Porcine rotavirus VP8 protein-based IgG and IgA antibody detection kit as well as preparation method and application thereof

Abstract

The invention discloses an IgG and IgA antibody detection kit based on porcine rotavirus VP8 protein as well as a preparation method and application of the IgG and IgA antibody detection kit. The kit comprises an elisa plate, positive control serum, negative control serum, an elisa secondary antibody, a sample diluent, a 20-time concentrated washing solution, a substrate solution A, a substrate solution B and a stop solution, wherein the elisa plate is coated with VP8 truncated protein of a porcine rotavirus epidemic strain expressed by mammalian cells. According to the kit, the IgG antibody and the IgA antibody of the porcine rotavirus can be detected at the same time under the condition that only one reagent (enzyme-labeled antibody) is added, and evaluation on the titer of the whole antibody is facilitated. The kit disclosed by the invention is high in sensitivity, high in specificity, good in stability, convenient to operate and relatively high in universality, and has a good market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to an IgG and IgA antibody detection kit based on porcine rotavirus VP8 protein, a preparation method and application thereof. BACKGROUND

[0002] Rotavirus (RV) belongs to the family of Reoviridae and the genus of Rotavirus, and has a shape like a wheel, is icosahedral symmetry, and has a dense hexagonal core of nucleic acid without a capsule membrane. The virus can cause diarrhea of various newborn animals and young animals, is an important enteric pathogen, and is also a common cause of gastroenteritis of suckling pigs and weaned piglets.

[0003] Porcine rotavirus (PoRV) is one of the important enteric pathogens causing diarrhea of suckling pigs and weaned piglets. It is particularly susceptible in intensive pig farms, and the incubation period of the virus infection in piglets is very short, generally 1 day, and symptoms appear. Piglets often show listlessness, reduced exercise, some vomiting, and a significant decrease in food intake. In the later stage of the disease, piglets have severe diarrhea or dysentery, and the diarrhea is mostly yellow, and the diarrhea of severe cases is black and red. Dehydrated and emaciated pigs have slow growth. If mixed infection or secondary infection with other pathogens occurs, the mortality of piglets can even reach 100%, which seriously affects the healthy development of the pig industry in China and causes serious economic losses to the animal husbandry. In recent years, more and more piglets have died due to infection with porcine rotavirus, and large pigs and breeding pigs often have latent infection. Once infected with porcine rotavirus, the growth and development of pigs will be seriously affected, and even adult pigs that have recovered will stop growing, resulting in a significant increase in the culling rate of pig farms. Enzyme-linked immunosorbent assay (ELISA) has the characteristics of high sensitivity, good specificity, simple operation, and high-throughput rapid detection of a large number of samples, and is suitable for disease detection. At present, protein expression systems mainly include Escherichia coli expression system, insect cell expression system, mammalian cell expression system, etc. The proteins expressed by prokaryotic cells such as Escherichia coli do not have post-translational modification processes, which affect the activity of the proteins, and the expression products are prone to form inactive inclusion body state and need to be refolded, which is difficult to promote in clinical practice. Although the insect cell expression system has eukaryotic modification ability, its glycosylation mode is different from that of mammalian cells, and strict quality control is required. The proteins expressed by mammalian cells can be modified by complex glycosylation, sulfation, etc., and have a structure closer to that of natural proteins and better immunogenicity. Therefore, it is particularly important to develop an antibody ELISA detection kit using mammalian cell-expressed and purified proteins as coating antigens for the diagnosis and prevention of porcine rotavirus disease.

[0004] PoRV is a segmented double-stranded RNA virus, and its genome mainly encodes six structural proteins (VP1, VP2, VP3, VP4, VP6 and VP7) and six non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6). The virion is composed of three layers of capsid proteins, the innermost layer is composed of VP2 and a small amount of VP1 and VP3 proteins, and the three together wrap the viral genomic RNA, which is indispensable for viral gene transcription and replication. The surface of the virion of PoRV has three antigen proteins: group antigen, neutralizing antigen and hemagglutinin antigen. The group antigen is related to various structural proteins, mainly determined by the inner capsid protein VP6, which is the central core capsid protein, accounts for 51% of the viral structural proteins, has high conservation, and is an important antigen for clinical detection. According to its VP6 antigen, rotavirus can be divided into A, B, C, D, E, F, G and other seven serogroups. According to epidemiological investigation, the porcine diarrhea rotavirus is mainly A group. The neutralizing antigen is mainly determined by the outer shell glycoprotein VP7, which forms the smooth surface of the virus; the hemagglutinin antigen is determined by the outer capsid protein VP4, which forms the sharp peaks of the virus particles. VP7 and VP4 are the main determinants of viral serotypes, which have important significance in the detection of pathogens and the production of neutralizing antibodies and immunoprotective processes. According to the antigen characteristics of the two, PoRV can be divided into G and P two serotypes, among which G9 and P

[23] are the most dominant genotypes. At the same time, VP4 protein can be decomposed into VP5 protein and VP8 protein after trypsin treatment, and the infection ability of the virus can be significantly enhanced. VP5 protein can increase the permeability of the cell membrane, thereby promoting the entry of the virus into the cell, while VP8 protein interacts with glycan receptors to enhance the adhesion ability of the virus. And VP8 protein contains the main antigenic sites of VP4 protein, and has good immunogenicity and reactivity, which has the potential to be used as an indirect ELISA coating antigen, and can be used for serological diagnosis to detect the antibodies produced by porcine rotavirus and provide detection methods for disease prevention and control. SUMMARY

[0005] In view of the above prior art, the purpose of the present application is to provide an indirect ELISA antibody detection kit based on porcine rotavirus VP8 truncated protein, which can detect IgG and IgA antibodies of porcine rotavirus at the same time, realize the detection of piglet infection or immune effect of porcine rotavirus, the detection sample of IgG antibody includes but is not limited to serum, plasma, milk and the like, and the detection sample of IgA antibody includes but is not limited to serum, plasma, milk and the like. At the same time, without affecting the overall performance of the kit, the signal of the kit can be reduced, which is beneficial to the evaluation of the actual antibody titer.

