CN117783523A - Rabbit hemorrhagic disease virus 2 type structural protein VP60 antibody enzyme-linked immunosorbent assay kit - Google Patents
Rabbit hemorrhagic disease virus 2 type structural protein VP60 antibody enzyme-linked immunosorbent assay kit Download PDFInfo
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Abstract
The invention discloses a rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody enzyme-linked immunosorbent assay kit. The kit comprises an ELISA plate, positive control serum, negative control serum, an ELISA secondary antibody, a sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the ELISA plate is coated with a rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition. The antigen epitope polypeptide composition is one or more than two of polypeptide shown in a sequence 1 in a sequence table, polypeptide shown in a sequence 2 in the sequence table, polypeptide shown in a sequence 3 in the sequence table or polypeptide shown in a sequence 4 in the sequence table. The kit uses the chemically synthesized antigen peptide coated ELISA plate, has less antigen consumption and high sensitivity and specificity, and can efficiently detect whether the rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody exists. The kit disclosed by the invention has the advantages of high sensitivity, good specificity, convenience in operation and good market prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody enzyme-linked immunosorbent assay kit.
Background
Rabbit hemorrhagic disease (Rabbit haemorrhagic disease, RHD), commonly known as rabbit plague, rabbit hemorrhagic fever, is an acute, virulent, highly contagious infectious disease of rabbits first discovered by China in 1984. The mortality rate of the disease is 50-90%, the OIE classifies RHD as B-type infectious disease, and China also classifies the RHD as animal-type disease. The pathogen of the disease is rabbit hemorrhagic disease virus Rabbit haemorrhagic disease virus, RHDV, belonging to the genus Leporis of the family Caliciviridae. The RHDV genome is a single-stranded positive strand RNA consisting of 7437 nucleotides. The RHDV genome contains 2 ORFs. In addition to genomic RNA, RHDV also contains a 2.2kb subgenomic. ORF1 encodes a multimeric protein. The polyprotein is further broken down by viral proteases into a capsid protein (65 KD) which is the major structural protein of the virus, referred to as VP60, and a plurality of non-structural proteins. The VP60 gene is the only structural protein gene of RHDV and plays a main role in inducing the immune response of the organism.
The rabbit hemorrhagic disease epidemic situation caused by the novel RHDV virus infection is reported for the first time in the county of Sichuan province in 2020. This new RHDV virus is also known as RHDV type 2, and was first detected as occurring by a farm in france 2010. Compared with classical RHDV strains, the RHDV type 2 has wider infection range and greater harm to young rabbits, and is an important pathogen for jeopardizing rabbit raising industry. Similar to classical RHDV, the capsid protein of RHDV2 is mainly VP60 protein, which is the main target for inducing immune response. Rabbit hemorrhagic disease virus type 2 has higher seropositive rate detected in various farms in China, and in serological diagnosis methods for the disease, a hemagglutination inhibition test, an enzyme-linked immunosorbent assay and the like are common detection methods.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting a rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody, which utilizes rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide as a coating antigen, establishes an indirect ELISA method with good specificity, sensitivity and repeatability, and is used for detecting whether rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody is contained in rabbit serum.
In order to achieve the above purpose, the rabbit hemorrhagic disease virus type 2 epitope polypeptide composition with excellent performance is firstly screened and obtained, and the rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition provided by the invention is one or any combination of more than two of the polypeptides shown in the sequence 1 in the sequence table, the polypeptides shown in the sequence 2 in the sequence table, the polypeptides shown in the sequence 3 in the sequence table or the polypeptides shown in the sequence 4 in the sequence table. When the polypeptide composition is two of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2, the polypeptide shown in the sequence 3 and the polypeptide shown in the sequence 4, the mass ratio of the two polypeptides is (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1, a step of; when the polypeptide composition is three of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2, the polypeptide shown in the sequence 3 and the polypeptide shown in the sequence 4, the mass ratio of any three polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1, a step of;
when the polypeptide composition is composed of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2, the polypeptide shown in the sequence 3 and the polypeptide shown in the sequence 4, the mass ratio of the polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1:1.
the invention relates to a rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody enzyme-linked immunosorbent assay kit, which comprises an enzyme-linked reaction plate, positive control serum, negative control serum, enzyme-labeled secondary antibodies, sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the enzyme-linked reaction plate is coated with a rabbit hemorrhagic disease virus type 2 structural protein VP60 antigen epitope polypeptide composition.
