CN110208542A - A kind of the ELISA antigen detection kit and its detection method of porcine circovirus 2 type - Google Patents

A kind of the ELISA antigen detection kit and its detection method of porcine circovirus 2 type Download PDF

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CN110208542A
CN110208542A CN201910385538.1A CN201910385538A CN110208542A CN 110208542 A CN110208542 A CN 110208542A CN 201910385538 A CN201910385538 A CN 201910385538A CN 110208542 A CN110208542 A CN 110208542A
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porcine circovirus
type
serum
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缪芬芳
肖澄
董彦鹏
徐凤
胡静雅
车巧林
周琳
耿晓眉
谢洁
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Jiangsu Nannong High Science Co Ltd
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

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Abstract

The present invention provides a kind of ELISA antigen detection kits of porcine circovirus 2 type, and the effect detection method that can solve existing vaccine is time-consuming and laborious, and need immune technical problem.A kind of ELISA antigen detection kit of porcine circovirus 2 type, it is characterised in that: ELISA antigen detection kit includes coating plate, positive quality control serum, negative quality controlled serum, control sample, ELIAS secondary antibody, sample diluting liquid, cleaning solution, developing solution and terminate liquid；Plate is coated with by porcine circovirus 2 type antigen coat, porcine circovirus 2 type antigen is the porcine circovirus 2 type totivirus albumen of porcine circovirus 2 type inactivated culture after purification；Control sample is that porcine circovirus 2 type refers to vaccine, and porcine circovirus 2 type is that porcine circovirus 2 type inactivated culture is mixed with adjuvant with reference to vaccine.Protein content in vaccine is compared by the kit with control sample, it is not necessary that experimental animal is immunized.

Description

A kind of the ELISA antigen detection kit and its detection method of porcine circovirus 2 type
Technical field
The present invention relates to detection kit technical fields, and in particular to a kind of ELISA antigen detection of porcine circovirus 2 type Kit and its detection method.
Background technique
Porcine circovirus 2 type (Procinecircovirus type2, PCV2) is to cause postweaning multisystemic failure comprehensive The main pathogen of simulator sickness (Postweaningmultisystemic wasting syndrome, PMWS).PMWS is occurred mainly in The weanling pig of 5-12 week old, clinical symptoms are mainly shown as progressive emaciation, respiratory symptom, fever, pale and jaundice Deng.Aetology and serosurvey cause serious economic damage studies have shown that the disease is widely present in many countries, to pig breeding industry It loses.
PCV2 belongs to circovirus section Circovirus, is a kind of sub-thread covalently closed circular DNA virus, and genome contains 1766~1768nt, structure is simple, mainly contains 2 reading frames (ORFs): ORF1 encode virus replication GAP-associated protein GAP Rep and Rep ' participates in virus replication；ORF2 encoding virus coat proteins Cap, be virus structural proteins, with virus it is pathogenic and exempt from Epidemic focus is related.
Currently, vaccine immunization is considered as the effective means of prevention and control Porcine circovirus desease, the popularization of commercialized vaccine Important function has been played using for effective prevention and control China PCV2 epidemic situation.Domestic goods PCV2 vaccine is numerous, the effect inspection of vaccine It surveys and generally uses Immunization method, this method classics but take time and effort, and animal immune is essential link.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of ELISA antigen detection kit of porcine circovirus 2 type, energy The effect detection method for solving existing vaccine is time-consuming and laborious, and needs immune technical problem.
A kind of ELISA antigen detection kit of porcine circovirus 2 type, it is characterised in that: the ELISA antigen detection examination Agent box include coating plate, positive quality control serum, negative quality controlled serum, control sample, ELIAS secondary antibody, sample diluting liquid, cleaning solution, Developing solution and terminate liquid；
For the coating plate by porcine circovirus 2 type antigen coat, the porcine circovirus 2 type antigen is 2 porcine circovirus The porcine circovirus 2 type totivirus albumen of type inactivated culture after purification；
The control sample is that porcine circovirus 2 type refers to vaccine, and the porcine circovirus 2 type is pig annulus with reference to vaccine Viral 2 type inactivated cultures are mixed with adjuvant.