[0006] The present application is realized by the following technical solutions: An indirect ELISA antibody detection kit based on porcine rotavirus VP8 protein, comprising a porcine rotavirus VP8 truncated protein coated ELISA plate, the enzyme-linked reaction plate is a detachable 96-well enzyme-labeled plate; the porcine rotavirus VP8 truncated protein coated ELISA plate is prepared by using the VP8 truncated protein of the most dominant epidemic strain of porcine rotavirus expressed by mammalian cells as the coating antigen, and carbonate buffer as the coating liquid, and is placed at 2-8°C for 8-12 hours, so that the antigen is fully combined with the enzyme-linked reaction plate, then 300 μL / well of PBS buffer containing 10 mg / mL bovine serum albumin (BSA) pH 7.4 is added, and the enzyme-linked reaction plate is sealed and stored at 4°C after drying.

[0007] Further, the porcine rotavirus VP8 truncated protein is prepared by the following method: using the porcine rotavirus G9 subtype epidemic strain SC01 strain (China General Microbiological Culture Collection Center Preservation Number CGMCC No. 46200) isolated by our company as a template, using specific primers in the VP8 region for PCR amplification, and sequencing to obtain the expression gene of the porcine rotavirus VP8 protein; through antigen epitope prediction analysis and combined with existing related literature reports, the specific epitope (46-201 aa, SEQ ID NO. 1 in the sequence listing) region gene sequence (SEQ ID NO. 2 in the sequence listing) of the VP8 protein is selected, cloned into the pcDNA3.1 vector, and fused to express His tag, to construct the eukaryotic expression plasmid pcDNA3.1-PoRV-VP8, then transiently transfected into single-layer 293F cells in good condition, collected the cell expression supernatant, and subjected to Ni-NTA column protein separation and purification, to obtain the truncated porcine rotavirus VP8 truncated protein.

[0008] Further, the ELISA antibody detection kit further comprises the following components: sample diluent, concentrated washing solution, enzyme conjugate working solution, color developing solution, termination solution, positive control and negative control.

[0009] The positive control is antibody positive serum collected after immunization with a commercial porcine rotavirus inactivated vaccine, and is used after being appropriately diluted with the sample diluent.

[0010] The negative control is specific pathogen-free (SPF) porcine serum.

[0011] The enzyme-labeled secondary antibody has two parts, which are horseradish peroxidase-labeled rabbit anti-pig IgG antibody and horseradish peroxidase-labeled goat anti-pig IgA antibody. The horseradish peroxidase-labeled rabbit anti-pig IgG antibody is prepared by diluting the horseradish peroxidase-labeled rabbit anti-pig IgG (purchased from Sigma Company, product number A5670) as a stock solution by 1:50000. The horseradish peroxidase-labeled goat anti-pig IgA antibody is prepared by diluting the horseradish peroxidase-labeled goat anti-pig IgA (purchased from Abeam Company, product number AB112746) as a stock solution by 1:60000 (2 bottles, 12 mL / bottle).

[0012] The substrate solution A is a citrate phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea, and the substrate solution B is a 0.2 mg / mL tetramethyl benzidine solution, which are mixed in a ratio of 1:1 when used. The termination solution is a 2 mol / L sulfuric acid solution.

[0013] The kit also includes a sample diluent and a 20-fold concentrated washing solution; the sample diluent is a 0.01 M phosphate buffer containing 5 mg / mL casein, pH 7.4; and the concentrated washing solution is a 0.01 M phosphate buffer containing 0.8%~1.2% (mL / mL) Tween-20, pH 7.4.

[0014] The detection procedure of the kit of the present application is as follows: 1. Equilibrium: take the kit out of the refrigeration environment and place it at room temperature for 30 min for standby; mix the liquid reagents before use.

[0015] 2. Liquid preparation: dilute the concentrated washing solution with distilled water or deionized water by 20 times to obtain the washing buffer; 3. Setting: 2 negative control wells and 2 positive control wells, and the rest are sample wells.

[0016] 4. Pre-dilution of the sample to be tested: dilute the sample to be tested with the sample diluent according to a ratio of 1:50.

[0017] 5. Sample addition: add 100 μL of the diluted sample to be tested to each well according to the pre-set. The time span of the sample addition process should be as short as possible.

[0018] 6. Incubation: shake and mix, and place in a 37℃ incubator for 30 min.

[0019] 7. Plate washing: discard the reaction solution, add 300 μL of the diluted washing buffer to each well, soak for 15 s, discard the washing solution, and wash the plate continuously for 4 times, and then pat dry.

