CN1588076A - Indirect ELISA diagnostic reagent kit for pig B type circular virus antibody - Google Patents
Indirect ELISA diagnostic reagent kit for pig B type circular virus antibody Download PDFInfo
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Abstract
The invention discloses an indirect ELISA diagnosing reagent box for pig II type circular virus (PCV2) antibody. There arranged with antibody detecting board in the reagent box, enzyme combination liquid, positive comparison, negative comparison, sample diluter, 10X condensed detergent, developing liquid A and B, and stopping liquid, the detecting board of the reagent box is removable 96-apertures enzyme labeling board enclosed with PCV2 reconstructed enucleation localizing signal capsid protein (dCap), the enzyme combination liquid is goat-anti-pig IgG multi-clone antibody labeled with HRP, the positive comparison is pig PCV 2 standard positive serum, the negative comparison is pig standard negative serum. The sensitivity of the invention is high, simple, and easy to be wide applied.
Description
Technical field
The present invention relates to the fast diagnosis method and the utensil of a kind of antiviral antibody of pig, specifically is pig II type PCV-II (PCV2) antibody indirect ELISA diagnostic kit.
Background technology
Pig circular ring virus (Porcine circovirus PCV) is the animal virus of the present minimum of finding, the about 17nm of virion diameter, and for covalence closed, ring-type, Single-stranded DNA virus, PCV have two kinds of genotype: PCV1 and PCV2.PCV1 no pathogenicity, but extensively be present in the pig body and pig source continuous cell line, PCV2 has pathogenic to pig, mainly cause multisystem exhaustion syndrome behind the weaned piglet (Post-weaningmultisystemic wasting syndrome, PMWS).PMWS is a kind of new infectious disease, finds in Canada first in 1991, mainly betides 5-12 weanling pig in age in week, shows as progressive emaciation, expiratory dyspnea, ochrodermia, diarrhoea, jaundice.Subsequently, the U.S., Britain, Germany, France, Ireland, Czech, Spain, TaiWan, China, Holland, Switzerland, Argentina, Mexico, Japan, Denmark, Italy, Korea S, Hungary, states such as Thailand have reported in succession should disease, epidemiology survey shows that this disease also is widely current in China, and causes suitable serious economy loss.PCV2 is except causing PMWS, also can cause growing and fattening pigs dermatitis and nephrotic syndrome (Porcine dermatitis and nephropathy syndrome, PDNS), and with the breeding difficulty of farrowing sow, the congenital battle array of newborn piglet is quivered, and newborn piglet diarrhoea and hyperplasia necrotizing pneumonia are in close relations.According to the epidemiology of PMWS, clinical symptoms and cut open the inspection pathological change and can infect PCV2 and make tentative diagnosis still need be with laboratory diagnosis technology for detection PCV2 antigen and antibody thereof but make a definite diagnosis.The capsid protein of PCV2 is this viral main immunogens, thereby detects the antibody horizontal that PCV2 capsid protein antibody can reflect PCV2.To swinery PCV2 antibody horizontal, particularly the maternal antibody level is monitored; can definitely understand pig body immune level, hold the suitableeest immunity of eqpidemic disease opportunity, guarantee to obtain good immune effect; effectively the heavy economic losses that therefore causes is avoided in invasion and attack and the propagation of prevention and control PCV2.
At present, the PCV2 detection technique that adopts both at home and abroad mainly contains: viral separation and Culture, indirect immunofluorescence (IFA), immuno-enzymatic monolayer assay (IMPA), in situ hybridization (ISH), PCR (PCR) or the like.Though above-mentioned technology and method can detect PCV2 and antibody horizontal thereof, also obtain certain effect in practice, all there is the test operation complexity, length consuming time, need specific professional skill and instrument and equipment etc., often be limited to and carry out in the laboratory, be difficult in basic unit and popularize and promote.Therefore, develop to be applicable to quick, the easy PCV2 antibody test utensil of culturing basic unit, swinery PCV2 immune level monitored in real time, set up science, swinery eqpidemic disease immune programme for children flexibly, to the prevention of eqpidemic disease with control significant.
Summary of the invention
The purpose of this invention is to provide boar II type PCV-II (PCV2) antibody indirect ELISA diagnostic kit.
