PRRSV antibody time-resolved immunofluorescence detection kit and preparation method thereof

Abstract

The invention discloses a PRRSV antibody time-resolved immunofluorescence detection kit and a preparation method thereof. The kit includes a PRRSV antibody time-resolved immunofluorescence detection card; the detection card includes a PVC base plate and an absorption pad, a reaction membrane, a conjugation pad and a sample pad sequentially connected on the PVC base plate; the conjugation pad is loaded with a recombinant rGP5-M protein labeled with lanthanide ions and a sheep anti-chicken IgY labeled with lanthanide ions; the detection line T of the reaction membrane is coated with a recombinant rGP5-M protein, and the quality control line C is coated with a sheep anti-chicken IgY immunoglobulin. The present invention can quantitatively detect PRRSV antibodies, and has a positive correlation with the neutralizing antibody level, with a correlation coefficient of 0.955, and has high sensitivity, specificity and precision; the operation is simple and fast, which can greatly reduce human errors and increase the reliability of the test results.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products, and particularly relates to a PRRSV antibody time-resolved immunofluorescence detection kit and a preparation method thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by reproductive disorders in sows (abortion, premature birth, stillbirth and mummification), respiratory diseases in piglets and fattening pigs, which poses a serious threat to the pig industry and causes significant economic losses. Based on antigenic differences, PRRSV can be divided into European PRRSV (PRRSV 1) represented by Lelystad virus (LV) and American PRRSV (PRRSV 2) represented by VR2332 strain. The virus strain currently prevalent in my country is mainly PRRSV 2.

[0003] PRRSV was first discovered in commercial pigs in the United States in 1987, and the disease spread rapidly and broke out frequently around the world. my country isolated the virus in 1996. In the summer and autumn of 2006, highly pathogenic PRRSV (HP-PRRSV) characterized by a 30-amino acid deletion in NSP2 broke out in pig herds in many provinces in southern my country, causing a large number of pig deaths and causing significant economic losses to my country's pig industry. The prevention and control of PRRS is mainly based on vaccine immunization. The vaccines sold on the market are mainly attenuated vaccines, inactivated vaccines and a small amount of subunit vaccines. In order to evaluate the effectiveness of vaccines and monitor wild virus infections in pig farms, it is necessary to collect blood from different pig herds in pig farms for antibody testing. The antibody detection kits sold on the market are mainly two types of indirect ELISA kits. One is the nucleocapsid protein (N protein) encoded by the ORF7 gene. The N protein has strong immunogenicity and can stimulate the body to produce specific antibodies with a relatively long duration in the early stage after infection, but it has nothing to do with immune protection; the other is the GP5 protein encoded by the ORF5 gene. Although the GP5 protein can induce the body to produce neutralizing antibodies, the antigenic epitope (decoy epitope) that causes the strongest immune response has nothing to do with neutralizing antibodies, and the M protein of PRRSV can also induce the body to produce neutralizing antibodies. The antibody level measured by the existing kit is poorly correlated with the neutralizing antibody level, and cannot be used to evaluate the vaccine immunity level, let alone quantitatively detect the antibody level. Therefore, it is imperative to seek antigenic proteins that can evaluate the effectiveness of vaccines and establish accurate and quantitative experimental methods to replace the ELISA method.