[0020] 8. Add enzyme: Add 100 μL of horseradish peroxidase-labeled rabbit anti-pig IgG antibody or goat anti-pig IgA antibody to each well.

[0021] 9. Incubation: Place in a 37℃ incubator and react for 30 min.

[0022] 10. Washing the plate: Discard the reaction solution, add 300 µL of diluted washing buffer to each well, soak for 15 seconds, discard the washing buffer, wash the plate 4 times and then pat dry.

[0023] 11. Color development: Add 100 µL of substrate working solution to each well (the substrate working solution is obtained by mixing equal volumes of substrate solution A and substrate solution B, and should be prepared fresh before use), shake to mix well, and place in a 37℃ incubator to react for 15 min in the dark.

[0024] 12. Add 50 µL of colorimetric stop solution to each well and shake to mix and terminate the reaction.

[0025] 13. Measure the OD of each well. 450 nm The OD value (the reaction plate after adding the stop solution should be read within 15 minutes) 450 nm value).

[0026] The interpretation of IgG antibody test results: Negative control OD 450 nm The mean (N) should be ≤0.20; otherwise, it is invalid. Positive control OD 450 nm The mean (P) should be ≥1.0; otherwise, it is invalid. Simultaneously calculate the S / P value = (sample OD...) / ( ... 450 nm Value - Negative control OD 450 nm (value) / (positive control OD) 450 nm Value - Negative control OD 450 nm The presence or absence of antibodies is determined by calculating the S / P value of each sample. (1) S / P < 0.40, the sample is judged as antibody negative; (2) S / P ≥ 0.40, the sample is judged as antibody positive.

[0027] The interpretation of IgA antibody test results: Negative control OD 450 nm The mean (N) should be ≤0.20; otherwise, it is invalid. Positive control OD 450 nm The mean (P) should be ≥1.0; otherwise, it is invalid. Simultaneously calculate the S / P value = (sample OD...) / ( ... 450 nm Value - Negative control OD 450 nm (value) / (positive control OD) 450 nm Value - Negative control OD 450 nm The presence or absence of antibodies is determined by calculating the S / P value of each sample. (1) S / P < 0.30, the sample is judged as antibody negative; (2) S / P ≥ 0.30, the sample is judged as antibody positive.

[0028] The kit can be used for detecting porcine rotavirus antibodies to determine whether the detected animal is infected with porcine rotavirus or produces antibodies after immunization.

[0029] The application in preparing the kit for detecting whether the animal is infected with porcine rotavirus disease also belongs to the protection scope of the present application.

[0030] The positive effect of the present application is that the present application takes the isolated porcine rotavirus G9 subtype epidemic strain SC01 strain as a parent, successfully expresses and purifies VP8 truncated protein as a coating antigen through a mammalian cell system, establishes a porcine rotavirus specific IgG and IgA antibody ELISA detection method with high sensitivity, high specificity, good stability, good repeatability and easy preparation, and can be applied to large-scale clinical detection.

[0031] Meanwhile, the kit of the present application can realize simultaneous detection of IgG antibody and IgA antibody of porcine rotavirus by only adding one reagent (enzyme-labeled antibody), which is beneficial to the evaluation of the whole antibody titer. In addition, the kit of the present application is not only suitable for serum detection, but also suitable for plasma and milk detection, and has strong universality.

[0032] In summary, the kit uses VP8 truncated protein of porcine rotavirus to coat the enzyme-linked reaction plate, has high sensitivity, high specificity and good repeatability, and can effectively detect specific IgG and IgA antibodies produced after porcine rotavirus infection or immunization. The experimental results show that the kit has good repeatability, high specificity and high sensitivity. It can meet the needs of different levels of personnel, has broad market prospects and good economic and social benefits.

[0033] The porcine rotavirus ELISA diagnostic kit of the present application is used for detecting whether the animal is infected with porcine rotavirus or produces antibodies after immunization, which is beneficial to the establishment of the prevention and control system of porcine rotavirus in China. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Porcine rotavirus VP8 truncated protein expression and purification identification chart. DETAILED DESCRIPTION

[0035] The methods in the following examples are all conventional methods unless otherwise specified.

[0036] Example 1: Mammalian cell expression and purification of porcine rotavirus VP8 truncated protein The specific epitope (46-201 aa) in the VP8 gene of the preserved porcine rotavirus epidemic strain (China General Microbiological Culture Collection Center, Patent Application Publication No. CN120060159A) is selected, as shown in sequence 1 in the sequence table, and the coding gene as shown in sequence 2 in the sequence table is added with a start codon, a Kozak sequence, an Mlu I enzyme cutting site and a protective base at the front, and a terminator and a Nhe I enzyme cutting site and a protective base at the end, and fused with a His tag, cloned into a pcDNA3.1 vector, and a eukaryotic expression plasmid pcDNA3.1-PoRV-VP8 is constructed, and its correctness is verified by sequencing.

[0037] The pcDNA3.1-PoRV-VP8 plasmid is transiently transfected into single-layer 293F cells in good cell state, the cell expression supernatant is collected, Ni-NTA column protein separation and purification are performed, and the porcine rotavirus VP8 truncated protein is obtained, as shown in Figure 1 .