The ELISA plate is a detachable 96-hole ELISA plate; the polypeptides in the rabbit hemorrhagic disease virus 2-type structural protein VP60 antigen epitope polypeptide composition are obtained through chemical artificial synthesis.
The optimal preparation method and the condition of the ELISA plate are that the rabbit hemorrhagic disease virus type 2 structural protein VP60 antigen epitope polypeptide composition is dissolved in a carbonate solution with pH of 9.6, then is added into a 96-hole polystyrene ELISA plate, 200ng of polypeptide is placed in each hole for 8-12 hours at the temperature of 2-8 ℃ so that polypeptide antigen and the ELISA plate are fully combined, then PBS buffer solution containing 1% (g/ml) Bovine Serum Albumin (BSA) with pH of 7.4 is added according to 300 mu l/hole, sealing treatment is carried out for 2-3 hours at the temperature of 37 ℃, and after spin-drying, the ELISA plate is dried and then sealed and stored at the temperature of 4 ℃.
The positive control serum is rabbit serum collected after the inactivated rabbit hemorrhagic disease virus type 2 VP60 antigen is immunized; the negative control serum is rabbit serum free of Specific Pathogen (SPF), i.e., rabbit serum free of rabbit hemorrhagic disease type 2 virus, including healthy rabbit serum free of rabbit hemorrhagic disease type 2 virus.
The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-rabbit IgG antibody.
The substrate solution A is a citric acid phosphate buffer solution containing 0.06% (g/ml) of urea hydrogen peroxide, the substrate solution B is a tetramethyl benzidine solution with the concentration of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when the substrate solution A is used. The stop solution is 2mol/L sulfuric acid solution.
The kit also comprises a sample diluent and a 20-time concentrated washing liquid; the sample dilution was a phosphate buffer with a value of 7.4 of 0.01M, pH containing 0.5% (g/100 ml) casein; the concentrated washing solution is phosphate buffer solution with pH value of 7.4 and 0.01M containing Tween-20 with concentration of 0.8% -1.2% (ml/ml).
The detection program of the kit provided by the invention is as follows:
1. balance: taking the kit out of the refrigeration environment, and standing at room temperature for 30min for standby; the liquid reagent is mixed evenly before use.
2. Preparing liquid: diluting the concentrated washing solution with distilled water or deionized water for 20 times to obtain a washing buffer solution;
3. setting: 2 negative control wells and 2 positive control wells, the remainder being wells for samples to be tested.
4. Pre-diluting a sample to be tested: sample serum to be detected, negative control serum and positive control serum are subjected to a sample dilution according to a ratio of 1: 20.
5. Sample adding: each well was pre-set with 100. Mu.l of diluted test sample. The time span of the sample adding process should be as short as possible.
6. Incubation: shaking and mixing uniformly, and placing in a 37 ℃ incubator for reaction for 30min.
7. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, the mixture was immersed for 15 seconds, the washing solution was discarded, and the plate was washed continuously for 4 times and then dried by pipetting.
8. Adding enzyme: 100 μl horseradish peroxidase was added to each well to label the goat anti-rabbit IgG antibody.
9. Incubation: placing in a 37 ℃ incubator for reaction for 30min.
10. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, immersed for 15 seconds, the washing solution was thrown away, and the plate was washed continuously for 4 times and then dried by shaking.
11. Adding 100 μl of substrate working solution (substrate working solution A and substrate working solution B are mixed in equal amounts to obtain substrate working solution, and mixing immediately before use), shaking, mixing, placing in a 37 deg.C incubator, and reacting for 15min in dark place.
12. 50 μl of the color development stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well 450nm Value (the reaction plate with stop solution should read OD within 15 min) 450nm Values).
And (3) judging a detection result:
1. negative control OD 450nm The average value should be less than or equal to 0.15, otherwise, the method is ineffective.