Further, the porcine circovirus 2 type inactivated culture is made by following methods: by porcine circovirus 2 type poison Kind is inoculated in the culture bottle of PK cell, is set and is adsorbed 30 minutes at 37 DEG C, and cell maintenance medium is added, sets rotating and culturing at 37 DEG C, Revolving speed is that 10~12 turns/hour set freeze thawing 3 times at -20 DEG C after culture 4 days, harvests cell culture and keeps sample and makees viral level Measurement will be examined qualified virus liquid that β-propionyl lactone is added and be inactivated, make its final concentration of 0.1%, mix well immediately, 4 DEG C 20 hours, 37 DEG C shook 2 hours, obtained the porcine circovirus 2 type inactivated culture.
Further, the peridium concentration of the porcine circovirus 2 type antigen is 0.05-5 μ g/ml, and coating dilution is The carbonate buffer solution of 0.1mol/L, pH9.6.
Further, the positive quality control serum is that the porcine circovirus 2 type totivirus albumen is immune new as antigen Serum is acquired after western orchid White Rabbit, purifies gained, dilution ratio 1:1000-1:4000 through MAB SELECT；
The feminine gender quality controlled serum is the serum of healthy new zealand white rabbit through MAB SELECT gained after purification, thinner ratio Example is 1:50-1:500.
Further, the ELIAS secondary antibody is HRP- goat-anti rabbit (goat anti-rabbit igg of horseradish peroxidase-labeled), dilution Concentration is 1:1000-1:20000, and dilution is the bovine serum albumin containing 0.1-5% (W/V) and 0.02-0.05% (V/V) The phosphate buffer of ProClin300, wherein phosphate buffering liquid concentration is 0.01mol/L, pH7.2-7.4.
Further, the sample diluting liquid is bovine serum albumin and 0.02-0.05% (V/ containing 0.1-5% (W/V) V) the phosphate buffer of ProClin300, wherein phosphate buffering liquid concentration is 0.01mol/L, pH7.2-7.4.
Further, the concentrated cleaning solution that the cleaning solution is 10 times, 10 times of the concentrated cleaning solution are to contain 0.5% (V/V) phosphate buffer of Tween-20, wherein phosphate buffering liquid concentration is 0.1mol/L, pH7.2-7.4.
Further, the developing solution is tmb substrate developing solution.
Further, the terminate liquid is the H of 2mol/L2SO4。
The present invention also provides the detection methods of above-mentioned ELISA antigen detection kit, comprising the following steps:
(1) each column of the diluted measuring samples of 1:2, the diluted control sample of 1:2, every 50 μ of hole are separately added into coating plate l；
(2) the diluted negative column of quality controlled serum one of 1:2, the diluted positive matter of 1:2 are separately added into coating plate remaining columns Serum one is controlled to arrange, as control, every 100 μ l of hole；
(3) positive quality control serum is added into the measuring samples of coating plate, control sample column, every 50 μ l of hole covers coating Plate；
(4) coating plate is set 37 DEG C and is reacted 1 hour；
(5) liquid in hole is outwelled, is washed 3~5 times with cleaning solution, every 300 μ 1 of hole, last time pats dry plank after washing；
(6) goat anti-rabbit igg of 100 μ, 1 horseradish peroxidase-labeled is added in every hole, and ELISA Plate is set 37 DEG C and reacted 1 hour；
(7) liquid in hole is outwelled, is washed 3~5 times with cleaning solution, every 300 μ 1 of hole, last time pats dry plank after washing；
(8) 100 μ 1TMB substrate developing solutions of every hole addition, color development at room temperature 15 minutes；
(9) 50 μ, 1 terminate liquid is added in every hole, and OD is read in microplate reader in 10 minutes450nmValue；
(10) result calculates: negative quality controlled serum 1:2 dilutes control wells OD450nmValue < 0.4, positive quality control serum 1:2 are dilute Release control wells OD450nmValue > 1.0, the OD of vaccine to be checked and control sample measurement450nmValue carries out phase using RelPot4.0 software Effect RP value is calculated, when RP value is greater than 1.0 vaccines qualifications to be checked.
Wherein, cleaning solution is 1 times of work cleaning solution after being diluted with deionized water.