[0004] Time-resolved immunofluorescence analysis (TRFIA) is a new type of non-radioactive immunolabeling technology characterized by the use of rare earth ion (mainly lanthanide ion) complexes to label antigens or antibodies, nucleic acid probes and cells. Rare earth complexes have longer fluorescence lifetimes (more than 100μs) than ordinary organic fluorescent markers. By detecting fluorescence with time differences, the background can be well eliminated, greatly improving the sensitivity of the measurement. TRFIA also has the characteristics of simple label preparation, no radioactive contamination, good detection repeatability, and a wide range of standard curves. It can be used for quantitative detection of antibodies or antigens. At present, no reports have been retrieved on the correlation analysis between PRRSV antibody detection and PRRSV neutralizing antibody levels, as well as the use of TRFIA to detect PRRSV antibodies. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the primary purpose of the present invention is to provide a PRRSV antibody time-resolved immunofluorescence detection kit. The present invention transforms the optimized PRRSV GP5 protein sequence and M protein sequence into CHO cells in series for expression, and the expressed recombinant rGP5-M protein is purified and used to prepare a PRRSV antibody time-resolved immunofluorescence detection kit, which can be used to measure the antibody level of PRRSV, and the measured antibody level is significantly positively correlated with the neutralizing antibody level of PRRSV, with high detection sensitivity, simple operation and short time consumption.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned PRRSV antibody time-resolved immunofluorescence detection kit.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A PRRSV antibody time-resolved immunofluorescence detection kit, comprising a PRRSV antibody time-resolved immunofluorescence detection card;

[0009] The PRRSV antibody time-resolved immunofluorescence detection card comprises a PVC base plate and an absorption pad, a reaction membrane, a binding pad and a sample pad sequentially connected on the PVC base plate; the sample pad is partially pressed on the binding pad, the binding pad is partially pressed on the reaction membrane, and the absorption pad is partially pressed on the reaction membrane;

[0010] The binding pad is loaded with recombinant rGP5-M protein labeled with lanthanide ions and goat anti-chicken IgY labeled with lanthanide ions;

[0011] The reaction membrane is provided with a detection line T and a quality control line C, wherein the detection line T is coated with recombinant GP5-M protein, and the quality control line C is coated with sheep anti-chicken IgY immunoglobulin.

[0012] The recombinant rGP5-M protein comprises rGP5 protein and M protein.

[0013] The rGP5 protein is obtained by removing amino acids 1-29 (signal peptide and decoy epitope) from the 3' end of the PRRSV GP5 protein, and its amino acid sequence is shown below (also as SEQ ID NO.2); preferably, it is obtained by CHO cell expression and purification.

[0014] MEFGLSWVFLVALFRGVQCADSNSSSHLQLIYNLTICELNGTDWLNEKFDWAVETFVIFPALTHIVSYGALTTSHFLDTVGLVAVSTAGYCHGRYVLSSIYAVCALAALICFTIRLAKNCMSWRYSCTRYTNFLQDTKGKLYRWRSPVIIEKGGKVDVEGHLIDLKRVVLDGSAATPITKISAEQWGRP.

[0015] The M protein is the 110-168th position of the PRRSV M protein, and its amino acid sequence is shown in SEQ ID NO.3; it is preferably obtained by expressing in CHO cells.

[0016] The rGP5 protein and the M protein are expressed in tandem fusion, and thus, the amino acid sequence of the recombinant rGP5-M protein is preferably as shown below (also as SEQ ID NO.1).

[0017] MEFGLSWVFLVALFRGVQCADSNSSSHLQLIYNLTICELNGTDWLNEKFDWAVETFVIFPALTHIVSYGALTTSHFLDTVGLVAVSTAGYCHGRYVLSSIYAVCALAALICFTIRLAKNCMSWRYSCT RYTNFLQDTKGKLYRWRSPVIIEKGGKVDVEGHLIDLKRVVLDGSAATPITKISAEQWGRPLAPAHHVESAAGFHPITASDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGKRAVKRGVVNLVHHHHHH.

[0018] The material of the absorbent pad is preferably absorbent paper.

[0019] The lanthanide ion is Eu 3+ , Tb 3+ 、Sm 3+ and Dy 3+ At least one of, preferably Eu 3+ .

[0020] The material of the reaction membrane is preferably nitrocellulose membrane.

[0021] The material of the binding pad is preferably glass cellulose membrane, and more preferably 8964 glass cellulose membrane.

[0022] The material of the sample pad is preferably glass fiber, preferably RB65 polyester fiber membrane.