2. The positive control should have a value between 1.0 and 2.5 per test, otherwise it is ineffective.
3. Calculation of the critical value: critical value = 0.17 x positive control OD 450nm Average value of values.
Determination of OD by serum to be examined 450nm If the value is more than or equal to the critical value, judging the value as positive; determination of OD by serum to be examined 450nm Value of<The threshold value is judged as negative.
The kit provided by the invention can be used for detecting the rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody so as to judge whether the rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody exists in the detected animal.
The application of the kit in preparing the kit for detecting whether the rabbit hemorrhagic disease virus type 2 is infected or not also belongs to the protection scope of the invention;
the invention has the positive effects that: the invention adopts bioinformatics method to accurately analyze the epitope of rabbit hemorrhagic disease virus type 2 structural protein VP60, and screens out peptide segments suitable for ELISA detection from main epitope on VP60 protein. The peptide segment integrates antigen epitope and has the advantages of high sensitivity and strong specificity.
Meanwhile, an advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, the coating antigen used in the kit is a chemically synthesized polypeptide, so that the kit does not contain mixed proteins, has high purity, and further improves the efficiency of detecting the rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody.
In a word, the kit adopts an antigen peptide coating enzyme-linked reaction plate of a main antigen site of the chemically synthesized structural protein VP60, has the advantages of less antigen consumption, high sensitivity and strong specificity, and can effectively detect the structural protein VP60 antibody generated by the rabbit hemorrhagic disease virus type 2. Experimental results show that the kit provided by the invention has the advantages of good repeatability, strong specificity and high sensitivity. Can meet the needs of people of different levels, and has wide market prospect and good economic and social benefits.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of coated antigen of rabbit hemorrhagic disease virus 2-structural protein VP60 antibody ELISA kit
The experiment adopts a bioinformatics method to accurately analyze main epitope of rabbit hemorrhagic disease virus type 2 structural protein VP60, screens out proper peptide segments, synthesizes four peptides respectively by a full-automatic polypeptide synthesizer, and prepares updated and more complete coating antigen with purity of about 85% by respectively showing sequence 1, sequence 2, sequence 3 and sequence 4 in a sequence table, thereby improving the detection rate of positive antibody. The method for synthesizing the polypeptide may be a conventional method, and the present invention synthesizes four polypeptides of the present invention as coating precursors of the kit of the present invention using the following method.
The coated antigen of the present invention may be prepared using a Applied Biosystem full-automatic polypeptide synthesizer (model 433A). Fmoc (9-fluoroethylene carbonyl, 9-fluorenylmethoxycarbonyl) modified amino acid was used by Merrifield solid phase synthesis, using Rink Amide MBHA resin as solid phase carrier. The production process includes the steps of polypeptide antigen solid phase synthesis, polypeptide cleavage and identification, antigen purification, freeze drying and preservation. The following description will be given respectively:
1. solid phase synthesis of coated antigen
1. Preparation of synthetic reagents
The amino acid sequences of the synthesized coating antigen are shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.
SEQ ID NO：1：AIDHDRGNASFPGSSSSNVLELWYASAGSAADNPISQIA
SEQ ID NO：2：VAKSIYGVANGINQTTAGLFVMASGV
SEQ ID NO：3：FASVVRRTGDINAEAGSTNGTQYGAGS
SEQ ID NO：4：VATTSVVTTENASTSIATAGIGG
Appropriate Fmoc modified amino acids (available from NOVA) were prepared according to the coating antigen sequence and the scale of synthesis and added to the corresponding Carridge. The resin 5g was weighed according to the required synthesis scale, placed in a reaction chamber, and the upper and lower lids were screwed down and labeled, and the name, lot number, TARE of the reaction chamber and the weight of the resin were recorded. The reaction chamber was loaded into the synthesizer. The appropriate amounts of synthetic reagents were formulated and placed into corresponding reagent bottles, including 100% NMP, 3% AIM (caproyl imidazole), 35% PIP (piperidine), 100% MeOH (methanol), etc.
