CN108802384B - ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide - Google Patents
ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide Download PDFInfo
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Abstract
The invention discloses a porcine circovirus type 2 synthetic peptide ELISA antibody detection kit. The kit comprises an enzyme label plate, positive control serum, negative control serum and an enzyme-labeled secondary antibody, wherein the enzyme label plate is coated with a porcine circovirus type 2 epitope polypeptide composition. The antigen epitope polypeptide composition is one or a combination of two of a polypeptide shown in a sequence 1 in a sequence table and a polypeptide shown in a sequence 2 in the sequence table. The invention adopts indirect ELISA, uses chemically synthesized antigen peptide to coat the ELISA plate, has less antigen dosage and high sensitivity and specificity, and can efficiently detect whether the porcine circovirus type 2 antibody exists. The kit has the advantages of high sensitivity, good specificity, convenient operation and good market prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an ELISA antibody detection kit for porcine circovirus type 2 synthetic peptides.
Background
Porcine Circovirus (PCV) is the smallest animal virus currently found and is one of the important members of the genus circovirus of the family circoviridae. According to its antigenicity, nucleotide sequence and pathogenicity, it can be divided into two genotypes (genotypes): types 1 and 2, i.e., PCV1 and PCV 2. PCV1 is nonpathogenic and widely exists in pig bodies and pig-derived continuous cell lines, PCV2 is one of the pathogens which are extremely harmful to the pig industry in the world at present, brings huge economic loss to the pig industry all over the world, and can cause Postweaning Multisystemic Wasting Syndrome (PMWS), piglet congenital tremor, Pig Dermatitis and Nephrotic Syndrome (PDNS), pig respiratory syndrome (PRDC), sow reproductive disorder and intestinal diseases, which are Porcine circovirus diseases (PCVDs), researches show that PCV2 can proliferate in the Porcine lymphatic system, can cause apoptosis of immune cells, cause immunosuppression and cause body resistance reduction, thereby inducing mixed infection and secondary infection of various bacteria and viruses and huge economic loss in the pig industry.
PCV is an unencapsulated, single-stranded circular DNA virus, mature virions are assembled into a spherical particle with a diameter of 17-20 nm by 60 nucleocapsid protein (Cap) subunits, the molecular weight is 580kDa, the sedimentation coefficient is 52S, the genome of the virus is wrapped in the particle, the whole length is about 1760bp, the particle contains 11 reading frames (ORFs), the neck-loop structure related to rolling-circle replication (RCR) is provided, and the particle contains two main Open Reading Frames (ORFs): ORF1 and ORF2, encoding the viral replication associated protein (rep) and capsid protein (cap), respectively. Rep has high amino acid homology (314aa, about 36kDa), and Cap protein (233aa, about 27kDa) is the only structural protein and main antigen, and can induce the body to generate effective immune protection response. PCV2, when cultured in vitro, replicates in PK-15 cells and swine-derived immune cells, but does not produce Cytopathic effects (CPE).
The PCV2 antibodies are detected by many serological methods, and currently, ifa (infectious immunofluorescent assay), ipma (immunoperoxidase monolayer assay), ELISA and the like are mainly used, wherein ELISA methods are more commonly used because of their convenience, rapidity and large-scale detection, and mainly include indirect ELISA, competitive ELISA and double-antigen sandwich ELISA. In recent years, with the continuous and deep research of epitopes, synthetic peptides are not only used for the research of circovirus epitope vaccines, but also used for the screening of envelope antigens in ELISA reactions, and the research shows that the PCV2Cap protein contains a plurality of epitopes, such as 92-103 aa, 156-162 aa, 175-192 aa, 195-201 aa, 231-233 aa and the like, which are all key epitopes, and on the basis of the epitope research, an indirect ELISA detection kit for detecting porcine circovirus type 2 antibodies based on the synthetic peptide envelope antigens is established.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting porcine circovirus type 2 antibodies, which utilizes porcine circovirus type 2 epitope polypeptide as a coating antigen to establish an indirect ELISA method with good specificity, sensitivity and repeatability and is used for detecting whether porcine circovirus type 2 antibodies are contained in porcine serum.
