?L/well). Three test groups are set up, the first group is incubated at 37? C. for 30 min, the second group is incubated at 37? C. for 45 min, and the third group is incubated at 37? C. for 60 min. [0062] (2) The OD450 nm value of the positive and negative milk in each group is measured, and the P/N value is calculated to determine the optimal incubated time of the secondary antibody, and the results are shown in Table 6.

TABLE-US-00006 TABLE 6 Determination of optimal secondary antibody incubated time Enzyme-labeled secondary antibody incubated time 37? C. 60 37? C. 45 37? C. 30 Groups min min min Positive milk OD.sub.450 nm 0.972 0.825 0.735 0.984 0.863 0.721 0.983 0.889 0.724 Positive milk OD.sub.450 nm 0.980 0.859 0.727 mean value Negative milk OD.sub.450 nm 0.110 0.105 0.095 0.113 0.102 0.098 0.111 0.104 0.097 Negative milk OD.sub.450 nm 0.111 0.104 0.097 mean value P/N value 8.799 8.286 7.517

[0063] As shown in Table 6, a secondary antibody incubation solution at 37? C. for 60 minutes was optimal.

Example 9: Determination of the Optimal Substrate Chromogenic Time

[0064] (1) The ELISA test is carried out under the determined optimal conditions, and three test groups are set up after adding TMB (with 100 ?L/well), the first group was incubated at room temperature for 10 min, the second group was incubated at room temperature for 15 min, and the third group was incubated at room temperature for 20 min; [0065] (2) After the chromogenic process was completed, 100 ?L of stop solution is added to each well to terminate the chronography, the OD 450 nm value was read, and the P/N value is calculated to determine the optimal substrate chromogenic time, and the results are shown in Table 7.

TABLE-US-00007 TABLE 7 Determination of TMB chromogenic time TMB chromogenic time 37? C. 10 37? C. 15 37? C. 20 Groups min min min Positive milk OD.sub.450 nm 0.737 0.972 1.112 0.728 0.982 1.106 0.724 0.988 1.103 Positive milk OD.sub.450 nm 0.730 0.981 1.107 mean value Negative milk OD.sub.450 nm 0.097 0.110 0.212 0.098 0.113 0.214 0.097 0.111 0.119 Negative milk OD.sub.450 nm 0.097 0.111 0.182 mean value P/N value 7.497 8.808 0.589

[0066] As shown in Table 7, the chromogenic time at 37? C. for 15 min was optimal.

Example 10: Preparation and Use Method of ELISA Detection Method

[0067] (1) Coating: The VP6 fusion recombinant protein is diluted to 3.5 ?g/mL using a 0.05 mol/L Na.sub.2CO.sub.3NaHCO.sub.3 buffer solution and 100 ?L of the diluted protein solution was added to each well, and adsorbed at 4? C. for 14 h. After the coating was completed, the liquid in the wells was discarded, and 250 ?L of PBS wash solution containing 0.05% Tween-20 was added to each well. The wells were washed three times, with the final wash being tapped dry; [0068] (2) Blocking: 200 ?L of PBS blocking solution containing 5% BSA and 0.05% Tween-20 is added to each well, and blocked at 37? C. for 1 h. After blocking, the liquid in the wells was discarded, 250 ?L of wash solution was added to each well. The wells were washed three times, with the final wash being tapped dry; [0069] (3) Primary antibody incubation: dilute the milk sample to be tested (1:100) or serum sample to be tested (1:200) with sample diluent, 100 ?L/well, and incubated at 37? C. for 60 min. The liquid in the wells is discarded, 250 ?L of wash solution is added to each well. The wells were washed five times, with the final wash being tapped dry; [0070] (4) Secondary antibody incubation: dilute the enzyme-labeled secondary antibody (1:10000) with antibody diluent, 100 ?L/well, incubated at 37? C. for 60 min, discard the liquid in the well, 250 ?L of wash solution was added to each well. The wells were washed five times, with the final wash being tapped dry; [0071] (5) Color rendering and reading: 100 ?L of a single-component TMB substrate chromogenic solution is added to each well, and the reaction was allowed to proceed in dark at 37? C. for 15 minutes. Subsequently, 100 ?L of stop solution is added to each well, and the OD value at 450 nm was read using a microplate reader.

Example 11: Determination of the Critical Value

[0072] The detection method prepared in Example 8 are used to ELISA detection for 24 porcine RV-negative milk and 30 porcine RV-negative serum preserved in the laboratory, and each sample is repeated twice. The OD.sub.450 nm reading of each sample is determined and the S/P value is calculated.

[0073] The OD.sub.450 nm absorbance value of 24 known negative milk is detected, and the mean OD.sub.450 nm absorbance value of 24 negative milk is 0.154 and the SD value is 0.035, and in order to ensure a 99% confidence interval, the cut off value is the mean value plus 3 times the SD value, and the final cut off value calculated is 0.262.

[0074] The OD.sub.450 nm absorbance value of 30 known negative serums is detected, and the mean OD.sub.450 nm absorbance value of 30 negative serums is 0.271 and the SD value is 0.015, in order to ensure a 99% confidence interval, the cut off value is the mean value plus 3 times the SD value, and the final cut off value calculated is 0.319.

Example 12: Specific Test

[0075] (1) According to the optimized ELSIA method, the standard positive serum of swine fever virus, porcine epidemic diarrhea virus, porcine PRRS virus and foot-and-mouth disease virus (type O) preserved in this laboratory are detected, and PRoV positive and negative serum control is set up to determine whether the PRoV indirect ELISA detection method established in this test had cross-reactivity with antibodies from other major porcine viral pathogens, the results are shown in FIG. 3.

[0076] As shown in FIG. 3, the standard positive serum S/P values of swine fever virus, porcine epidemic diarrhea virus, porcine PRRS virus, and foot-and-mouth disease virus (type 0) were all less than 0.319, which shows that PRoV was negative, indicating that the ELISA detection method for VP6 fusion recombinant protein IgA antibody based on against Porcine rotavirus group A established by this invention has good specificity.

Example 13: Sensitivity Detection

[0077] 3 PRoV-positive milk were diluted from the ratio of 1:10 and detected using an established indirect ELISA method with a positive and negative control. The sensitivity of this ELISA method is analyzed by measuring the OD.sub.450 nm value, and the results are shown in FIG. 4.

[0078] The results showed that all 3 positive milk were still positive at a 400 times dilution (OD.sub.450 nm value ?0.262). It was indicated that the ELISA detection method for VP6 fusion recombinant protein IgA antibody based on against Porcine rotavirus group A established by this invention has high sensitivity.

Example 14: Repeatability Test

[0079] (1) Three batches of PRoVVP6 fusion recombinant protein were coated at different times, 3 positive and 3 negative milks were performed by ELISA detection, 4 replicates were set up in the batch, the OD.sub.450 nm value is determined, and the mean value, standard deviation and coefficient of variation were calculated to determine the reproducibility within the batchs and the reproducibility between batches, the results are shown in Table 8.

TABLE-US-00008 TABLE 8 Repeatability test for ELISA detection method for VP6 fusion recombinant proteins IgA antibody CV range for 6 milk median value reproducibility within the batches 7%-16% 13% reproducibility between batches 3%-6% 4%

[0080] As shown in Table 8, the ELISA detection method for VP6 fusion recombinant protein IgA antibody based on against Porcine rotavirus group A variant strains established by this invention has good repeatability.