The object of the present invention is achieved like this: the pig II type PCV-II reorganization nls-defected capsid protein (dCap) that adopts prokaryotic expression is as antigen coated removable 96 hole ELISA Plate, prepare the enzyme conjugates working fluid with the anti-pig IgG of horseradish peroxidase-labeled goat, directly detect PCV2 antibody in the sample with indirect elisa method.Specific as follows:
1. the preparation of antibody test plate: the 0.05M Tris-HCl damping fluid with pH8.5 is made coating buffer; the dilution of pig II type PCV-II reorganization nls-defected capsid protein is 0.1-10 μ g/ml; add in the polystyrene micropore plate by 100 μ l/ holes; 37 ℃ 2 hours; 4 ℃ of bags are spent the night again; dry; contain 1% bovine serum albumin(BSA) (BSA) by the adding of 100 μ l/ holes; 37 ℃ of sealings of the phosphate buffer of PH7.4 2 hours; washing dries; added 20% sucrose phosphate buffer room temperature protection again 3 hours, put the hothouse drying after, preserve in the packaging bag that contains drying agent of packing into.
2. the preparation of enzyme conjugates working fluid: prepare horseradish peroxidase (HRP with ultrapure water, RZ 〉=3.0) solution, added 4 ℃ of lucifuges of 0.06M sodium periodate solution 1 hour, add 160mM ethylene glycol, room temperature reaction 30 minutes, add 1 hour 2-8 ℃ of dialysed overnight of acetate buffer solution of PH4.4, change liquid twice.The anti-pig IgG of the goat of purifying is added in the enzyme of above-mentioned activation, be transferred to bag filter, in the carbonic acid buffer of 0.05mM PH9.6, dialyse, 4 ℃ of stirrings spend the night (changing liquid twice).Dislysate is drawn in the centrifuge tube, adds the NaBH that newly joins
4Liquid, 4 ℃ were reacted 2 hours.The saturated ammonium sulfate solution that adds equivalent, 4 ℃ 30 minutes, 4 ℃ centrifugal 20 minutes, abandon supernatant, drain.Precipitation is dissolved in a small amount of PBS, and in the bag filter of packing into, to 0.02M PBS (PH7.4) dialysis, 4 ℃ are spent the night.Liquid in the dislysate is drawn in the centrifuge tube, centrifugal, with the supernatant sucking-off, add equivalent glycerine, mixing ,-20 ℃ of preservations.Again containing 0.1%BSA, 0.05% Tween-20,0.01% merthiolate, the phosphate buffer of pH7.4 is by 1: the anti-pig IgG polyclonal antibody of goat of the horseradish peroxidase-labeled that the 200-5000 dilution is frozen is as the enzyme conjugates working fluid.
3. the preparation of other reagent: sample diluting liquid is the 0.01M phosphate buffer (pH7.4) that contains 0.05% Tween-20; 10 * concentrated cleaning solution is the 0.1M phosphate buffer (pH7.4) that contains 0.5% Tween-20; Colour developing liquid A is tetramethyl benzidine (TMB) solution of 0.2mg/ml, and colour developing liquid B is the citric acid-phosphate buffer that contains 0.5 ‰ hydrogen peroxide ureas; Stop buffer is the 2M sulfuric acid solution.
4. the preparation of the positive, negative control: will screen the pig II type PCV-II standard positive serum (OD that obtains
450nm〉=1.00) add a small amount of red pigment and also press 1000U/ml adding penicillin and streptomysin, aseptic filtration is as positive control; Pig standard female serum (OD with the screening acquisition
450nm≤ 0.150), press 1000U/ml and add penicillin and streptomysin, aseptic filtration is as negative control.
5. its trace routine is: 1) dilution of 10 * concentrated cleaning solution is cleansing solution for 10 times; 2) serum to be checked is done dilution in 1: 100 with sample diluting liquid, add in the antibody test plate by 100 μ l/ holes, establish blank (only adding 100 μ l sample diluting liquids), negative control (PCV2 negative serum), positive control (PCV2 standard positive serum) simultaneously, hatched 20-45 minute for 37 ℃, dry; 3) every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; 4) every hole adds 100 μ l enzyme conjugates working fluids (blank does not add), hatches 20-45 minute for 37 ℃, dries; 5) every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; 6) add 50 μ l colour developing liquid A and 50 μ l colour developing liquid B successively, 37 ℃ of lucifuges were hatched 5-15 minute; 7) add 50 μ l stop buffers, under the 450nm wavelength, read every hole absorbance value (OD with microplate reader
450nmValue).