In order to achieve the purpose, the porcine circovirus type 2 epitope polypeptide composition with excellent performance is obtained by screening, and the porcine circovirus type 2 epitope polypeptide composition provided by the invention is one or a combination of two of a polypeptide shown in a sequence 1 in a sequence table and a polypeptide shown in a sequence 2 in the sequence table. When the polypeptide composition is a polypeptide shown in a sequence 1 and a polypeptide shown in a sequence 2, the mass ratio of the two polypeptides is (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1: 1.
the polypeptide sequence shown in the sequence 1 is from left to right from an amino terminal to a carboxyl terminal, and the carboxyl of the 17 th amino acid from the amino terminal and the epsilon amino of the 19 th amino acid form peptide bond connection.
Wherein, the epsilon K is a peptide bond formed by an epsilon-site amino group of lysine, and the specific principle is as follows:
each amino acid comprising at least one "-COOH" (carboxyl) and one "-NH group3"(amino group), the carboxyl group and the amino group bonded to the α carbon atom are referred to as" α carboxyl group "and" α amino group ". The general peptide synthesis reaction is a reaction in which the" α amino group "of the previous amino acid and the" α carboxyl group "of the next amino acid are reacted to form an amide bond (peptide bond).
Lys (lysine) is a basic amino acid with an amino group at the epsilon position in addition to the normal "α amino group" and "α carboxyl group", as shown in the following formula.
ε K specifies that the amino groups involved in the peptide-forming reaction are "ε amino" rather than "α amino" and, because of their long arm structure, are used to link epitopes that need to be used together but do not affect each other.
The porcine circovirus type 2 epitope polypeptide is also within the protection range of the invention, and the porcine circovirus type 2 epitope polypeptide is a polypeptide shown as a sequence 1 in a sequence table or a polypeptide shown as a sequence 2 in the sequence table.
The ELISA antibody detection kit for the porcine circovirus type 2 synthetic peptide comprises an enzyme-linked reaction plate, positive control serum, negative control serum and an enzyme-labeled secondary antibody, wherein the enzyme-linked reaction plate is coated with a porcine circovirus type 2 epitope polypeptide composition.
The enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the polypeptides in the porcine circovirus type 2 epitope polypeptide composition are obtained by chemical artificial synthesis.
The optimal preparation method and conditions of the enzyme-linked reaction plate are that the porcine circovirus type 2 epitope polypeptide composition is dissolved in a carbonate solution with the pH value of 9.6, then the mixture is added into a 96-hole polystyrene enzyme-linked reaction plate, 100ng of polypeptide (wherein 50ng of polypeptide shown in a sequence 1 in a sequence table and 50ng of polypeptide shown in a sequence 2 in the sequence table) is added into each hole, the mixture is placed at the temperature of 2-8 ℃ for 8-12 hours, so that the polypeptide antigen is fully combined with the enzyme-linked reaction plate, then PBS buffer solution containing 1% (g/ml) Bovine Serum Albumin (BSA) with the pH value of 7.4 is added into each hole according to 300 mu l, the mixture is subjected to sealing treatment at the temperature of 37 ℃ for 2-3 hours, and after being dried, the enzyme-linked reaction plate is sealed and stored.
The positive control serum is porcine serum collected after the porcine circovirus type 2 inactivated virus is immunized; the negative control serum is porcine serum free of specific pathogens (SPF, porcine circovirus free).
The enzyme-labeled secondary antibody is a horse radish peroxidase-labeled rabbit anti-pig IgG antibody.
The kit also comprises a substrate solution A, a substrate solution B and a stop solution, wherein the substrate solution A is a citrate phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide, the substrate solution B is a tetramethylbenzidine solution of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when in use. The stop solution is a 2mol/L sulfuric acid solution.