[0023] The PRRSV antibody time-resolved immunofluorescence detection kit also includes one or both of a serum diluent and a standard positive serum.

[0024] The serum diluent is a phosphate buffer containing surfactant S21 and Proclin300; preferably, it is a 0.01M, pH 7.4 phosphate buffer containing 0.8% (v / v) surfactant S21 and 0.03% (v / v) Proclin300.

[0025] The standard positive serum is the serum of clean-grade experimental pigs immunized with PRRSV attenuated vaccine.

[0026] Compared with the prior art, the present invention has the following advantages and effects:

[0027] (1) The present invention discloses a time-resolved immunofluorescence detection kit for PRRSV antibodies, which can be used for quantitative detection and has a linear relationship in the range of 2 to 640 TU / ml.

[0028] (2) The technical solution provided by the present invention can be used to evaluate the titer of PRRSV vaccine antibodies. The detection value is positively correlated with the reciprocal mean value of the PRRSV neutralizing antibody (SN) titer, with a correlation coefficient of 0.955, which has a good correlation.

[0029] (3) The present invention discloses a time-resolved immunofluorescence detection method and kit for PRRSV antibodies, which can be used for the rapid detection of PRRSV antibodies, and only takes 15 minutes from sample addition to result output. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the structure of a reagent card with a cartridge provided by the present invention; wherein, 1-PVC base plate, 2-sample pad, 3-binding pad, 4-reaction membrane, 5-absorption pad, 6-cartridge, 7-sample addition hole, T is the detection line, and C is the quality control line.

[0031] Figure 2 This is a schematic diagram of the rGP5-M sequence provided by the present invention.

[0032] Figure 3This is the SDS-PAGE electrophoresis diagram of the rGP5-M protein provided by the present invention; wherein the left lane is the rGP5 protein purified by a nickel column, and the right lane is the protein Marker.

[0033] Figure 4 This is a western blotting identification result diagram of the rGP5 protein provided by the present invention; wherein lane 1 is rGP5-M protein, and lane M is protein Marker.

[0034] Figure 5 It is the standard curve of PRRSV antibody level. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0036] Unless otherwise specified, all reagents and raw materials used in the present invention can be purchased from the market or prepared by known methods.

[0037] Example 1

[0038] Preparation of recombinant rGP5-M protein

[0039] 1) Gene synthesis

[0040] According to the ORF5 and ORF6 gene sequences of PRRSV (GenBank: EF112445), the antigenic epitopes of GP5 and M proteins encoded by ORF5 and ORF6 genes were analyzed by the biological software DNAStar, the GP5 protein sequence (SEQ ID NO.2) without the signal peptide and decoy epitope was removed and replaced with the IgG signal peptide sequence, the M protein had two antigenic epitopes in similar positions (SEQ ID NO.3), and the codons were optimized according to the preference of CHO cells to obtain the gene sequences SEQ ID NO.5 and SEQ ID NO.6, and the gene sequence SEQ ID NO.4 was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] 2) Screening and identification of CHO cell lines stably expressing recombinant rGP5-M protein

[0042] Design 1 pair of primers:

[0043] Upstream amplification primer PRRSV-F: 5'-GAC GCGGCCGC ATGGAGTTTGGCTTGAGCT G-3' (SEQ ID NO.7);

[0044] Downstream primer PRRSV-R: 5'-GTC GGATCCTCAGTGGTGGTGGTGATGGT-3' (SEQ ID NO. 8);

[0045] The underlined part is the introduced restriction site, and Not I and BamH I restriction sites were introduced into PRRSV-F and PRRSV-R, respectively; at the same time, in order to facilitate the purification of the expressed recombinant protein, 6 histidine coding bases were introduced after the M protein, and the sequence was: CACCATCACCACCACCAC (SEQID NO.9). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0046] The synthesized gene sequence SEQ ID NO.4 was used as a template and PCR amplification was performed with primers PRRSV-F / PRRSV-R. The amplified PCR product was recovered by gel electrophoresis and then recovered by Not I / BamH I double enzyme digestion and electrophoresis. The product was connected with the eukaryotic expression vector pIRESpuro3 treated in the same way. The recombinant plasmid pIRES-rGP5-M was obtained by transformation and identification of the recombinant plasmid according to conventional methods.