Microbial resources information
SH plants of (deposit number CGMCC of porcine circovirus 2 type (Porcine circovirus2, PCV2) of the present invention No.2389 is provided by Agricultural University Of Nanjing professor Jiang Ping), which is Chinese commodity porcine circovirus 2 type inactivated vaccine The production of (SH plants) kind of a poison, porcine circovirus 2 type inactivated vaccine (SH plants) is approved as by The Ministry of Agriculture of the People's Republic of China, MOA Two class novel chiral synthons (Ministry of Agriculture announces No. 1448,2010.08.27 publication).
The present invention is by ELISA antigen detection kit detection vaccine potency, the kit and based on the kit Protein content in vaccine is compared by detection method with control sample, it is not necessary that experimental animal is immunized, on the one hand, time saving It is laborsaving, cost is reduced, on the other hand, the efficacy test of vaccine can be made to reduce the dependence to experimental animal, it is dynamic to reduce test " 3R " principle of animal experiment has been respected in influence of the difference of object to efficacy test itself.
Detailed description of the invention
Fig. 1 is the Western Blot identification of embodiment PCV2 totivirus albumen after purification, wherein PM is colored pre-dyed egg White matter molecular criteria amount, 1 is PK cell culture, and 2 be PCV2 totivirus albumen.
Fig. 2 is that the SDS-PAGE of embodiment positive quality control serum is identified, wherein PM is colored pre-dyed protein molecule standard Amount, 1 is rabbit anteserum before purification, and 2 be rabbit anteserum after purification.
Specific embodiment
A kind of embodiment 1: ELISA antigen detection kit of porcine circovirus 2 type
A kind of ELISA antigen detection kit of porcine circovirus 2 type, ELISA antigen detection kit include coating plate, Positive quality control serum, negative quality controlled serum, control sample, ELIAS secondary antibody, sample diluting liquid, cleaning solution, developing solution and terminate liquid；
Plate is coated with by porcine circovirus 2 type antigen coat, porcine circovirus 2 type antigen is porcine circovirus 2 type inactivation training The porcine circovirus 2 type totivirus albumen of feeding object after purification；
Control sample is that porcine circovirus 2 type refers to vaccine, and porcine circovirus 2 type is porcine circovirus 2 type with reference to vaccine Inactivated culture is mixed with adjuvant.
The preparation and preparation explanation that kit respectively forms:
1, porcine circovirus 2 type seed culture of viruses the preparation of porcine circovirus 2 type inactivated culture: is inoculated in length by 5% (V/V) It in culture bottle at good single layer PK cell, sets and is adsorbed 30 minutes at 37 DEG C, the DMEM containing 2% newborn bovine serum is added and maintains Liquid sets rotating and culturing at 37 DEG C, and revolving speed is 10~12 turns/hour, is observed 1~2 time daily, and cell answers well-grown, cultivates 4 Afterwards, freeze thawing 3 times at -20 DEG C are set, cell culture is harvested and keeps sample and make viral assay, qualified virus liquid will be examined to be added β-propionyl lactone inactivation, make its final concentration of 0.1%, mix well immediately, 4 DEG C 20 hours, 37 DEG C shake 2 hours, obtain pig Circovurus type 2 inactivated culture.
2, the preparation of porcine circovirus 2 type totivirus albumen: by porcine circovirus 2 type inactivated culture molecular sieve SepharoseTM4 Fast Flow are purified, and the PCV2 totivirus albumen of purifying measures concentration, protein concentration 320 with BCA μ g/ml, while the PCV2 totivirus albumen of purifying is subjected to Western Blot identification, go out to have the purpose item of specificity in 27Kd Band.
3, it is coated with plate: the PCV2 totivirus albumen of purifying being diluted to 2 μ with 0.1mol/L carbonate buffer solution (pH9.6) 96 hole elisa Plates are added in g/ml, and 100 holes μ l/ pat dry after washing 3 times with 1 times of work cleaning solution after 37 DEG C are incubated for 2 hours, The closing of 5% (W/V) skimmed milk is added, 200 holes μ l/, 37 DEG C are incubated for 2 hours, are washed 3 times with 1 × cleaning solution, 25 DEG C air-dry.