The kit also includes a sample diluent and a concentrated wash (20-fold); the sample diluent was 0.01M, pH value 7.4 phosphate buffer containing 0.5% (g/ml) casein; the concentrated washing solution is 0.01M phosphate buffer solution with pH value of 7.4 and contains Tween-20 with concentration of 0.8-1.2% (ml/ml).
The detection program of the kit of the invention is as follows:
1. balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2. Preparing liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3. setting: 2 negative control holes and 2 positive control holes, and the rest are sample holes to be detected.
4. Pre-diluting a sample to be detected: and diluting the serum of the sample to be detected, the negative control serum and the positive control serum by using a sample diluent according to the proportion of 1: 200.
5. Sample adding: each well was pre-diluted with 100. mu.l of the test sample. The time span of the sample application process should be as short as possible.
6. And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 30 min.
7. Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing buffer solution into each well, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
8. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
9. And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
10. Washing the plate: discarding the reaction solution, adding 300 mu l of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
11. Adding 100 μ l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, and is prepared at present), shaking, mixing, placing in 37 deg.C incubator, and reacting for 15min in dark.
12. 50. mu.l of chromogenic stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well450nmValue (reaction plate with stop solution should read OD within 15min450nmValue).
And (3) judging a detection result:
1. negative control OD450nmThe average value should be less than or equal to 0.15, otherwise it is not effective.
2. Each test value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid.
3. Calculation of the critical value: critical value 0.18 × positive control OD450nmValue average.
Determination of OD in serum to be examined450nmIf the value is larger than or equal to the critical value, judging the value as positive; determination of OD in serum to be examined450nmValue of<The critical value is judged to be negative.
The kit can be used for detecting the porcine circovirus type 2 antibody.
The application in the preparation of the kit for detecting whether the porcine circovirus type 2 disease is infected or not and detecting the antibody level of the porcine circovirus type 2 vaccine after immunization also belongs to the protection range of the invention;
the invention has the positive effects that: the invention adopts a bioinformatics method to accurately analyze the epitope of the porcine circovirus type 2Cap protein and screen out peptide fragments suitable for ELISA detection. The peptide segments shown in the sequence 1 and the sequence 2 concentrate the epitope, and have the advantages of high sensitivity and strong specificity.
Furthermore, when the composition consisting of the peptide fragments of the sequence 1 and the sequence 2 is used as a coating antigen, the effect is greatly better than that of singly using one polypeptide.
Meanwhile, the advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, because the coating antigen used in the kit is chemically synthesized polypeptide, the kit does not contain foreign protein and has high purity, the efficiency of detecting the porcine circovirus type 2 antibody is further improved.
In a word, the kit adopts the antigen peptide chemically synthesized from the main antigen site of the porcine circovirus type 2Cap protein to coat the enzyme-linked reaction plate, has the advantages of small antigen dosage, high sensitivity and strong specificity, and can effectively detect the Cap protein antibody generated after the porcine circovirus type 2 is infected or immunized. The experimental result shows that the kit has good repeatability, strong specificity and high sensitivity. Can meet the requirements of personnel at different levels, and has wide market prospect and good economic and social benefits.
The diagnosis kit for the porcine circovirus type 2 enzyme-linked immunosorbent assay is used for detecting the antibody of Cap protein generated after the immunization of porcine circovirus type 2 vaccines.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of envelope antigen of porcine circovirus type 2 synthetic peptide ELISA antibody detection kit
The invention researches the variation condition of the main antigenic sites of the porcine circovirus type 2 by sequence determination of the domestic porcine circovirus type 2 recent epidemic strains and combining with the sequence of the porcine circovirus vaccine strain, counts the variation frequency of the main variation amino acid sites, and analyzes and predicts the foot-and-mouth disease antigenic sites with the assistance of a computer, and chemically synthesizes possible antigenic site peptide segments, namely uses different amino acids at the sites according to the statistical variation frequency aiming at the easy variation sites, thereby obtaining a plurality of candidate polypeptide antigens covering all the possible variation sites at present. And screening the candidate polypeptide antigens through a large number of serological tests to obtain the porcine circovirus type 2 epidemic virus polypeptide antigen which can react with most of positive sera and has good sensitivity and specificity.