[0047] 3) Construction of a CHO cell line stably expressing rGP5-M

[0048] The above-mentioned recombinant plasmid pIRES-rGP5-M was extracted and transfected into CHO-K1 cells, and pressure screening was carried out using F12 culture medium containing 6 μg / 1 mL puromycin and 10% v / v fetal bovine serum (fetal bovine serum and F12 culture medium were purchased from Gibico). Cell colonies were obtained after 2 weeks of screening, and a small amount of cells were taken to extract nucleic acid for PCR identification. The correct cell colonies were identified and then single-cell cloning was performed (clones were obtained by culturing one cell in each well of a 96-well cell culture plate for 2-3 rounds), and the cell line stably expressing rGP5-M protein was obtained and named CHO-rGP5-M cell line.

[0049] 4) Purification of recombinant rGP5-M protein

[0050] CHO-rGP5-M cell line in 75cm 2 The cells were cultured in F12 medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 After 3 days of culture in the incubator, the supernatant of 5 bottles of cultured cells was collected by centrifugation for protein purification. Nickel ion affinity chromatography was used for protein purification. After purification, SDS-PAGE and Western Blotting were used to identify a single band (such as Figure 2 and 3 The BCA method was used to determine the protein content after purification, and the concentration of the purified rGP5-M protein was 1.8 mg / mL.

[0051] Example 2

[0052] A PRRSV antibody time-resolved immunofluorescence detection kit (such as Figure 1 As shown), comprising a PRRSV antibody time-resolved immunofluorescence detection card and card shell 6.

[0053] The PRRSV antibody time-resolved immunofluorescence detection card includes a PVC base plate 1 and a sample pad 2, a binding pad 3, a reaction membrane 4 and an absorption pad 5 arranged on the PVC base plate from left to right. The sample pad is partially pressed on the binding pad, the binding pad is partially pressed on the reaction membrane, and the absorption pad is partially pressed on the reaction membrane. The binding pad 3 is loaded with PRRSV rGP5 protein labeled with lanthanide ions and sheep anti-chicken IgY labeled with lanthanide ions. The reaction membrane 4 is provided with a detection line T and a quality control line C. The detection line T is coated with recombinant rGP5-M protein. The quality control line C is coated with sheep anti-chicken IgY immunoglobulin.

[0054] The card shell 6 is arranged outside the time-resolved immunofluorescence detection card for PRRSV antibody detection. The card shell 6 is provided with a sample adding hole 7 and an observation hole. The sample adding hole 7 is located above the sample pad 2 and is used for adding the sample to the sample pad. The observation hole is located at the reaction membrane 4 and is used to observe the display results of the detection line T and the quality control line C.

[0055] The above-mentioned PRRSV antibody time-resolved immunofluorescence detection kit also includes a box body, a serum diluent bottle containing a serum diluent, a serum bottle containing a standard positive serum, and a serum bottle containing a standard negative serum. The time-resolved immunofluorescence detection card for PRRSV antibody detection, the serum diluent bottle containing a serum diluent, the serum bottle containing a standard positive serum, and the serum bottle containing a standard negative serum are placed inside the box body.

[0056] The preparation method of the above-mentioned PRRSV antibody time-resolved immunofluorescence detection kit comprises the following steps:

[0057] (1) Preparation of sample pad:

[0058] Prepare a sample pad working solution containing 1% (v / v) S9 surfactant, 0.5% (v / v) Triton X-100, and 0.05% (v / v) sodium azide in Tris (0.01M, pH 8.0); add 40 mL / sheet of RB65 polyester fiber membrane, spread each sheet evenly with a roller, dry at 37°C for 24 h, cut into 23 mm × 300 mm sizes with a cutter, put them into an aluminum film bag, add desiccant, and seal for use.