4, positive quality control serum:
(1) by the porcine circovirus 2 type totivirus albumen purified and Freund's complete adjuvant/incomplete Freund's adjuvant 1:1 Mixing and emulsifying is configured to immunogene；The new zealand white rabbit of subcutaneous multi-point injection immune health, first immunisation (help completely by Freund Agent) it carries out within 2 weeks afterwards the 2nd time immune (incomplete Freund's adjuvant), the measurement of blood sampling in 4 weeks antibody titer after head exempts from, if pig in rabbit anteserum Circovirus 2 type antibody is not up to 1:3200 and then carries out that (incomplete Freund's adjuvant) is immunized for the third time, blood sampling measurement serum after 2 weeks Antibody titer can separate and collect rabbit-anti porcine circovirus 2 type hyper-immune serum when antibody titer reaches 3200.
(2) purify this rabbit anteserum: the PBS of MAB SELECT filler affinity column pH7.2 balances 10 columns Then volume is added the rabbit-anti porcine circovirus 2 type hyper-immune serum that precipitating is abandoned in centrifugation, washs miscellaneous egg with the PBS of 10 column volumes It is white, it is finally eluted with the glycine-HCl of the 0.1M pH2.9 of 5 column volumes, collects eluent, obtain the rabbit-anti pig of purifying The hyper-immune serum of circovurus type 2 hyper-immune serum, purifying carries out SDS-PAGE Purity.
(3) qualified rabbit-anti porcine circovirus 2 type hyper-immune serum is subjected to 1:1000 dilution, obtains positive quality control Serum.
5, negative quality controlled serum: healthy rabbit anteserum is acquired, serum is centrifugated, by the sterile mixing of the serum of each rabbit, through MAB Negative serum progress is diluted for 1:50 times, obtains negative quality controlled serum by SELECT filler affinity chromatography column purification.
6, ELIAS secondary antibody: the goat anti-rabbit igg of horseradish peroxidase-labeled, diluted concentration 1:1000-1:20000 are dilute The phosphate buffer that liquid is the bovine serum albumin containing 0.1-5% (W/V) and 0.02-0.05% (V/V) ProClin300 is released, Wherein phosphate buffering liquid concentration is 0.01mol/L, pH7.2-7.4.
7, sample diluting liquid: the bovine serum albumin containing 0.1-5% (W/V) and 0.02-0.05% (V/V) ProClin300 Phosphate buffer, wherein phosphate buffering liquid concentration be 0.01mol/L, pH7.2-7.4.
The preparation of phosphate buffer: NaCl8.0g, KH are taken2PO4-2H2O0.2g、Na2HPO4-2H2O2.9g and KCl0.2g is dissolved in sterilizing distilled water, is settled to 1000ml；Then 5mg bovine serum albumin, 0.5ml ProClin300 is added, It mixes.
8, NaCl80g, KH 10 × cleaning solution: are taken2PO4-2H2O2g、Na2HPO4-2H2O29g, KCl2g, Tween-205ml, Sterilizing distilled water is added, is settled to 1000ml.
9, TMB developing solution:
A liquid: Na is weighed2HPO414.6g, citric acid 9.33g, 30%H2O22ml adds distilled water to 1000ml, adjusts pH value extremely 5.0~5.4；
B liquid: 3,3 ', 5,5 '-tetramethyl biphenyl diamines (TMB) 20mg, dehydrated alcohol 10ml are taken to add distilled water extremely 1000ml；
Tmb substrate liquid: A liquid and B liquid mixed in equal amounts are sub-packed in brown bottle.
10, terminate liquid: distilled water 1778ml is taken, H is slowly added to2SO4(96%) 222ml, packing.
11, control sample:
It is 2.25 × 10 that porcine circovirus 2 type inactivated culture, which is adjusted to viral level,5.0TCID50/ ml, with water solubility Adjuvant is mixed and made into control sample in 9:1 ratio.
The preparation of water-soluble adjuvant: weighing carbomer 50g, is added distilled water 1000mL, 115 DEG C high pressure sterilization 30 minutes, In gelatin, it is placed at room temperature for.It is slowly added to 5% sodium hydroxide solution of sterilizing before use, it is made to be converted into water-soluble character, is added Tocopherol, 4-8 DEG C of preservation.
Control sample is according to referring to epidemic disease in " porcine circovirus 2 type inactivated vaccine (SH, II) manufacture with examine Trial Regulation " Seedling quality standard carries out character, steriling test, safety verification, efficacy test.
(1) steriling test is tested by existing " Chinese veterinary pharmacopoeia " annex, asepsis growth.