[0038] Example 2: Preparation of porcine rotavirus positive and negative serum 1. Preparation of negative control (1) Preparation of animals: 3 piglets of 3 days old, corresponding to the blood sampling of sows within 7 days before delivery for porcine epidemic diarrhea virus, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, porcine blue ear virus, classical swine fever virus, and porcine circovirus type 2 antibody screening, and healthy piglets produced by all negative sows are selected; (2) Serum preparation and subpackaging: blood is collected from the anterior vena cava of the pig, and the blood is placed in a sterilized clean triangular flask. The separated serum is transferred to a centrifuge bottle, centrifuged at 3000 r / min for 5 minutes, and the supernatant is taken. The supernatant is mixed and filtered through a 0.22 μm filter membrane to remove bacteria, and is quantitatively subpackaged at 1.5 mL / tube; (3) Preparation of negative control: the negative serum is diluted 40 times with a sample diluent, mixed uniformly, filtered through a 0.22 μm filter membrane to remove bacteria, and quantitatively subpackaged at 1.5 mL / tube.

[0039] 2. Preparation of positive control (1) Preparation of animals: 3 piglets of 3 days old, corresponding to the blood sampling of sows within 7 days before delivery for porcine epidemic diarrhea virus, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine delta coronavirus, porcine blue ear virus, classical swine fever virus, and porcine circovirus type 2 antibody screening, and healthy piglets produced by all negative sows are selected; (2) Take commercial pig rotavirus inactivated vaccine, according to the instructions, 3 pigs were injected with vaccine 1 mL in the neck muscle, 14 days later, the method and dose were the same as the first time, every 7 days after the second immunization, blood was collected, and the collected serum was detected for pig rotavirus antibody by neutralization method; (3) The serum titer was determined by neutralization method, and the pigs with pig rotavirus neutralizing antibody not less than 1:128 were selected for positive serum preparation; (4) The experimental pigs meeting the conditions were subjected to anterior vena cava blood collection, the blood of each pig was preserved in a sterilized triangular flask, the separated serum was transferred to a centrifuge bottle, centrifuged at 3000 r / min for 5 minutes, the supernatant was taken, and the supernatant was mixed and filtered through a 0.22 μm filter membrane to remove bacteria, and quantitatively packaged, 1.5 mL / tube; (5) The qualified positive serum was diluted by sample diluent to 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, each dilution was detected by the prepared kit, and the highest dilution multiple of IgG antibody OD 450 nm value≥1.2 and IgA antibody OD 450 nm value≥1.0 was selected, the positive serum was diluted according to the dilution multiple by sample diluent, mixed uniformly, filtered through a 0.22 μm filter membrane to remove bacteria, and quantitatively packaged, 1.5 mL / tube.

[0040] Example 3: Establishment of pig rotavirus IgG antibody detection kit 1. Determination of optimal antigen coating concentration and secondary antibody working concentration (1) The optimal antigen coating concentration and the optimal secondary antibody working concentration were determined by matrix method. The purified VP8 truncated protein was diluted to 4.0 μg / mL, 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL, 0.25 μg / mL and 0.125 μg / mL in turn, and 100 μL / well was coated on one horizontal row of ELISA plate for each concentration; (2) The HRP-labeled rabbit anti-pig IgG antibody was diluted by antibody diluent to 1:20000, 1:40000, 1:50000 and 1:60000 in turn, and 100 μL / well was added to two vertical columns for each dilution, forming a square; (3) The test was repeated three times, and the antigen concentration and secondary antibody dilution degree with the maximum ratio of the average OD 450 nm value of positive serum to the average OD 450 nm value of negative serum (P / N value) were selected as the optimal antigen coating concentration and the optimal secondary antibody working concentration, and the results were shown in Table 1.

[0041] Table 1 Determination of optimal antigen coating concentration and secondary antibody working concentration

[0042] Note: The data in the table is the OD of positive serum 450 nm The mean and the OD of negative serum 450 nm The ratio of the mean (P / N value).

[0043] From Table 1, the protein concentration is diluted to 0.25-4 μg / mL, and the dilution of the enzyme-labeled secondary antibody is in the range of 1:20000-60000, which can achieve a higher P / N value. Among them, the P / N value obtained when the protein concentration is 1.0 μg / mL and the dilution of the enzyme-labeled secondary antibody is 1:50000 is the highest, indicating that this dose combination is the optimal combination.

[0044] 2. Determination of the optimal antigen coating condition The optimal antigen coating concentration and the optimal secondary antibody working concentration obtained are used as the test conditions. Set 37℃ incubation for 1 h, 37℃ incubation for 2 h and 4℃ incubation overnight (8-12 h) for three test groups for ELISA detection, calculate P / N value (P / N value = positive sample OD 450 nm mean / negative sample OD 450 nm mean), and select the maximum P / N value as the optimal antigen coating condition. The results are shown in Table 2.

[0045] Table 2 Determination of the optimal antigen coating condition

[0046] From Table 2, the coating condition of 4℃ overnight has obvious advantages in P / N value compared with the scheme of 37℃ coating for 1-2 h, so the protein is coated by the method of 4℃ overnight.

[0047] 3. Determination of the optimal blocking condition The enzyme-labeled plate is coated with the optimal antigen coating concentration and the optimal coating condition. Set 37℃ blocking for 1 h, 37℃ blocking for 2 h and 4℃ blocking overnight for three test groups. The OD 450 nm values of positive and negative serum of each group are measured, and the P / N value is calculated to determine the optimal blocking time, and the results are shown in Table 3.