2. Detection of synthesizer state
Checking 433A whether the polypeptide synthesis instrument runs normally, and running a Run Self Test program after starting up, wherein the instrument automatically checks whether various indexes are normal. Additionally, it was checked whether nitrogen was sufficient and the system gauge pressure was normal (433A normal gauge pressure 10.2 psi). The performance of the instrument should be known prior to synthesis, so the flow rate of each synthetic reagent is measured. 433A synthesizer: sending Flow Rate1-18 to synthesizer, selecting Main Menu-Module Test-find Module A, moduleD, moduleI, moduleI, module A according to Prer or next) -measure or observe according to Start-according to more, if the Flow is not proper, regulating the lower valve pressure until the requirement is met (specific detection requirement see Table 1 below).
TABLE 1 flow rate detection Standard Meter for polypeptide synthesizer
| Reagent(s) | Bottle number | Module | Standard range |
| 35％Piperidine | 1 | A | 1.0～1.2ml |
| 3％AIM | 4 | D | 1.0～1.2ml |
| 100％MeOH | 9 | I | 3.5～4.0ml |
| DIC | 8 | I | 0.45～0.55g |
| 100％NMP | 10 | A | 2.6～2.8ml |
3. Coated antigen synthesis begins
The amino acid sequence to be synthesized in the procedure of 433A synthesizer was sent Std Fmoc 1.0Sol DIC90 to the synthesizer. File-New-Sequence-edit Sequence of synthetic peptide, save. File-New-Run, check Chemistry for Std Fmoc 1.0Sol DIC 90; whether Sequence is a stored name; setting the Cycles; and (5) preserving. And finally, sending the obtained product to a synthesizer.
Main Menu-Cycle Monitor-begin, start running.
4. Coated antigen synthesis
The Fmoc group is removed, the electron-withdrawing effect of the fluorene ring system of the Fmoc group enables the 9-H to be acidic and easy to be removed by weak base, piperidine (PIP) is used for attacking the 9-H during reaction, beta is eliminated to form dibenzofluorenene, and the dibenzofluorenene is easy to be attacked by secondary cyclic amine to form a stable addition product. After removal of the Fnov group, the "-NH2" group is exposed for the synthesis reaction. The activated next Fmoc group protected amino acid and 1-Hydroxybenzotriazole (HOBT) were then added to the reactor.
The above polypeptide sequence is synthesized by sequentially repeating the synthesis steps from the C terminal to the N terminal according to a specific sequence (the synthesizer automatically completes the synthesis according to the program, and the specific circulation steps are shown in the following table 2). And (5) observing and recording the dosage and the running condition of the reagent during the process.
TABLE 2 coating antigen Synthesis cycle procedure
5. End of coating antigen synthesis
The synthesizer will automatically stop after the coated antigen synthesis is completed and the peptide resin (peptide now also attached to the resin) is essentially washed clean. Then the reactor is taken down from the polypeptide synthesizer, the peptide resin is washed 3 times by using 100 percent methanol, and then the peptide resin is dried in a fume hood, and then the peptide resin is completely transferred into a brown polyethylene bottle and is put into a refrigerator with the temperature of minus 20 ℃ and sealed by a sealing film for standby.
2. Cleavage and identification of coating antigen
1. Cleavage of polypeptide antigen
The polypeptide obtained by the above reaction is bound to the solid phase carrier by chemical bond, and the polypeptide must be separated from the solid phase carrier by acidolysis with a specific organic strong acid. The acid hydrolysis also removes the protecting groups on the functional groups of each amino acid. The method comprises the following steps:
the synthesized polypeptide resin (referring to the peptide also attached to the resin) was removed from the freezer and placed in a 2L round bottom flask, 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), 10ml Tripropylsilane (TIS) and magnetic stirrer were added to the flask in a fume hood, and the flask was then stably placed on a magnetic stirrer and stirred at room temperature for 1h until the reaction was complete. After the reaction is finished, the TFA in the crude product is removed by continuous evaporation for 30-120 min by using a rotary evaporator with a cold trap. And then, repeatedly cleaning the crude product of the polypeptide antigen by using Dimethylformamide (DMF), and finally, filtering the mixed resin by using a sand core funnel to obtain the coating antigen.
2. Identification of coating antigen
After the synthesis of the polypeptide antigen, qualitative and quantitative analysis is carried out by using matrix-assisted laser desorption/flight time mass spectrometry (MODAL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC), and the synthesized peptide is identified by using common amino acid analysis.