[0059] (2) Preparation of conjugate pad:

[0060] 1 mg of nano-lanthanide ion microspheres (purchased from PerkinElmer) were ultrasonically dispersed, diluted 10 times with buffer MES (0.1 M, pH 5), and added to a 2 mL EP tube; 10 mg of activator EDC and 10 mg of activator NHS were added, mixed, and activated for 15 min to 30 min; then centrifuged at 14000 r / min for 15 min; the supernatant was discarded, 1 mL of coupling buffer (0.1 M MES, pH 5) was added, and then evenly dispersed with ultrasound, and PRRSV rGP5-M protein or goat anti-chicken IgY was added dropwise while stirring. 100 μg, mix at room temperature for 1 hour, add blocking agent (0.01M, pH 7.4 phosphate buffer and 1% (w / v) BSA) to block for 1 hour, centrifuge at 14000r / min for 15min, remove the supernatant, add PB (0.01M, pH 7.4) to resuspend and disperse evenly, that is, the labeled PRRSVrGP5-M recombinant fusion protein and the labeled goat anti-chicken IgY.

[0061] Cut the glass fiber membrane 8964 into a size of 10 mm × 300 mm, mix the labeled PRRSV rGP5-M and the labeled goat anti-chicken IgY in a ratio of 1:10 (V:V), spray them onto the cut 8964 using a gold spray film scribing instrument at 3 μL / cm and 0.02 MPa, place them in a drying room at a temperature of (18-26) ° C and a humidity of ≤30% and dry overnight (16-18) h. After drying, quickly put them into an aluminum film bag, add a desiccant, seal them, and store them at 2-8 ° C for use.

[0062] (3) Preparation of reaction membrane:

[0063] PRRSV rGP5-M recombinant fusion protein and IgY were diluted to a concentration of 1 mg / mL with PB (0.01 M, pH 7.4) containing 3% (w / v) sucrose as the test line and quality control line solutions, and lines were drawn on the nitrocellulose membrane. The quality control line and the test line were marked with a marker at one end of the membrane. The distance between the quality control line and the test line was 5 mm, the distance between the T line and the lower edge of the nitrocellulose membrane was 10 mm, and the streaking concentration was 1 μL / cm.

[0064] After completion, place the sheet in a drying room with a temperature of 18-26°C and a humidity of ≤30% to dry overnight (16-18 hours). After drying, quickly put it into an aluminum film bag, add desiccant, seal it, and store it at 2-8°C for later use.

[0065] (4) Preparation of absorbent pad:

[0066] Cut the absorbent paper into 25mm×300mm size and set aside.

[0067] In the process of establishing the time-resolved immunofluorescence detection method, positive and negative standard serum was prepared to establish the effectiveness verification of the kit, the positive and negative judgment standard and to avoid human operation errors. The present invention prepared a standard PRRSV antibody positive control serum and a standard PRRSV antibody negative control serum for the optimization of experimental conditions and the determination of detection results.

[0068] (5) Preparation of serum diluent;

[0069] Prepare 0.01 M, pH 7.4 phosphate buffer containing 0.8% (v / v) surfactant S21 and 0.03% (v / v) Proclin 300 preservative as serum diluent for later use.

[0070] (6) Preparation of standard positive serum:

[0071] 1) Clean-grade experimental pigs (purchased from the Experimental Animal Center of Southern Medical University) aged 30-60 days were selected. One dose of PRRSV attenuated vaccine (JXA1-R) (purchased from Guangdong Animal Vaccine Supply Station) was injected intramuscularly. After an interval of 4 weeks, the same dose was used for the second immunization. Blood was collected from the anterior vena cava, serum was separated, and serum titer was detected by enzyme-linked immunosorbent assay. The antibody titer can reach more than 1:1000.