[0048] Table 3 Determination of the optimal blocking condition

[0049] From Table 3, the scheme of 37℃ blocking for 2 h has obvious advantages in P / N value, so the enzyme-labeled plate is treated by the method of 37℃ blocking for 2 h.

[0050] 4. Determination of the optimal serum dilution and reaction time (1) Under the optimal conditions, the positive and negative serum samples were diluted by 1:25, 1:50, 1:100 and 1:200, and 100 μL was added to each well.

[0051] (2) After the diluted serum was added, three groups were set up. The first group was incubated at 37°C for 30 min, the second group was incubated at 37°C for 45 min, and the third group was incubated at 37°C for 60 min.

[0052] (3) The OD values of the positive and negative serum samples in each group were measured three times, and the P / N values were calculated to determine the optimal serum dilution and reaction time. The results are shown in Table 4. 450 nm

[0053] Table 4. Optimization of serum dilution and reaction time

[0054] As shown in Table 4, within the range of serum dilution of 1:25-100 and reaction time of 30-60 min, a high P / N value can be achieved. The highest P / N value is obtained when the serum dilution is 1:50 and the reaction time is 30 min, indicating that this combination is the optimal combination.

[0055] 5. Determination of the optimal reaction time of the secondary antibody and the optimal substrate color development time (1) Under the optimal conditions, the secondary antibody was added (100 μL / well) and three groups were set up. The first group was incubated at 37°C for 30 min, the second group was incubated at 37°C for 45 min, and the third group was incubated at 37°C for 60 min. (2) After the substrate solution (A and B mixed at a ratio of 1:1, 100 μL / well) was added, three groups were set up. The first group was reacted at room temperature for 10 min, the second group was reacted at room temperature for 15 min, and the third group was reacted at room temperature for 20 min. (3) After color development, 50 μL of stop solution was added to each well to stop the color development, and the OD 450 nm values were read. The P / N values were calculated to determine the optimal reaction time of the secondary antibody and the optimal substrate color development time. The results are shown in Table 5.

[0056] Table 5. Determination of the optimal reaction time of the secondary antibody and the optimal substrate color development time

[0057] ​From Table 5, it can be seen that when the secondary antibody reaction time is in the range of 30-60 min and the substrate coloration time is in the range of 10-20 min, a higher P / N value can be achieved, wherein the secondary antibody reaction time is 30 min and the substrate coloration time is 15 min, the P / N value obtained is the highest, indicating that this combination is the optimal combination.

[0058] 6. Determination of critical value of detection result According to the optimized detection method, 40 laboratory-preserved porcine rotavirus negative sera were subjected to IgG antibody ELISA detection, and each sample was repeated three times. The OD 450 nm value of each serum was determined, and the S / P value was calculated. The S / P value=(sample serum OD 450 nm value-negative control serum OD 450 nm value) / (positive control serum OD 450 nm value-negative control serum OD 450 nm ).

[0059] The mean (X) and standard deviation (SD) of the S / P values of the 40 sera were calculated. When the sample S / P value≥X+3SD, it was determined to be positive; when the sample S / P valueX+3SD, it was determined to be negative.

[0060] The results showed that when the sample S / P value≥0.40, it was positive; when the sample S / P value<0.40, it was negative.

[0061] The mean (N) of the OD 450 nm of the negative control should be ≤0.20, otherwise it is invalid. The mean (P) of the OD 450 nm of the positive control should be ≥1.0, otherwise it is invalid. At the same time, the S / P value=(sample serum OD 450 nm value-negative control serum OD 450 nm value) / (positive control serum OD 450 nm value-negative control serum OD 450 nm ) was calculated. By calculating the S / P value of each sample, the presence or absence of antibody was determined.

[0062] (1) S / P<0.40, determined to be antibody negative; (2) S / P≥0.40, determined to be antibody positive.

[0063] Example 4: Establishment of porcine rotavirus IgA antibody detection kit In order to facilitate the implementation of the IgA antibody and IgG antibody of porcine rotavirus synchronous detection, the inventors only replace the rabbit anti-pig IgG enzyme-labeled antibody (replace the goat anti-pig IgA enzyme-labeled antibody) when establishing the porcine rotavirus IgA antibody detection kit, and only optimize the dilution of the goat anti-pig IgA enzyme-labeled antibody and the positive and negative critical value, and the specific test method is the same as that in Example 3, which will not be repeated here. The optimization results are as follows: The goat anti-pig IgA enzyme-labeled antibody is directly used after being diluted by 1:60,000; The positive and negative critical value: the sample S / P value is greater than or equal to 0.30, which is positive; and the sample S / P value is less than 0.30, which is negative.

[0064] Example 5: Preparation and use method of ELISA detection kit One kind of porcine rotavirus VP8 truncated protein IgG and IgA antibody detection kit comprises: (1) 96-hole detachable polystyrene enzyme-linked reaction plate coated with mammalian cell expressed and purified porcine rotavirus VP8 truncated protein antigen (coating concentration, 1.0 μg / mL, 2x96 holes).

[0065] (2) Positive control serum: porcine rotavirus antibody positive serum collected after immunization of commercial porcine rotavirus inactivated vaccine, which is appropriately diluted by sample diluent and used as positive control serum of the kit (1 tube, 1.5 mL / tube).