3. Coated antigen purification
The cyclized polypeptide antigen is ultrafiltered by using a circulating tangential filter membrane package (Tangential Flow Device circulating tangential filter membrane package produced by PALL company and a peristaltic pump matched with the circulating tangential filter membrane package), the polypeptide antigen can not pass through a filter membrane with a certain aperture as a macromolecule, and small molecular impurities formed or introduced in the early synthesis process and the later cyclization reaction can pass through the filter membrane. Then sterilizing by a filter with the aperture of 0.2 mu m, subpackaging the obtained solution into sterile plastic bottles, and labeling. The label is marked with the name, number, production lot number, concentration, production date, shelf life and preservation condition of the polypeptide, and the polypeptide is packaged and stored at-20 ℃ or-40 ℃ for standby.
4. Freeze drying of coated antigen
In order to facilitate long-term storage and transport, the coated antigen needs to be freeze-dried to obtain the polypeptide in a solid state. And (3) placing the pre-frozen coated antigen on a Labconco freeze dryer for drying to obtain the coated antigen in a solid state. And (5) labeling after packaging. The label is marked with the name, number, production lot number, concentration, date of production, shelf life and storage conditions of the polypeptide.
Example 2 preparation of Rabbit hemorrhagic disease Virus 2-type structural protein VP60 antibody ELISA kit
The rabbit hemorrhagic disease virus 2 type structural protein VP60 antibody enzyme-linked immunosorbent assay kit comprises:
(1) A 96-hole removable polystyrene enzyme-linked reaction plate coated with rabbit hemorrhagic disease virus type 2 antigen; 2X 96 wells.
(2) Positive control serum: the rabbit serum collected after the inactivated rabbit hemorrhagic disease virus type 2 VP60 antigen is immunized is used as positive control serum (1 tube, 1.5 ml/tube) of the kit.
(3) Negative control serum: rabbit serum without Specific Pathogen (SPF) was used as negative control serum for the kit (1 tube, 1.5 ml/tube).
(4) Enzyme-labeled secondary antibody: is prepared by taking horseradish peroxidase labeled goat anti-rabbit IgG (purchased from Earth Ox company) as stock solution and diluting the stock solution by 1:10000, and 2 bottles (12 ml/bottle).
(5) Sample dilution: 1 bottle (24 ml/bottle) of phosphate buffer with a value of 7.4 of 0.01M, pH containing 0.5% (g/100 ml) casein.
(6) Substrate solution A: phosphate buffer (1 bottle, 12 ml/bottle) of citric acid containing 0.6mg/ml urea hydrogen peroxide
(7) Substrate solution B: a solution of 0.2mg/ml Tetramethylbenzidine (TMB) (1 bottle, 12 ml/bottle).
(8) Stop solution: 2mol/L sulfuric acid solution (1 bottle, 12 ml/bottle).
(9) 20-fold concentrated washing solution: phosphate buffer (50 ml/bottle, 2 bottles) containing Tween-20 at a concentration of 0.8% -1.2% (ml/ml) and having a pH of 7.4.
Serum dilution plates (2, 96 wells/block) can also be included in the kit as needed for dilution of serum samples.