[0072] 2) Blood was collected from the anterior vena cava, and the serum was separated after precipitation. The supernatant was centrifuged at 3000 r / min for 15 min, and 0.01% thimerosal was added for preservation. The supernatant was packaged and stored at -20°C.

[0073] (7) Preparation of standard negative serum:

[0074] Clean-grade experimental pigs (purchased from the Experimental Animal Center of Southern Medical University) were used as blood collection pigs. Before blood collection, they were in good condition, with normal body temperature, appetite, and defecation. They were not immunized with any vaccine and had no other diseases. After disinfection, blood was collected from the anterior vena cava, serum was separated, and 1 / 10,000 thimerosal was added for preservation. 0.5 mL was dispensed into sterile tubes and stored at -20°C.

[0075] (8) Assembly of PRRSV antibody time-resolved immunofluorescence detection kit.

[0076] Lay the PVC base plate 1 with NC film flat on the work surface, stick the binding pad 4, sample pad 5 and absorption pad 2 respectively, cut them into 4.0 mm wide test strips with a strip cutter, put each test strip into a plastic card shell, put each reagent card in an aluminum film bag, add a 1g desiccant bag, and heat seal to make an immunofluorescence chromatography test card.

[0077] Example 3

[0078] Use the kit for detection.

[0079] 1) Tear open the aluminum foil bag seal of the time-resolved immunofluorescence test card for PRRSV antibody detection, take out the test card, and place it horizontally.

[0080] 2) Use a pipette to draw 20 μL of the sample to be tested and add it to the serum diluent and mix well.

[0081] 3) Pipette 100 μL of the mixed sample and add it to the sample well of the test card.

[0082] The specific operation methods are as follows: (Method 1 / Method 2 are optional for testing)

[0083] Method 1: Quick test: Select the "Quick Test" mode and put the IC card of the corresponding project into the card reading area of ​​the detector.

[0084] Click on the batch, the instrument will automatically read the IC card information, wait for the test card to react at room temperature for 10 minutes, then insert it into the instrument card slot steadily (with the sample hole end facing the card slot entrance), push it all the way in, click on the "measure" option, start the test, and display and print the results;

[0085] Method 2: Standard test: Select the "standard measurement" mode, put the IC card of the corresponding project into the card reading area of ​​the detector, click batch, the instrument automatically reads the IC card information, insert the test card smoothly into the card slot of the instrument (with the sample hole end facing the card slot entrance), push it all the way in, click the "measurement" option, the instrument automatically times, and when the timing ends, the automatic test display and print results.

[0086] 2. Analysis of test results

[0087] 1) The test is considered valid when the ratio of the OD value of the positive control serum (PC) to the fluorescence intensity value of the negative control serum (NC) must be greater than 2.0.

[0088] PC / NC ≥ 2.1;

[0089] Positive Cut Off = NC*2.1;

[0090] Where NC = negative control serum T / C value;

[0091] PC = positive control serum T / C value.

[0092] 2) When parallel testing is performed, the final T / C value used is the average of the two parallel tests.

[0093] When the T / C value of the serum sample is lower than the positive cut off value, the sample is negative for PRRSV antibodies.

[0094] When the T / C value of the serum sample is higher than the negative cut off value, the sample is positive for PRRSV antibodies.

[0095] 3) Linear range

[0096] PRRSV positive serum was diluted with negative serum in a 2-fold gradient; the test was repeated 3 times for each concentration; the reaction lasted for 10 minutes after adding the sample, and the test paper was placed in a time-resolved immunofluorescence quantitative analyzer to read the T / C value, and the curve fitting and concentration calculation were performed using the built-in standard curve.

[0097] The test results show that the kit has a good linear fitting relationship in the detection of 2-640TU / mL, r>0.9900. When the concentration increases to 1280TU / mL, the linear fitting r value is less than 0.9900. Therefore, the maximum detection range of this test can reach 640TU / mL. In the concentration range of 2-640TU / ml, the dose-response curve of the kit is Log(Y)=0.3045Log(X)+0.6789, R=0.9988(R2=0.9978), see Figure 5 .