(2) safety verification 14~21 age in days PCV2ELISA negative antibodies and PCV2 antigen negative sodium selenite 3, often Head intramuscular injection vaccine 4ml, is observed continuously 14, clinical response without exception.
(3) efficacy test
Piglet immunological attacks malicious method: with 14~21 age in days PRRSV ELISA negative antibodies, PCV2-ELISA negative antibody and The susceptible piglet of PCV2 antigen negative negative healthy 15, is divided into 3 groups, and every group 5, the 1st group of every incidence intramuscular injection vaccine 1ml, after two weeks by identical approach and dosage carry out the 2nd time inoculation, the 2nd group make it is nonimmune attack malicious control, the 3rd group is made blank control (nonimmune, non-to attack poison), equal isolated rearing observation.It weighs to all pigs within 5 weeks after head exempts from.1st, 2 group (is contained with SH plants of PCV2 106.0TCID50/ ml) attack, every pig collunarium 1ml, intramuscular injection 2ml, isolated rearing.Attack three groups of all pigs on the the 4th, 7 after poison The keyhole hemocyanin that 4 point inoculations at two oxters of pig and two stern positions are emulsified with incomplete Freund's adjuvant respectively (KLH/ICFA, 0.5mg/ml), each point inoculation 1ml (4ml/), while intraperitoneal inoculation thioglycollate medium, 10ml/ head； After attacking poison intraperitoneal inoculation thioglycollate medium, 10ml/ head are distinguished again within the 11st day and 19 days.It is observed continuously 25 after attacking poison, It is slaughtered after weighing on the 25th, dissect.Determined according to body temperature, opposite daily gain and virus antigen detection result.Control sample Product immune group has no apparent clinical manifestation, nonimmune to attack malicious group and have 2 the thick unrest of coat occur, appetite stimulator, disappear from the 7th day It is thin.It attacks after poison to cut open for 25th and kills all test pigs, carry out pathological anatomy, observe pathological change, it is nonimmune to attack malicious group and have 2 appearance bright Aobvious lesion, including lymph node exception swelling, section flat-white；Lungs swelling, elasticity attenuating etc..Take out inguinal lymph nodes group It knits and carries out immunohistochemistry detection PCV2 antigen, Immunization control group does not have 4 positives, and control sample immune group protection efficiency is 100% (table 1).
1. immune swine protest test result of table
Mouse immuning test method: with 5~6 week old PCV2ELISA negative antibodies, Healthy female cleaning grade Balb/c mouse 10 Only, it is divided into 2 groups, every group 5.1st group of intradermal vaccination, every 0.2ml carry out the 2nd by identical approach and dosage after two weeks Secondary inoculation；2nd group is not inoculated with, and makees blank control.The equal isolated rearing observation of each group mouse.It takes a blood sample within 5 weeks after head exempts from, separates serum, Measure PCV2 ELISA antibody titer in serum.The results are shown in Table 2, and control group mice serum is below 1:50, sample vaccine Immune group antibody mean titre is 1:2080.
2 mouse efficacy test result of table
Group 1 2 3 4 5 Mean titre
Blank control group < 1:50 < 1:50 < 1:50 < 1:50 < 1:50 < 1:50
Sample sets 1:800 1:3200 1:3200 1:1600 1:1600 1:2080
Embodiment 2: the detection method of kit
The detection method of above-mentioned ELISA antigen detection kit, comprising the following steps:
(1) each column of the diluted measuring samples of 1:2, the diluted control sample of 1:2, every 50 μ of hole are separately added into coating plate l；
(2) the diluted negative column of quality controlled serum one of 1:2, the diluted positive matter of 1:2 are separately added into coating plate remaining columns Serum one is controlled to arrange, as control, every 100 μ l of hole；
(3) positive quality control serum is added into the measuring samples of coating plate, control sample column, every 50 μ l of hole covers coating Plate；
(4) coating plate is set 37 DEG C and is reacted 1 hour；
(5) liquid in hole is outwelled, is washed 3~5 times with cleaning solution, every 300 μ 1 of hole, last time pats dry plank after washing；
(6) goat anti-rabbit igg of 100 μ, 1 horseradish peroxidase-labeled is added in every hole, and ELISA Plate is set 37 DEG C and reacted 1 hour；
(7) liquid in hole is outwelled, is washed 3~5 times with cleaning solution, every 300 μ 1 of hole, last time pats dry plank after washing；
(8) 100 μ 1TMB substrate developing solutions of every hole addition, color development at room temperature 15 minutes；
(9) 50 μ, 1 terminate liquid is added in every hole, and OD is read in microplate reader in 10 minutes450nmValue；
(10) result calculates: negative quality controlled serum 1:2 dilutes control wells OD450nmValue < 0.4, positive quality control serum 1:2 are dilute Release control wells OD450nmValue > 1.0, the OD of vaccine to be checked and control sample measurement450nmValue carries out phase using RelPot4.0 software Effect RP value is calculated, when RP value is greater than 1.0 vaccines qualifications to be checked.