The preparation method of the 96-hole removable polystyrene enzyme-linked reaction plate coated with the rabbit hemorrhagic disease virus type 2 virus antigen comprises the following steps: 1. the polypeptide antigen prepared in example 1 was dissolved in a carbonate solution at pH 9.6 and then added to a 96-well polystyrene enzyme-linked reaction plate with 200ng of polypeptide per well (set up 14 sets of treatments in which ZMR2001 has 200ng of polypeptide shown in sequence 1 per well; 200ng of the polypeptide shown in SEQ ID No. 2 per well of ZMR 2002; 200ng of the polypeptide shown in SEQ ID No. 3 per well of ZMR 2003; the polypeptide shown in 200ng sequence 4 in each hole of ZMR2004, the polypeptide shown in 100ng sequence 1 and the polypeptide shown in 100ng sequence 2 in each hole of ZMR2005, the polypeptide shown in 100ng sequence 1 and the polypeptide shown in 100ng sequence 3 in each hole of ZMR2006, the polypeptide shown in 100ng sequence 1 and the polypeptide shown in 100ng sequence 4 in each hole of ZMR2007, the polypeptide shown in 100ng sequence 3 and the polypeptide shown in 100ng sequence 2 in each hole of ZMR 2002008, the polypeptide shown in 100ng sequence 4 in each hole of ZMR2009, the polypeptide shown in 100ng sequence 4 in each hole of ZMR20010, the polypeptide shown in 100ng sequence 3 in each hole of ZMR20010, the polypeptide shown in 66.7ng sequence 2 in each hole of ZMR20011, the polypeptide shown in 66.7ng sequence 2 in each hole of ZMR20012, the polypeptide shown in 66.7ng sequence 3 in each hole of ZMR20012, the polypeptide shown in 66.7 in each hole in each 6.7ng sequence 4 in each hole of ZMR2007, the polypeptide shown in each 66.7 in each hole in each 66.7ng sequence 4 in each ZMR20013, the polypeptide shown in each 66.7 in each 6, the polypeptide shown in each 66.7 in each hole in each 6.7ng sequence 4 in each hole in each 3 in each 6.7, the polypeptide in each 6.7, polypeptide in each 6, in each 6.7, in each 6, 7, 6 7, 6 7 shown in each polypeptide shown in each hole shown in each hole each well shown in each well each well sequence 6 each shown in each well sequence each shown in each shown in each sequence each 200 each the a the, then adding PBS buffer solution containing 1% (g/ml) Bovine Serum Albumin (BSA) pH7.4 into the mixture according to 300 μl/well, sealing the mixture at 37 ℃ for 2-3 hours, drying the mixture, and sealing the mixture at 4 ℃ for storage after the enzyme-linked reaction plate is dried.
Example 3 sensitivity test of Rabbit hemorrhagic disease Virus type 2 structural protein VP60 antibody ELISA kit
1. Application method of rabbit hemorrhagic disease virus 2-type structural protein VP60 antibody enzyme-linked immunosorbent assay kit
1. Balance: taking the kit out of the refrigeration environment, and standing at room temperature for 30min for standby; the liquid reagent is mixed evenly before use.
2. Preparing liquid: diluting the concentrated washing solution with distilled water or deionized water for 20 times to obtain a washing buffer solution; 3. setting: 2 negative control wells and 2 positive control wells, the remainder being wells for samples to be tested.
4. Pre-diluting a sample to be tested: sample serum to be detected, negative control serum and positive control serum are subjected to a sample dilution according to a ratio of 1: 20.
5. Sample adding: each well was pre-set with 100. Mu.l of diluted test sample. The time span of the sample adding process should be as short as possible.
6. Incubation: shaking and mixing uniformly, and placing in a 37 ℃ incubator for reaction for 30min.
7. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, the mixture was immersed for 15 seconds, the washing solution was discarded, and the plate was washed continuously for 4 times and then dried by pipetting.
8. Adding enzyme: 100 μl horseradish peroxidase was added to each well to label the goat anti-rabbit IgG antibody.
9. Incubation: placing in a 37 ℃ incubator for reaction for 30min.
10. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, immersed for 15 seconds, the washing solution was thrown away, and the plate was washed continuously for 4 times and then dried by shaking.
11. Adding 100 μl of substrate working solution (substrate working solution A and substrate working solution B are mixed in equal amounts to obtain substrate working solution, and mixing immediately before use), shaking, mixing, placing in a 37 deg.C incubator, and reacting for 15min in dark place.
12. 50 μl of the color development stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well 450nm Value (the reaction plate with stop solution should read OD within 15 min) 450nm Values).
And (3) judging a detection result:
1. negative control OD 450nm The average value should be less than or equal to 0.15, otherwise, the method is ineffective.
2. The positive control should have a value between 1.0 and 2.5 per test, otherwise it is ineffective.
3. Calculating a critical value: critical value = 0.17 x positive control OD 450nm Average value of values.
Determination of OD by serum to be examined 450nm If the value is more than or equal to the critical value, judging the value as positive; determination of OD by serum to be examined 450nm Value of<The threshold value is judged as negative.