[0066] (3) Negative control serum: specific pathogen-free pig serum, which is used as negative control serum of the kit (1 tube, 1.5 mL / tube).

[0067] (4) Enzyme-labeled secondary antibody: rabbit anti-pig IgG labeled with horseradish peroxidase (purchased from sigma company, item number A5670) as stock solution, which is diluted by 1:50,000 to prepare (2 bottles, 12 mL / bottle).

[0068] Goat anti-pig IgA labeled with horseradish peroxidase (purchased from Abeam company, item number AB112746) as stock solution, which is diluted by 1:60,000 to prepare (2 bottles, 12 mL / bottle).

[0069] (5) Sample diluent: 0.01 M, pH 7.4 phosphate buffer solution containing 5 mg / mL casein (1 bottle, 24 mL / bottle).

[0070] (6) Substrate solution A: citric acid phosphate buffer solution containing 0.6 mg / mL hydrogen peroxide urea (1 bottle, 12 mL / bottle) (7) Substrate solution B: 0.2 mg / mL tetramethyl benzidine solution (1 bottle, 12 mL / bottle).

[0071] (8) Stop solution: 2 mol / L sulfuric acid solution (1 bottle, 12 mL / bottle).

[0072] (9) 20-fold concentrated wash solution: 0.01 M phosphate buffer solution containing 0.8%~1.2% (mL / mL) Tween-20, pH 7.4 (2 bottles, 50 mL / bottle).

[0073] According to the needs, the kit can also have serum dilution plates (2 pieces, 96 wells / piece) for dilution of serum samples.

[0074] The preparation method of the 96-well detachable polystyrene enzyme-linked reaction plate coated with the purified porcine rotavirus VP8 truncated protein antigen expressed by mammalian cells is as follows: the VP8 truncated protein antigen prepared in Example 1 is dissolved in a carbonate solution with pH 9.6, then added to the 96-well polystyrene enzyme-linked reaction plate, the coating concentration is 1.0 μg / mL, 100 μL / well, placed at 4°C for 8~12 h, so that the antigen is fully combined with the enzyme-linked reaction plate, then 300 μL / well of PBS buffer solution containing 10 mg / mL bovine serum albumin (BSA) with pH 7.4 is added, and the blocking treatment is carried out at 37°C for 2 h, after spinning dry, the enzyme-linked reaction plate is dried and then stored at 4°C in a sealed state.

[0075] II. A method for using a porcine rotavirus VP8 truncated protein-based IgG and IgA antibody detection kit 1. Equilibrium: take the kit out of the refrigerated environment, and place it at room temperature for equilibrium for 30 min for standby; mix the liquid reagents before use.

[0076] 2. Liquid preparation: dilute the concentrated wash solution with distilled water or deionized water by 20 times to obtain the wash buffer.

[0077] 3. Setting: 2 negative control wells and 2 positive control wells, and the rest are sample wells.

[0078] 4. Pre-dilution of the sample to be tested: dilute the sample to be tested according to a ratio of 1:50 using the sample diluent.

[0079] 5. Sample addition: add 100 μL of the diluted sample to be tested to each well according to the pre-set. The time span of the sample addition process should be as short as possible.

[0080] 6. Incubation: shake and mix, and place in a 37°C incubator for reaction for 30 min.

[0081] 7. Plate washing: discard the reaction solution, add 300 μL of the diluted wash buffer to each well, soak for 15 s, discard the wash solution, and continuously wash the plate for 4 times, and then pat dry.

[0082] 8. Add enzyme: Add 100 μL of horseradish peroxidase-labeled rabbit anti-pig IgG antibody or goat anti-pig IgA antibody to each well.

[0083] 9. Incubation: Place in a 37℃ incubator and react for 30 min.

[0084] 10. Washing the plate: Discard the reaction solution, add 300 µL of diluted washing buffer to each well, soak for 15 seconds, discard the washing buffer, wash the plate 4 times and then pat dry.

[0085] 11. Color development: Add 100 µL of substrate working solution to each well (the substrate working solution is obtained by mixing equal volumes of substrate solution A and substrate solution B, and should be prepared fresh before use), shake to mix well, and place in a 37℃ incubator to react for 15 min in the dark.

[0086] 12. Add 50 µL of colorimetric stop solution to each well and shake to mix and terminate the reaction.

[0087] 13. Measure the OD of each well. 450 nm The OD value (for reaction plates with added stop solution, should be read within 15 minutes) 450 nm value).

[0088] Among them, the samples to be tested for IgG antibody detection include, but are not limited to, serum, plasma, and breast milk, while the samples to be tested for IgA antibody detection include, but are not limited to, serum, plasma, and breast milk.

[0089] Among them, the interpretation of IgG antibody test results: Negative control OD 450 nm The mean (N) should be ≤0.20; otherwise, it is invalid. Positive control OD 450 nm The mean (P) should be ≥1.0; otherwise, it is invalid. Simultaneously calculate the S / P value = (sample OD...) / ( ... 450 nm Value - Negative control OD 450 nm (value) / (positive control OD) 450 nm Value - Negative control OD 450 nm The presence or absence of antibodies is determined by calculating the S / P value of each sample. (1) S / P < 0.40, is judged as antibody negative; (2) S / P ≥ 0.40, is judged as antibody positive.