[0098] 4. Minimum detection limit

[0099] Twenty negative sera were tested, and the mean T / C was calculated and substituted into the dose-response curve, which showed that the minimum detection limit of this method was 1.25 TU / mL.

[0100] 5. Precision

[0101] Take 1 sample of each serum collected from pigs immunized with PRRSV attenuated vaccine at 0, 2, 3, 4 and 5 weeks, and repeat the test 10 times for each serum using the test card of this method, calculate the coefficient of variation of the test concentration, and use the P / N value of the test result of the IDEXX test kit as a reference. The results are shown in Table 1, from which it can be seen that the coefficient of variation detected by this kit is less than 10%, so this detection method has a high precision.

[0102] Table 1 Precision test results

[0103]

[0104] Example 4

[0105] Correlation analysis between time-resolved immunofluorescence detection of PRRSV antibodies and neutralizing antibodies

[0106] 120 samples of swine sera immunized with PRRSV vaccine were collected for correlation analysis between the test antibody and neutralizing antibody. The neutralization test of PRRSV antibody was carried out according to the fixed virus amount dilution serum method. The correlation between the test value of the time-resolved immunofluorescence detection kit of PRRSV antibody and the neutralizing antibody titer was determined by statistical analysis. At the same time, a comparative test was carried out with imported ELISA kits coated with GP5 protein. The calculation formula of the correlation coefficient is as follows:

[0107]

[0108] x is the reciprocal of the average value of the neutralizing antibody titer of the serum sample; y is the test value of the antibody detection kit of the serum sample; n is the segmented group of the test value of the kit. If r>0, it means it is positively correlated; r<0, it means it is negatively correlated. Generally speaking, after taking the absolute value, 0-0.09 means no correlation, 0.1-0.3 means weak correlation, >0.3-0.5 means moderate correlation, and >0.5-1.0 means strong correlation.

[0109] The serum to be tested was inactivated at 56°C for 30 min, and diluted to 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256 with modified RPMI-1640 medium without serum. 50 μL of the serum to be tested at different dilutions was taken and diluted with an equal volume of 200 TCID 50 / 0.1mL virus solution was fully mixed, incubated at 37℃ for 1h, and then inoculated into Marc145 monolayer cells (the original cell culture medium was discarded); at the same time, serum toxicity control to be tested, negative and positive serum control, virus control and normal cell control were set up, of which the virus control was 200 TCID 50 , 20 TCID 50 , 2 TCID 50 , 0.2 TCID 50 The culture medium was set at 37°C and 5% CO. 2 Culture in an incubator, observe the cytopathic effect (3-4 days), make records, and calculate the neutralization titer according to the Reed-Muench method.

[0110] The test results of the time-resolved immunofluorescence detection kit are shown in Table 2. Among the 120 serum samples, the average value of 26 sera with PRRSV antibody values ​​greater than 640TU / mL was 742.74, and the corresponding reciprocal average value of serum neutralizing antibody titer was 36.52; the average value of 48 sera with PRRSV antibody values ​​between 320 and 640TU / mL (excluding 320) was 536.73TU / mL, and the corresponding reciprocal average value of serum neutralizing antibody titer was 15.47; the average value of 24 sera with PRRSV antibody values ​​between 160 and 320 was 256.34, and the corresponding reciprocal average value of serum neutralizing antibody titer was 7.32; the average value of 22 sera with PRRSV antibody values ​​less than 160 was 122.37TU / mL, and the corresponding reciprocal average value of serum neutralizing antibody titer was 2.27. The results show that the serum neutralizing antibody titer decreases with the decrease of the test value of the PRRSV antibody time-resolved immunofluorescence detection kit. Biostatistical analysis showed that the test value of the time-resolved immunofluorescence detection kit was positively correlated with the reciprocal mean value of the PRRSV neutralizing antibody (SN) titer, with a correlation coefficient of 0.955, showing a good correlation.