The synthetic peptide antigen of the invention which is screened and identified can be prepared by a Merrifield solid phase synthesis method by using an American PSI300 full-automatic polypeptide synthesizer, wherein an amino acid modified by 9-fluorenylmethyloxycarbonyl (Fmoc) is adopted, and a solid phase carrier is Rink Amide MBHA resin from Tianjin south China. The production process generally comprises solid phase synthesis of polypeptide antigen, cleavage of polypeptide, purification of antigen and aseptic storage.
First, solid phase synthesis of envelope antigen
1. Preparation of synthetic starting materials
The synthetic envelope antigen amino acid sequence is as follows:
SEQ ID NO:1：
RLSTIGRYTVKRTTVRT-εK-GTGSITQGDRVAVILYDPYNVYSSRDDNFTVKATALT；
wherein, the epsilon K is a peptide bond formed by an epsilon-site amino group of lysine and a carboxyl group of a 17 th-site amino acid.
SEQ ID NO:2：
YHSKYFPTRPVIDKTIDYFQPANKRNQLGNIKIYEADRINLKDPPLFPKTMYVGFRGF；
Appropriate Fmoc-modified amino acids (purchased from gille, shanghai, biochemistry) were prepared according to the envelope antigen sequence and synthesis scale and added to the corresponding amino acid reagent bottles. Rink Amide MBHA resin was also weighed as required, placed in the reaction chamber, the upper and lower caps were tightened, the label was attached, and the name of the synthesized peptide, the batch number, the tare weight of the reaction chamber, and the weight of the resin were recorded. The reaction chamber was loaded into the synthesizer. Preparing synthetic reagents, including 100% of N-methylpyrrolidone (NMP), 3% of Acetyl Imidazole (AIM), 35% of piperidine (PIP), 100% of methanol and the like, and placing the synthetic reagents into corresponding reagent bottles.
2. Synthesizer state detection
The PSI300 polypeptide synthesizer was checked for proper operation. In addition, it is checked whether N2 is sufficient and the system gauge pressure is normal. The flow rate of each synthesis reagent is measured because the performance of the instrument is known prior to synthesis. A to RV, B to RV, C to RV, D to RV, E to RV, F to RV, A to AA, C to AA were run, respectively. And (4) measuring or observing, and if the flow is not proper, adjusting the pressure of a lower valve or modifying parameters of each solution until the requirements are met.
3. Initiation of Synthesis of synthetic peptide antigens
Editing Sequence according to the synthesis Sequence in the Program of the synthesizer, editing specific synthetic Program, and finally combining the Sequence and the Program into a runfile for storage. And opening the running file, setting the preparation quantity of the amino acid bottles, and starting the running.
4. Synthesis of synthetic peptide antigens
The polypeptide sequence is synthesized by starting from the C-terminal to the N-terminal, and repeating the following steps in sequence according to a given sequence:
(1) deprotection reaction: placing the amino resin in NMP solution containing 15-30% of piperidine by volume percent, and reacting at 20-28 ℃ for 25-40 min to remove Fmoc protective groups on the amino resin;
(2) washing: drying with nitrogen, and washing amino resin with NMP;
(3) condensation reaction: adding HOBT, DIC and Fmoc protected amino acid, and reacting at 20-28 deg.C for 0.5-2.5 h;
(4) washing: drying with nitrogen, and washing amino resin with NMP;
(5) and (3) blocking reaction: adding 1.5-4% by weight of an NMP solution of acetyl imidazole according to volume percentage, and reacting for 20-40 min at the temperature of 20-28 ℃.
5 synthetic peptide antigen Synthesis termination
The synthesizer will automatically stop after the synthesis of the antigen is finished. And then taking the reactor off the polypeptide synthesizer, washing the polypeptide resin for 3 times by using 100% methanol, then drying the polypeptide resin in a fume hood, transferring the polypeptide resin into a brown bottle, putting the brown bottle into a refrigerator with the temperature of 20 ℃ below zero, and sealing a sealing film for later use.