2. Sensitivity test to known positive serum
The rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody ELISA kit prepared according to the method of example 2 was used (ZMR 2001, ZMR2002, ZMR2003, ZMR2004, ZMR2005, ZMR2006, ZMR2007, ZMR2008, ZMR2009, ZMR20010, ZMR20011, ZMR20012, ZMR20013, ZMR20014, ZMR 20015), 30 parts of rabbit hemorrhagic disease virus type 2 inactivated antigen immune rabbit serum was subjected to a sensitivity test according to the method of using the above-mentioned rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody ELISA kit, the test results were shown in Table 3, the sensitivity of the ZMR2001 kit to 30 parts of known positive serum was 50.0%, the sensitivity of the ZMR2002 kit to 30 parts of known positive serum was 60.0%, the sensitivity of the ZMR2003 kit to 30 parts of known positive serum was 30.0%, the sensitivity of the ZMR2004 kit to 30 parts of known positive serum was 23.3%, the sensitivity of the kit for lot No. ZMR2005 to 30 known positive sera was 70.0%, the sensitivity of the kit for lot No. ZMR2006 to 30 known positive sera was 70.0%, the sensitivity of the kit for lot No. ZMR2007 to 30 known positive sera was 73.3%, the sensitivity of the kit for lot No. ZMR2008 to 30 known positive sera was 70.0%, the sensitivity of the kit for lot No. ZMR2009 to 30 known positive sera was 73.3%, the sensitivity of the kit for lot No. ZMR20010 to 30 known positive sera was 43.3%, the sensitivity of the kit for lot No. ZMR20011 to 30 known positive sera was 80.0%, the sensitivity of the kit for lot No. ZMR20012 to 30 known positive sera was 83.3%, the sensitivity of the kit for lot No. ZMR20013 to 30 known positive sera was 83.3%, the sensitivity of the kit for lot No. ZMR20014 to 30 known positive sera was 83.3%, and the sensitivity of the kit for lot No. ZMR 20035 to 30 known positive sera was 93.3%.
TABLE 3 sensibility detection result of rabbit hemorrhagic disease virus 2 structural protein VP60 antibody ELISA detection kit
3. Minimum detection limit test
3 parts of rabbit serum positive for the VP60 antibody of the structural protein 2 of the rabbit hemorrhagic disease virus are selected, 1:20-1:320 times dilution is carried out, three batches of the ELISA kit (batch ZMR 20015) for the VP60 antibody of the structural protein 2 of the rabbit hemorrhagic disease virus prepared in the embodiment 2 are used, the double dilution of the rabbit serum is detected according to the use method of the ELISA kit for the VP60 antibody of the structural protein 2 of the rabbit hemorrhagic disease virus, and the result shows that the kit can detect the positive serum diluted 1:160 times, wherein, the table 4 is a batch of experimentsAs a result. In table 4, positive control: the rabbit serum is collected after the inactivated rabbit hemorrhagic disease virus type 2 VP60 antigen is immunized and used as positive control serum (1 tube, 1.5 ml/tube) of the kit. Negative control: rabbit serum without Specific Pathogen (SPF) was used as negative control serum for the kit (1 tube, 1.5 ml/tube). Critical value (Cut-off value) =0.17×positive control OD 450nm Average value of values
TABLE 4 detection results of lowest detection limit test of rabbit hemorrhagic disease virus 2 structural protein VP60 antibody ELISA detection kit
Example 4, specificity test of Rabbit hemorrhagic disease Virus type 2 structural protein VP60 antibody ELISA kit
40 healthy rabbit serum (supplied by Miao England Biolabs pharmaceutical Co., ltd.), 2 positive serum for Rabbit hemorrhagic disease type 1 (supplied by Miao England Co., ltd.), 2 positive serum for Rabbit Pasteurella (supplied by Miao England Co., ltd.) were each tested according to the method of using the rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody ELISA kit described in example 3 using the three kits of example 2.