[0111] Table 2 Time-resolved immunofluorescence detection kit test values ​​and reciprocal averages of neutralizing antibody titers

[0112]

[0113] The same serum was tested in parallel using an imported ELISA kit coated with GP5 protein, and the results are shown in Table 3. Among the 120 serum samples, the average value of 43 sera with PRRSV antibody S / P values ​​greater than 3.5 was 3.645, and the corresponding reciprocal average value of serum neutralizing antibody titer was 22.29; the average value of 32 sera with PRRSV antibody S / P values ​​between 3.5 and 3.0 was 3.261, and the corresponding reciprocal average value of serum neutralizing antibody titer was 9.41; the average value of 27 sera with PRRSV antibody S / P values ​​between 2.5 and 3.0 was 2.842, and the corresponding reciprocal average value of serum neutralizing antibody titer was 6.36; the average value of 18 sera with PRRSV antibody S / P values ​​less than 2.5 was 1.627, and the corresponding reciprocal average value of serum neutralizing antibody titer was 2.64. The results show that the serum neutralizing antibody titer decreases as the test value of the ELISA detection kit decreases. Biostatistical analysis revealed that the test value of the ELISA test kit was positively correlated with the reciprocal mean value of the PRRSV neutralizing antibody (SN) titer, with a correlation coefficient of 0.828, which was significantly lower than that of the time-resolved immunofluorescence detection kit prepared by the present invention.

[0114] Table 3 ELISA test values ​​and reciprocal averages of neutralizing antibody titers

[0115]

[0116] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A PRRSV antibody time-resolved immunofluorescence detection kit, characterized in that: Includes PRRSV antibody time-resolved immunofluorescence test card; The PRRSV antibody time-resolved immunofluorescence detection card comprises a PVC base plate and an absorption pad, a reaction membrane, a binding pad and a sample pad sequentially connected on the PVC base plate; the sample pad is partially pressed on the binding pad, the binding pad is partially pressed on the reaction membrane, and the absorption pad is partially pressed on the reaction membrane; The binding pad is loaded with recombinant rGP5-M protein labeled with lanthanide ions and goat anti-chicken IgY labeled with lanthanide ions; The reaction membrane is provided with a detection line T and a quality control line C, wherein the detection line T is coated with recombinant GP5-M protein, and the quality control line C is coated with sheep anti-chicken IgY immunoglobulin; The recombinant rGP5-M protein comprises rGP5 protein and M protein; The amino acid sequence of the rGP5 protein is shown in SEQ ID NO.2; The amino acid sequence of the M protein is shown in SEQ ID NO.3; The amino acid sequence of the recombinant rGP5-M protein is shown in SEQ ID NO.

1.

2. The PRRSV antibody time-resolved immunofluorescence detection kit according to claim 1, characterized in that: Also included is one or both of a serum diluent and a standard positive serum.

3. The PRRSV antibody time-resolved immunofluorescence detection kit according to claim 2, characterized in that: The serum diluent is a 0.01M, pH 7.4 phosphate buffer solution containing 0.8% (v / v) surfactant S21 and 0.03% (v / v) Proclin 300; The standard positive serum is the serum of clean-grade experimental pigs immunized with PRRSV attenuated vaccine.

4. The PRRSV antibody time-resolved immunofluorescence detection kit according to claim 1 or 2, characterized in that: The lanthanide ion is Eu 3+ , Tb 3+ 、Sm 3+ and Dy 3+ At least one of .

5. The PRRSV antibody time-resolved immunofluorescence detection kit according to claim 1 or 2, characterized in that: The absorbent pad is absorbent paper; The material of the reaction membrane is nitrocellulose membrane; The material of the binding pad is glass cellulose membrane; The sample pad is made of glass fiber.

Images