Second, cleavage and identification of synthetic peptide antigens
1. Cleavage of synthetic peptide antigens
Preparing a lysate according to a volume ratio (trifluoroacetic acid (TFA)/Triisopropylsilane (TIS)/phenol/H2O ═ 85/8/6/1), taking out the synthesized polypeptide resin from a refrigerator, placing the polypeptide resin into a round-bottom flask, adding the prepared lysate and a magnetic stirrer into the flask in a fume hood, then placing the flask on a magnetic stirrer stably, and continuously stirring for 1H at room temperature until the reaction is complete. After the reaction is finished, continuously evaporating for 30-120 min by using a rotary evaporator with a cold trap to remove TFA in the crude product. And collecting and precipitating the polypeptide by using ether, washing a crude product of the polypeptide antigen for multiple times by using Dimethylformamide (DMF), and finally filtering the mixed resin by using a sand core funnel to obtain the polypeptide antigen.
2. Identification of synthetic peptide antigens
After the polypeptide antigen is synthesized, qualitative and quantitative analysis is carried out by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC).
Thirdly, purification and sterilization of synthetic peptide antigen
The synthetic peptide antigen is ultrafiltered by a circulating tangential filtration membrane at the temperature of 20-28 ℃ (Tangentialflow Device circulating tangential filtration membrane and a peristaltic pump matched with the Tangentialflow Device circulating tangential filtration membrane), the polypeptide antigen is a filtration membrane through which macromolecules can not pass through a certain aperture, and micromolecular impurities formed or introduced in the early synthesis process and the later cyclization reaction can pass through the filtration membrane. Then sterilizing through a filter with the pore diameter of 0.2 mu m, subpackaging the finally obtained solution into sterile plastic bottles, and labeling. The name, serial number, production lot number, concentration, production date, storage life and storage condition of the polypeptide are marked on the label, and the label is subpackaged and stored at-20 ℃ or-40 ℃ for later use.
Fourthly, freeze drying of the envelope antigen
For ease of transport and long term storage requirements, the polypeptide antigen is freeze-dried to obtain the polypeptide in a solid state. And taking out the pre-frozen polypeptide antigen, and drying on a Labconco freeze dryer to obtain the polypeptide antigen in a solid state. And simultaneously labeling. The name, number, production lot number, concentration, production date, shelf life and storage conditions of the polypeptide are indicated on the label.
Example 2 preparation of ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide
The porcine circovirus type 2 synthetic peptide ELISA antibody detection kit comprises:
(1) a 96-hole detachable polystyrene enzyme-linked reaction plate coated with porcine circovirus type 2 antigen; 2X 96 wells.
(2) Positive control serum: porcine serum collected after porcine circovirus type 2 inactivated virus immunization is used as positive control serum (1 tube, 1.5 ml/tube) of the kit.
(3) Negative control serum: is Specific Pathogen Free (SPF) pig serum as negative control serum for the kit (1 tube, 1.5 ml/tube).
(4) Enzyme-labeled secondary antibody: the preparation method is characterized in that horseradish peroxidase-labeled rabbit anti-pig IgG (purchased from sigma company, product number A5670) is used as a stock solution to be diluted by 1:30000 to prepare 2 bottles (12 ml/bottle).
(5) Sample diluent: 1 vial (24 ml/vial) of 0.01M, pH value 7.4 phosphate buffer containing 0.5% (g/100ml) casein.
(6) Substrate solution A: is citrate phosphate buffer solution (1 bottle, 12 ml/bottle) containing 0.6mg/ml urea hydrogen peroxide
(7) Substrate solution B: a 0.2mg/ml solution of Tetramethylbenzidine (TMB) (1 vial, 12 ml/vial).
(8) Stopping liquid: 2mol/L sulfuric acid solution (1 bottle, 12 ml/bottle).