The results of the specific tests of the kit are shown in the following table (table 5), and the test results of 40 healthy rabbit serum show that the specificity of the ZMR2001 kit is 100.0%, the specificity of the ZMR2002 kit is 100.0%, the specificity of the ZMR2003 kit is 100.0%, and the specificity of the ZMR2004 kit is 100.0%. The specificity of the ZMR2005 kit was 100.0%, the specificity of the ZMR2006 kit was 100.0%, the specificity of the ZMR2007 kit was 100.0%, the specificity of the ZMR2008 kit was 100.0%, the specificity of the ZMR2009 kit was 100.0%, the specificity of the ZMR20010 kit was 100.0%, the specificity of the ZMR20011 kit was 100.0%, the specificity of the ZMR20012 kit was 100.0%, the specificity of the ZMR20013 kit was 100.0%, the specificity of the ZMR20014 kit was 100.0%, and the specificity of the ZMR20015 kit was 100.0%. The detection results of 2 rabbit hemorrhagic disease type 1 positive serum and rabbit pasteurella positive serum are negative, so that the specificity of fifteen kits for detecting the 4 relevant pathogenic positive serum is 100%.
TABLE 5 specific detection results of rabbit hemorrhagic disease virus 2 structural protein VP60 antibody ELISA kit
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Claims (9)
1. The rabbit hemorrhagic disease virus type 2 structural protein VP60 antigen epitope polypeptide composition is one or more than two of polypeptide shown in a sequence 1 in a sequence table, polypeptide shown in a sequence 2 in the sequence table, polypeptide shown in a sequence 3 in the sequence table or polypeptide shown in a sequence 4 in the sequence table.
2. The rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition of claim 1, wherein: when the polypeptide composition is two of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2, the polypeptide shown in the sequence 3 and the polypeptide shown in the sequence 4, the mass ratio of the two polypeptides is (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1, a step of; when the polypeptide composition is three of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2, the polypeptide shown in the sequence 3 and the polypeptide shown in the sequence 4, the mass ratio of any three polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1, a step of; when the polypeptide composition is composed of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2, the polypeptide shown in the sequence 3 and the polypeptide shown in the sequence 4, the mass ratio of the polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1:1.
3. an enzyme-linked immunosorbent assay kit for a rabbit hemorrhagic disease virus type 2 structural protein VP60 antibody, which comprises an enzyme-linked reaction plate, positive control serum, negative control serum, enzyme-labeled secondary antibodies, substrate liquid A, substrate liquid B and stop solution, wherein the enzyme-linked reaction plate is coated with the rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition according to claim 1 or 2.
4. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the ELISA plate is a detachable 96-hole ELISA plate; the rabbit hemorrhagic disease virus 2 type structural protein VP60 antigen epitope polypeptide composition is obtained by chemical artificial synthesis of each polypeptide.
5. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the method for obtaining the ELISA plate comprises the steps of dissolving the rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition in 100 mu l of carbonate solution with the pH of 9.6, adding the solution into a 96-hole polystyrene ELISA plate, placing 200ng of the rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition in each hole at the temperature of 2-8 ℃ for 8-12 hours, fully combining the rabbit hemorrhagic disease virus type 2 structural protein VP60 epitope polypeptide composition with the ELISA plate, adding PBS buffer with the pH of 0.01g/ml bovine serum albumin 7.4 according to 300 mu l/hole, sealing at the temperature of 37 ℃ for 2-3 hours, spin-drying, and sealing at the temperature of 4 ℃ for storage after the ELISA plate is dried.
6. The enzyme-linked immunosorbent assay kit according to claim 3, wherein:
the positive control serum is serum obtained by immunizing rabbits by taking the rabbit hemorrhagic disease virus type 2 structural protein VP60 antigen as an immunogen; the negative control serum is rabbit serum without specific pathogen.
7. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled goat anti-rabbit IgG antibody.
8. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the substrate solution A is a citric acid phosphate buffer solution containing 0.0006g/ml of urea hydrogen peroxide, the substrate solution B is a tetramethyl benzidine 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 2mol/L sulfuric acid solution.
9. A kit according to claim 3, wherein: the kit also comprises a sample diluent and a 20-time concentrated washing liquid; the sample diluent is phosphate buffer solution with 0.01mol/L, pH value of 7.4 and containing 0.00005g/ml casein; the concentrated washing liquid is phosphate buffer solution with the pH value of 7.4 and 0.01mol/L of Tween-20 with the volume percentage concentration of 0.8-1.2%.
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