[0090] Among them, the interpretation of IgA antibody test results: Negative control OD 450 nm The mean (N) should be ≤0.20; otherwise, it is invalid. Positive control OD 450 nm The mean (P) should be ≥1.0; otherwise, it is invalid. Simultaneously calculate the S / P value = (sample OD...) / ( ... 450 nm Value - Negative control OD 450 nm (value) / (positive control OD) 450 nm Value - Negative control OD 450 nmThe S / P value is calculated, and the presence or absence of the antibody is determined. (1) S / P < 0.30, negative; (2) S / P ≥ 0.30, positive.

[0091] Example 6: Sensitivity test Three positive porcine rotavirus serum samples were diluted from 1:25, and the established indirect ELISA method was used to detect IgG and IgA antibodies, with positive and negative controls. The OD 450 nm The sensitivity of the kit was analyzed by calculating the S / P value, and the results are shown in Table 6.

[0092] Table 6 Sensitivity test results of the IgG and IgA antibody detection kit based on porcine rotavirus VP8 protein

[0093] The results show that the IgG antibody detection of the three positive serum samples is still positive (S / P ≥ 0.40) at a dilution of 1600 times. At the same time, the IgA antibody detection of the three positive serum samples is still positive (S / P ≥ 0.30) at a dilution of 800 times, indicating that the established porcine rotavirus IgG and IgA antibody detection kit has high sensitivity.

[0094] Example 7: Specificity test According to the optimized ELSIA method, 30 healthy pig serum samples, 2 classical porcine pestivirus positive serum samples, 2 porcine foot-and-mouth disease virus O positive serum samples, and 2 porcine foot-and-mouth disease virus A positive serum samples were detected to determine whether the established detection method has cross-reaction with antibodies of other major porcine viral pathogens.

[0095] The specificity detection results of the IgG and IgA antibody kit are shown in the following table (Table 7). The detection results of 30 healthy pig serum samples show that the specificity of the kit is 100.0%. The detection results of 2 classical porcine pestivirus positive serum samples, 2 porcine foot-and-mouth disease virus O positive serum samples, and 2 porcine foot-and-mouth disease virus A positive serum samples are all negative, so the specificity of the kit for detecting these 6 related pathogen positive serum samples is 100%.

[0096] Table 7 Specificity test results of the IgG and IgA antibody detection kit based on porcine rotavirus VP8 protein

[0097] Example 8: Reproducibility test Three batches of mammalian cell-expressed and purified porcine rotavirus VP8 truncated protein were coated at different times, and 3 positive serum samples and 3 negative serum samples were detected by ELISA according to the use method, with 4 repeats within the batch.450 nm The S / P value was calculated, and the average value, standard deviation and coefficient of variation were calculated to determine the repeatability within the batch and the repeatability between batches, and the results are shown in Table 8.

[0098] Table 8 Results of repeatability test of the IgG and IgA antibody detection kit based on porcine rotavirus VP8 protein

[0099] From Table 8, it can be seen that the porcine rotavirus IgG and IgA antibody detection kit established by the application has good repeatability.

[0100] Example 9: Coincidence rate test At present, there is a commercial IgG antibody detection kit for porcine rotavirus, and the recognized detection method for porcine rotavirus is virus neutralization test (VNT), and therefore the coincidence rate test is carried out using the commercial antibody detection kit, the self-developed kit and the virus neutralization test.

[0101] 1. Virus neutralization test (VNT) method (1) Materials: porcine serum to be tested (40 portions), porcine milk to be tested (30 portions), inactivated at 56°C for 30 min, provided by Zhongmu Industry Co., Ltd. The test virus is MARC-145 cell virus of porcine rotavirus, and the titer is adjusted to 400 TCID 50 / 0.1 mL, stored at -20°C for standby.

[0102] (2) Method: After inactivation of serum, the serum (milk) was diluted in a 1.5 mL EP tube with DMEM culture solution to make 2-fold dilution, and the dilution degrees were 1:2, 1:4, 1:8……1:1024, and the virus liquid was diluted with DMEM culture solution to 200 TCD 50 / 0.1 mL. Mix equal volume of different dilution of serum (milk) and diluted virus liquid, at the same time, mix equal volume of DMEM and diluted virus liquid as positive control, shake well and incubate at 37℃, 5% CO2 incubator for 1 h. Carefully wash the monolayer of MARC-145 cells in 96-well cell culture plate with 200 μL / well PBS for 3 times, discard the PBS. Add 100 μL serum (milk) and virus mixture to each well, 4 replicates for each dilution, at the same time, add 100 μL DMEM and 100 μL mixture of DMEM and diluted virus liquid as negative control and positive control, respectively, incubate at 37℃, 5% CO2 incubator for 1 h. Carefully wash the monolayer of MARC-145 cells with 200 μL / well PBS for 3 times, discard the PBS, add 100 μL DMEM medium containing 10 μg / mL trypsin to each well using an 8-channel pipette, incubate at 37℃, 5% CO2 incubator for 48-72 h to observe cytopathic effect (CPE). The last dilution with 90-100% CPE inhibition is considered as the virus neutralization test (VNT) titer, less than 1:8 is negative.