(9)20 times of concentrated washing solution: is 0.01M phosphate buffer solution (50 ml/bottle, 2 bottles) with pH value of 7.4 and containing Tween-20 with concentration of 0.8-1.2% (ml/ml).
Serum dilution plates (2, 96 wells/block) may also be present in the kit for dilution of serum samples, as desired.
The preparation method of the 96-hole detachable polystyrene enzyme-linked reaction plate coated with the porcine circovirus type 2 antigen comprises the following steps: 1. the polypeptide antigen prepared in example 1 is dissolved in a carbonate solution with pH of 9.6, and then added to a 96-well polystyrene enzyme-linked reaction plate, wherein 100ng of polypeptide is added to each well (when a single peptide is used as a coating antigen, 100ng of polypeptide shown in sequence 1 and 100ng of polypeptide shown in sequence 2; when the polypeptide shown in sequence 1 and the polypeptide shown in sequence 2 are mixed to be used as the coating antigen, 50ng of polypeptide shown in sequence 1 in a sequence table and 50ng of polypeptide shown in sequence 2 in a sequence table), the mixture is placed at 2-8 ℃ for 8-12 hours to enable the polypeptide antigen to be fully combined with the enzyme-linked reaction plate, then PBS buffer solution containing 1% (g/ml) albumin (BSA) and pH7.4 is added according to 300 mu l/well, the mixture is sealed at 37 ℃ for 2-3 hours, and the mixture is dried, and then the enzyme-linked reaction plate is sealed and stored at 4 ℃.
Example 3 sensitivity test of ELISA antibody detection kit for synthetic peptide of porcine circovirus type 2
Use method of porcine circovirus type 2 synthetic peptide ELISA antibody detection kit
1. Balancing: taking out the kit from the refrigeration environment, and standing at room temperature for balancing for 30min for later use; the liquid reagents were mixed well before use.
2. Preparing liquid: diluting the concentrated washing solution by 20 times of distilled water or deionized water to obtain a washing buffer solution;
3. setting: 2 negative control holes and 2 positive control holes, and the rest are sample holes to be detected.
4. Pre-diluting a sample to be detected: and diluting the serum of the sample to be detected, the negative control serum and the positive control serum by using a sample diluent according to the proportion of 1: 200.
5. Sample adding: each well was pre-diluted with 100. mu.l of the test sample. The time span of the sample application process should be as short as possible.
6. And (3) incubation: shaking and mixing evenly, placing in an incubator at 37 ℃ and reacting for 30 min.
7. Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing buffer solution into each well, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
8. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
9. And (3) incubation: the reaction mixture was placed in an incubator at 37 ℃ and reacted for 30 min.
10. Washing the plate: discarding the reaction solution, adding 300 mu l of diluted washing buffer solution into each hole, soaking for 15s, throwing away the washing solution, continuously washing the plate for 4 times, and then drying by beating.
11. Adding 100 μ l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, and is prepared at present), shaking, mixing, placing in 37 deg.C incubator, and reacting for 15min in dark.
12. 50. mu.l of chromogenic stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well450nmValue (reaction plate with stop solution should read OD within 15min450nmValue).
And (3) judging a detection result:
1. negative control OD450nmThe average value should be less than or equal to 0.15, otherwise it is not effective.
2. Each test value of the positive control should be between 1.0 and 2.5, otherwise, the positive control is invalid.
3. Calculation of the critical value: critical value 0.18 × positive control OD450nmValue average.
Determination of OD in serum to be examined450nmIf the value is larger than or equal to the critical value, judging the value as positive; determination of OD in serum to be examined450nmValue of<The critical value is judged to be negative.
Second, sensitivity test to known positive serum
The sensitivity test of 30 porcine circovirus type 2 infected porcine sera (animals with PDNS and abortion symptoms and positive disease material identified by PCV2-Cap protein amplified by RT-PCR) was performed by using the porcine circovirus type 2 synthetic peptide ELISA antibody detection kit prepared according to the method of example 2 (the polypeptide shown in sequence 1, the polypeptide shown in sequence 2 or the mixed peptide is used as coating antigen), and the test results are shown in Table 1, the sensitivity of the kit with sequence 1 as coating antigen to 30 known positive sera is 86.7%, the sensitivity of the kit with sequence 2 as coating antigen to 30 known positive sera is 83.3%, and the sensitivity of the kit with mixed 2 peptides as coating antigen to 30 known positive sera is 96.7%.