6. the criterion that detects sample is: with sample OD to be checked
450nmValue and standard female OD
450nmThe ratio (P/N) of value is judged to the positive more than or equal to 2.2, and is negative smaller or equal to 1.7.Be suspicious, must heavily examine that the P/N value is judged to the positive more than or equal to 2.0 when resurveying, and is negative less than 2.0 between 1.7-2.2.
The good effect that the present invention is useful is: simple to operate, everybody can operate, can better satisfy different levels personnel's needs, arrive individual breed etc. as eqpidemic disease monitoring, customs quarantine control, health and epidemic prevention, intensive culture, be easy to apply on a large scale, have vast market prospect and bigger economical, societal benefits.The present invention has following advantage:
(1) high specificity, the susceptibility height.PCV2 antibody indirect ELISA diagnostic kit is that the basis is prepared from the reorganization dCap albumen of gene engineering expression; Reorganization dCap albumen is that non-totivirus antigen, security are good, does not contain irrelevant foreign protein, only with pig II type PCV-II positive serum specific bond, not with other eqpidemic disease positive serum generation cross reaction of pig such as PCV1 positive serum.Have good resistance originality, therefore have very high specificity and susceptibility.
(2) easy and simple to handle quick.When using the PCV2 antibody assay kit to detect PCV2 antibody, need not to join other reagent in addition, sample need not aseptic process, is the decidable testing result in 1-2 hour by the kit explanation.
(3) result judges image, accurate, reliable.PCV2 antibody indirect ELISA diagnostic kit shows testing result with the depth of colour developing, and it is positive promptly to detect the blue colour developing of hole appearance, is yellow after the termination, and colourless negative, the result judges image.Adopt the machine-readable number of microplate reader, reduce subjectivity, accurately and reliably.
(4) cost is low, small investment.But PCV2 antibody indirect ELISA diagnostic kit batch detection settles at one go, and is with low cost, small investment, instant effect.
Description of drawings
Fig. 1 is that PCV2 antibody indirect ELISA diagnostic kit detects principle schematic, among the figure; 1 is ELISA Plate, and 2 is PCV2 dCap antigen, and 3 is pig PCV2 antibody or serum to be checked, and 4 is the anti-pig IgG polyclonal antibody of HRP mark goat.
Fig. 2 is the antigen active qualification result of reorganization PCV2 dCap albumen, among the figure: be the SDS-PAGE analysis of recombinant protein, B is that the Western Blot of recombinant protein identifies, 1 is the e. coli bl21 mycoprotein, and 2 is the abduction delivering product of empty carrier pGEX-4T-1, and 3 do not induce mycoprotein for recombinant plasmid, 4 induce the 4h expression product for recombinant plasmid, 5 is the reorganization GST-dCap fusion of purifying, and 6 is the reorganization dCap albumen after the fibrin ferment cutting, and M. is protein Marker.
Embodiment
Technical scheme of the present invention is: a kind of PCV2 antibody indirect ELISA diagnostic kit, kit is provided with the antibody test plate, enzyme conjugates working fluid, positive control, negative control, sample diluting liquid, 10 * concentrated cleaning solution, colour developing liquid A, colour developing liquid B and stop buffer.The check-out console of PCV2 antibody indirect ELISA diagnostic kit is for wrapping by the removable 96 hole ELISA Plate of PCV2 reorganization dCap albumen, the enzyme conjugates working fluid is the anti-pig IgG polyclonal antibody of HRP mark goat, positive control is a pig PCV2 standard positive serum, and negative control is a pig standard female serum.Be prepared as follows:
(1) preparation of PCV2 reorganization dCap albumen
According to PCV2 Cap gene order design Auele Specific Primer (upstream primer: 5 '-GC
GGATCCAATGGCATCT TCAACAC-3 '; Downstream primer: 5 '-CCG
CTCGA GTTAAGGGTTAAGTGGG-3 '), utilize from pig PCV2 genome, increase capsid protein gene sequence (fragment of the Cap gene 3 ' end 579bp) (see figure 3) of stoning positioning signal of round pcr, glutathione-S-transferase gene (GST) downstream that is inserted into prokaryotic expression carrier pGEX-4T-1 makes up the pGEX-PCV2-dCap expression vector, and transformed into escherichia coli BL21, through 0.1mM IPTG abduction delivering after 5 hours, use the ultrasonic treatment bacterium, the fusion (GST-dCap) that utilizes commercial GST affinity column purifying to express, remove the GST label with the fibrin ferment enzymolysis again, obtain pig II type PCV-II reorganization nls-defected capsid protein (Fig. 2), this albumen has kept the antigenicity of complete capsid protein, can with pig II type PCV-II positive serum specific bond (Fig. 2).Measure the protein concentration of GST-dCap fusion and reorganization dCap albumen respectively, be used for the preparation of indirect ELISA diagnostic reagent kit antibody test plate.