Wherein, cleaning solution is 1 times of work cleaning solution after being diluted with deionized water.
Experimental example 1: specific detection
PCV2, PEDV, HPS, PRRSV, PK- are detected with established porcine circovirus 2 type ELISA antigen detection kit 15, PCV1 analyzes the specificity of this method.These samples are only subjected to 1:2 Cigarette dilution detection, testing result (table 3) display adds It is lower to enter PCV2 antigen its OD450nm value for detecting, and other Pathogen tests its OD450nm values is all larger than 1.0, shows to be built Vertical porcine circovirus 2 type ELISA antigen detection kit has good specificity.
3 specific detection result of table
Experimental example 2: repeatability detection
Repetitive test in 2.1 batches
In established porcine circovirus 2 type ELISA antigen detection kit, including 3 batches, 1805001, 1805002, in 1805003,1805001 batches of 5 boxes of porcine circovirus 2 type ELISA antigen detection kit are randomly selected, to pig circle 2 type inactivated vaccine of circovirus virus (SH plants, II) (lot number) carries out the measurement of vaccine antigen relative effectivenes, calculates standard deviation and variation within batch Coefficient the results are shown in Table 4.
4 batches, table interior repetitive test testing results.
Repetitive test between 2.2 batches
In established porcine circovirus 2 type ELISA antigen detection kit, including 3 batches, 1805001, 1805002, in 1805003,1805001,1805002,1805003 batches of each 1 boxes of solid phase competitive ELISA kit are randomly selected, To (SH plants, II) the progress vaccine antigen relative effectivenes measurements of same a collection of porcine circovirus 2 type inactivated vaccine, calculates standard deviation and criticize Between the coefficient of variation, the results are shown in Table 5.
5 batches of repetitive test testing results of table.
Experimental result standard deviation is 0.08 in batch, the coefficient of variation 6.45%, and experimental result standard deviation is 0.058 between batch, The coefficient of variation is 4.62%, shows that the ELISA detection method that this research is established has preferable repeatability.
Experimental example 3: effect detection method comparative experiments
The detection method of the ELISA antigen detection kit of porcine circovirus 2 type is compared with mouse efficacy test method
The 10 batches of porcine circovirus 2 type inactivated vaccines (SH plants, II) for selecting different potency respectively are detected, as a result such as table Shown in 6, with the raising of mouse efficacy test mean titre, RP value is also gradually increased, and relative effectivenes RP value and mouse effect are examined The Mean antibody titer tested has good parallel sexual intercourse, therefore can be used to substitute mouse efficacy test method.
The testing result of 6 two kinds of effect detection methods of table.

Claims (10)

1. a kind of ELISA antigen detection kit of porcine circovirus 2 type, it is characterised in that: the ELISA antigen detecting agent Box includes coating plate, positive quality control serum, negative quality controlled serum, control sample, ELIAS secondary antibody, sample diluting liquid, cleaning solution, shows Color liquid and terminate liquid；
The coating plate is gone out by porcine circovirus 2 type antigen coat, the porcine circovirus 2 type antigen for porcine circovirus 2 type The porcine circovirus 2 type totivirus albumen of culture living after purification；
The control sample is that porcine circovirus 2 type refers to vaccine, and the porcine circovirus 2 type is pig circular ring virus with reference to vaccine 2 type inactivated cultures are mixed with adjuvant.
2. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 1, it is characterised in that: institute It states porcine circovirus 2 type inactivated culture to be made by following methods: porcine circovirus 2 type seed culture of viruses is inoculated in the culture of PK cell It in bottle, sets and is adsorbed 30 minutes at 37 DEG C, cell maintenance medium is added, set rotating and culturing at 37 DEG C, revolving speed is 10~12 turns/hour, After culture 4 days, freeze thawing 3 times at -20 DEG C are set, cell culture is harvested and keeps sample and make viral assay, qualified disease will be examined β-propionyl lactone inactivation is added in venom, make its final concentration of 0.1%, mix well immediately, 4 DEG C 20 hours, 37 DEG C of shakings 2 are small When, obtain the porcine circovirus 2 type inactivated culture.
3. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 2, it is characterised in that: institute The peridium concentration for stating porcine circovirus 2 type antigen is 0.05-5 μ g/ml, and coating dilution is 0.1mol/L, the carbonate of pH9.6 Buffer.
4. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 3, it is characterised in that: institute Stating positive quality control serum is to acquire blood after new zealand white rabbit is immunized as antigen in the porcine circovirus 2 type totivirus albumen Clearly, gained, dilution ratio 1:1000-1:4000 are purified through MAB SELECT；
The feminine gender quality controlled serum is the serum of healthy new zealand white rabbit through MAB SELECT gained after purification, clear dilution ratio For 1:50-1:500.
5. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 4, it is characterised in that: institute State ELIAS secondary antibody be horseradish peroxidase-labeled goat anti-rabbit igg, diluted concentration 1:1000-1:20000, dilution be containing There are the bovine serum albumin of 0.1-5% (W/V) and the phosphate buffer of 0.02-0.05% (V/V) ProClin300, wherein phosphoric acid Salt buffer concentration is 0.01mol/L, pH7.2-7.4.
6. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 5, it is characterised in that: institute State the phosphoric acid that sample diluting liquid is the bovine serum albumin containing 0.1-5% (W/V) and 0.02-0.05% (V/V) ProClin300 Salt buffer, wherein phosphate buffering liquid concentration is 0.01mol/L, pH7.2-7.4.
7. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 6, it is characterised in that: institute The concentrated cleaning solution that cleaning solution is 10 times is stated, 10 times of the concentrated cleaning solution is the phosphoric acid containing 0.5% (V/V) Tween-20 Salt buffer, wherein phosphate buffering liquid concentration is 0.1mol/L, pH7.2-7.4.
8. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 7, it is characterised in that: institute Stating developing solution is tmb substrate developing solution.
9. a kind of ELISA antigen detection kit of porcine circovirus 2 type according to claim 8, it is characterised in that: institute State the H that terminate liquid is 2mol/L2SO4。
10. the detection method of ELISA antigen detection kit described in claim 9, comprising the following steps:
(1) each column of the diluted measuring samples of 1:2, the diluted control sample of 1:2, every 50 μ l of hole are separately added into coating plate；
(2) the diluted negative column of quality controlled serum one of 1:2, the diluted positive quality control blood of 1:2 are separately added into coating plate remaining columns A clear column, as control, every 100 μ l of hole；
(3) positive quality control serum is added into the measuring samples of coating plate, control sample column, every 50 μ l of hole covers coating plate；
(4) coating plate is set 37 DEG C and is reacted 1 hour；
(5) liquid in hole is outwelled, is washed 3~5 times with cleaning solution, every 300 μ 1 of hole, last time pats dry plank after washing；
(6) goat anti-rabbit igg of 100 μ, 1 horseradish peroxidase-labeled is added in every hole, and ELISA Plate is set 37 DEG C and reacted 1 hour；
(7) liquid in hole is outwelled, is washed 3~5 times with cleaning solution, every 300 μ 1 of hole, last time pats dry plank after washing；
(8) 100 μ 1TMB substrate developing solutions of every hole addition, color development at room temperature 15 minutes；
(9) 50 μ, 1 terminate liquid is added in every hole, and OD is read in microplate reader in 10 minutes450nmValue；
(10) result calculates: negative quality controlled serum 1:2 dilutes control wells OD450nmValue < 0.4, positive quality control serum 1:2 dilution pair According to hole OD450nmValue > 1.0, the OD of vaccine to be checked and control sample measurement450nmValue carries out opposite effect using RelPot4.0 software Power RP value calculates, when RP value is greater than 1.0 vaccines qualifications to be checked.
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