TABLE 1 sensitivity test results of ELISA antibody test kit for porcine circovirus type 2 synthetic peptide
| Kit batch number | Detection rate | Sensitivity of the composition |
| Sequence 1 Single peptide | 26/30 | 86.7％ |
| Sequence 2 Monopeptide | 25/30 | 83.3％ |
| 2 peptide mixture peptides | 29/30 | 96.7％ |
Third, the minimum detection limit test
Selecting 1 part of porcine serum positive to the porcine circovirus type 2 antibody, diluting by a multiple ratio of 1:20, 1: 40-1: 2560, detecting the porcine serum diluted by the multiple ratio by using the porcine circovirus type 2 synthetic peptide ELISA antibody detection kit prepared in the embodiment 2 (respectively using the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 or the mixed peptide as a coating antigen) according to the using method of the porcine circovirus type 2 synthetic peptide ELISA antibody detection kit, wherein the result shows that the kit prepared by using the polypeptide shown in the sequence 1 can detect the positive serum diluted by the 1:160 time, the kit prepared by using the polypeptide shown in the sequence 2 can detect the positive serum diluted by the 1:40 time, and the kit prepared by using the mixed peptide can detect the positive serum diluted by the 1:320 time. In table 2, positive control: porcine serum collected after porcine circovirus type 2 virus inactivated virus immunization is taken as a positive pair of the kitControl serum (1 tube, 1.5 ml/tube). Negative control: is Specific Pathogen Free (SPF) pig serum as negative control serum for the kit (1 tube, 1.5 ml/tube). The Cut-off value (Cut-off value) was 0.18 Xthe OD of the positive control450nmValue average.
TABLE 2 detection results of the minimum detection limit test of the ELISA antibody detection kit for porcine circovirus type 2 synthetic peptides
Example 4 specificity test of ELISA antibody detection kit for synthetic peptide of porcine circovirus type 2
Three lots of kits (ZPCV 2001, ZPCV 2002, ZPCV 2003) in which mixed polypeptides (50 ng of polypeptide of sequence 1, 50ng of polypeptide of sequence 2) were prepared as a coating source according to the method described in example 2 were used to detect 200 parts of healthy pig serum (supplied from Lanzhou biopharmaceutical factory, Meditherd Utility Co., Ltd.), 2 parts of positive serum for porcine foot-and-mouth disease A (supplied from Lanzhou biopharmaceutical factory, Meditherd Utility Co., Ltd.), 2 parts of positive serum for porcine foot-and-mouth disease O (supplied from Lanzhou biopharmaceutical factory, Meditherd Utility Co., Ltd.), 2 parts of swine fever positive serum (China veterinary institute) and 2 parts of positive serum for porcine reproductive and respiratory syndrome virus (PRRS) (China veterinary institute).
The results of the specific detection of the kit are shown in the following table (table 3). The detection results of 200 healthy pig sera show that the specificity of the ZPCV 2001 kit is 99.0%, the specificity of the ZPCV 2002 kit is 99.5%, and the specificity of the ZPCV 2003 kit is 99.0%. The detection results of 2 parts of Asia type A positive serum of the pig foot-and-mouth disease, 2 parts of O type positive serum of the pig foot-and-mouth disease, 2 parts of swine fever positive serum and 2 parts of positive serum of the porcine reproductive and respiratory syndrome virus (PRRS) are all shown to be negative, so that the detection specificity of the three kits to the 10 parts of the pig positive serum is 100 percent.