[0103] 2. Coincidence rate test results: The coincidence rate test results of pig serum detection (Table 9) showed that the sensitivity of the commercial porcine rotavirus IgG antibody ELISA detection kit for 40 test pig sera was 90.0%, the sensitivity of the virus neutralization test was 92.5%, 37 were consistent in the detection results of the two methods, and the coincidence rate of the commercial porcine rotavirus IgG antibody ELISA detection kit and the virus neutralization test was 92.5%.

[0104] The sensitivity of the self-developed porcine rotavirus IgG antibody ELISA detection kit for 40 test pig sera was 92.5%, the sensitivity of the virus neutralization test was 92.5%, 38 were consistent in the detection results of the two methods, the coincidence rate of the self-developed porcine rotavirus IgG antibody ELISA detection kit and the virus neutralization test was 95.0%, the result accuracy was higher, and higher than the coincidence rate of the commercial porcine rotavirus IgG antibody ELISA detection kit and the virus neutralization test, so it can be used for detection of IgG antibody of porcine rotavirus.

[0105] Table 9 Coincidence rate test results of commercial kit, self-developed porcine rotavirus IgG antibody detection kit and virus neutralization test

[0106] The coincidence rate test results of pig milk detection showed (Table 10) that the sensitivity of the self-developed pig rotavirus IgA antibody ELISA detection kit for 30 samples to be detected was 93.33%, and the sensitivity of the virus neutralization test was 96.67%. The results of 29 samples were consistent, the coincidence rate of the self-developed pig rotavirus IgA antibody ELISA detection kit and the virus neutralization test was 96.67%, the result accuracy was high, and therefore it could be used for detection of IgA antibody of pig rotavirus.

[0107] Table 10 Coincidence rate test results of self-developed pig rotavirus IgA antibody detection kit and virus neutralization test

Claims

1. A detection kit for IgG and IgA antibodies based on porcine rotavirus VP8 protein, characterized in that, The kit includes an enzyme-linked reaction plate coated with porcine rotavirus VP8 truncated protein and an enzyme-labeled secondary antibody.

2. The reagent kit according to claim 1, characterized in that, The amino acid sequence of the porcine rotavirus VP8 truncated protein is shown in SEQ ID NO.

1.

3. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 2, characterized in that: The enzyme-linked reaction (ELISA) plate coated with porcine rotavirus VP8 truncated protein was prepared using the porcine rotavirus VP8 truncated protein as the coating antigen and carbonate buffer as the coating solution. The coating concentration was 1.0 μg / mL, 100 μL / well. The plate was incubated at 2-8°C for 8-12 hours to allow the antigen to fully bind to the ELISA plate. Then, PBS buffer containing 10 mg / mL bovine serum albumin (BSA) at pH 7.4 was added at 300 μL / well. The plate was blocked at 37°C for 2-3 hours, spun dry, and stored at 4°C after drying.

4. The enzyme-linked immunosorbent assay kit according to claim 2, characterized in that: The enzyme-linked reaction plate is a detachable 96-well ELISA plate; the porcine rotavirus VP8 truncated protein is obtained through recombinant expression.

5. The reagent kit according to claim 7, characterized in that, The method for obtaining the porcine rotavirus VP8 truncated protein is as follows: the encoding gene of the VP8 truncated protein of the SC01 strain, the most dominant circulating strain of porcine rotavirus G9 subtype, is cloned into the pcDNA3.1 vector to obtain a recombinant expression vector expressing the His tag fused VP8 truncated protein, named pcDNA3.1-PoRV-VP8. Subsequently, pcDNA3.1-PoRV-VP8 is transiently transfected into a well-functioning monolayer of 293F cells, and the cell expression supernatant is collected. The supernatant is then purified by Ni-NTA column protein separation to obtain the porcine rotavirus VP8 truncated protein. The nucleotide sequence of the encoding gene of the VP8 truncated protein of the porcine rotavirus SC01 strain is shown in SEQ ID NO.2, and the amino acid sequence of the VP8 truncated protein of the SC01 strain is shown in SEQ ID NO.

1.

6. The enzyme-linked immunosorbent assay kit according to claim 2, characterized in that: The enzyme-labeled secondary antibodies consist of two samples: horseradish peroxidase-labeled rabbit anti-pig IgG antibody and horseradish peroxidase-labeled goat anti-pig IgA antibody.

7. The enzyme-linked immunosorbent assay kit according to claim 2, characterized in that: The kit also includes a positive control serum; the positive control is antibody-positive serum collected after immunization with a commercial porcine rotavirus inactivated vaccine, and used after appropriate dilution with sample diluent.

8. The enzyme-linked immunosorbent assay kit according to claim 2, characterized in that: The kit also includes a negative control serum; the negative control serum is porcine serum free of specific pathogens.

9. The enzyme-linked immunosorbent assay kit according to claim 2, characterized in that: The kit also includes substrate solution A, substrate solution B, and a stop solution; substrate solution A is citrate phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea, substrate solution B is a 0.2 mg / mL tetramethylbenzidine solution, and the stop solution is a 2 mol / L sulfuric acid solution.

10. The enzyme-linked immunosorbent assay kit according to claim 2, characterized in that: The kit also includes a sample diluent and a 20-fold concentrated wash buffer; the sample diluent is a 0.01 mol / L phosphate buffer containing 5 mg / mL casein at pH 7.4; the concentrated wash buffer is a 0.01 mol / L phosphate buffer containing 0.8%–1.2% Tween-20 at pH 7.4.

Images