TABLE 3 specificity test results of ELISA antibody test kit for porcine circovirus type 2 synthetic peptides
Example 5 compliance Rate test of porcine circovirus type 2 synthetic peptide ELISA antibody detection kit
The 3 kits in the embodiment 4 are randomly extracted into 1 kit, 48 immune pig sera and 48 pig sera from different pig farms are respectively detected by the kit and commercial antibody detection kits of the same type at home and abroad, the results are shown in table 4, the results show that the detection results of the 3 kits for 48 immune pig sera are consistent and are all positive, the results of the pig sera from the 48 different pig farms are slightly different, the overall coincidence rate of the kit and the domestic commercial kit is 91.7%, and the overall coincidence rate of the kit and the foreign commercial kit is 88.5%.
TABLE 4 detection results of ELISA antibody detection kit compliance rate of porcine circovirus type 2 synthetic peptide
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
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<120> ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide
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Claims (8)
1. A porcine circovirus type 2 synthetic peptide ELISA antibody detection kit comprises an enzyme-linked reaction plate coated with a porcine circovirus type 2 epitope polypeptide composition as an antigen, positive control serum, negative control serum and an enzyme-labeled secondary antibody; the porcine circovirus type 2 epitope polypeptide composition consists of a polypeptide shown as a sequence 1 in a sequence table and a polypeptide shown as a sequence 2 in the sequence table; wherein, the polypeptide shown in the sequence 1 is connected with the epsilon-site amino group of the 18 th site amino acid through a peptide bond formed by the carboxyl group of the 17 th site amino acid from the amino terminal.
2. The ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide according to claim 1, wherein the mass ratio of the polypeptide shown in the sequence 1 in the sequence table to the polypeptide shown in the sequence 2 in the sequence table is (0.5-1.5): (0.5 to 1.5).
3. The ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide according to claim 2, wherein the mass ratio of the polypeptide shown by the sequence 1 in the sequence table to the polypeptide shown by the sequence 2 in the sequence table is 1: 1.
4. The kit for detecting the porcine circovirus type 2 synthetic peptide ELISA antibody of claim 3, wherein the preparation method of the enzyme-linked reaction plate comprises the steps of dissolving the porcine circovirus type 2 epitope polypeptide composition in 100 μ l of a carbonate solution with pH of 9.6, adding the solution into a 96-hole polystyrene enzyme-linked reaction plate, placing 100ng of the porcine circovirus type 2 epitope polypeptide composition in each hole at 2-8 ℃ for 8-12 hours to fully combine the porcine circovirus type 2 epitope polypeptide composition with the enzyme-linked reaction plate, adding a PBS buffer solution containing 0.01g/ml of bovine serum albumin with pH of 7.4 into each hole according to 300 μ l, sealing the solution at 37 ℃ for 2-3 hours, drying the enzyme-linked reaction plate, and sealing and storing the enzyme-linked reaction plate at 4 ℃.
5. The ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide according to claim 3, wherein the positive control serum is porcine serum collected after immunization with porcine circovirus type 2 inactivated virus; the negative control serum is porcine serum without porcine circovirus.
6. The ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide according to claim 3, wherein the enzyme-labeled secondary antibody is a horse radish peroxidase-labeled rabbit anti-porcine IgG antibody.
7. The ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide, according to claim 3, characterized in that the kit further comprises a substrate solution A, a substrate solution B and a stop solution, wherein the substrate solution A is a citrate phosphate buffer solution containing 0.6mg/ml urea hydrogen peroxide, the substrate solution B is a tetramethylbenzidine solution of 0.2mg/ml, and the substrate solution A, the substrate solution B and the stop solution are mixed in a ratio of 1:1 when in use; the stop solution is a 2mol/L sulfuric acid solution.
8. The ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide according to claim 3, wherein the kit further comprises a sample diluent and a concentrated washing solution; the sample diluent is a phosphate buffer solution with a value of 7.4 and 0.01mol/L, pH containing 0.005g/ml casein; the concentrated washing solution is 0.01mol/L phosphate buffer solution containing 0.8-1.2% of Tween-20 by volume percentage and with the pH value of 7.4.
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