1 AATGGCATCT?TCAACACCCG?CCTCTCCCGC?ACCTTCGGAT?ATACTATCAA?GCGAACCACA
61 GTCAAAACGC?CCTCCTGGGC?GGTGGACATG?ATGAGATTCA?ATATTAATGA?CTTTCTTCCC
121 CCAGGAGGGG?GCTCAAACCC?CCGCTCTGTG?CCCTTTGAAT?ACTACAGAAT?AAGAAAGGTT
181 AAGGTTGAAT?TCTGGCCCTG?CTCCCCGATC?ACCCAGGGTG?ACAGGGGAGT?GGGCTCCAGT
241 GCTGTTATTC?TAGATGATAA?CTTTGTAACA?AAGGCCACAG?CCCTCACCTA?TGACCCCTAT
301 GTAAACTACT?CCTCCCGCCA?TACCATAACC?CAGCCCTTCT?CCTACCACTC?CCGCTACTTT
361 ACCCCCAAAC?CTGTCCTAGA?TTCCACTATT?GATTACTTCC?AACCAAACAA?CAAAAGAAAT
421 CAGCTGTGGC?TGAGACTACA?AACTGCTGGA?AATGTGGACC?ACGTAGGCCT?CGGCACTGCG
481 TTCGAAAACA?GTATATACGA?CCAGGAATAC?AATATCCGTG?TAACCATGTA?TGTACAATTC
541 AGAGAATTTA?ATCTTAAAGA?CCCCCCACTT?AACCCTTAA
(2) preparation of PCV2 antibody test plate
0.05M Tris-HCl damping fluid with pH8.5 is made coating buffer, the dilution of pig II type PCV-II reorganization nls-defected capsid protein is 0.1-10 μ g/ml, add removable 96 hole ELISA Plate by 100 μ l/ holes, 37 ℃ 2 hours, 4 ℃ of bags are spent the night again, with 37 ℃ of sealings of 1%BSA 2 hours, fully wash with PBS (pH7.4) cleansing solution that contains 0.05% Tween-20, dry.Added 20% sucrose phosphate buffer room temperature protection again 3 hours, put the hothouse drying after, be used for the assembling of PCV2 antibody indirect ELISA diagnostic kit.
(3) the anti-pig IgG Polyclonal Antibody Preparation of goat
Take the pig whole blood, separation of serum is slightly carried pig IgG with the saturated ammonium sulphate method.Obtain the pure product of pig IgG through DEAE cellulose ion-exchange chromatography purifying again.With the pig IgG albumen of purifying by 50～100 μ g/kg body weight subcutaneous and intramuscular injection immune health goat 3～4 times, after the last immunity 10 days, venous blood collection is measured its serum antibody titer at 1: 2000 when above with ELISA, hyper-immune serum is collected in heart blood sampling or arteria carotis bloodletting; With the anti-pig IgG antibody of saturated ammonium sulfate method purifying goat, do not repeat, again through DEAE cellulose ion-exchange chromatography purifying, collect and concentrate goat-anti pig polyclonal antibody, be used to prepare PCV2 antibody assay kit enzyme conjugates working fluid.
(4) preparation of enzyme conjugates working fluid
With improvement sodium periodate method horseradish peroxidase (HRP) enzyme is coupled to the anti-pig IgG polyclonal antibody of goat.With ultrapure water 1ml dissolving horseradish peroxidase (HRP, RZ 〉=3.0) 5mg solution, added 4 ℃ of lucifuges of 1ml 0.06M sodium periodate solution 1 hour, add 1ml 160mM ethylene glycol, room temperature reaction 30 minutes, the acetate buffer solution 2-8 ℃ dialysed overnight of adding PH4.4 is changed liquid twice.The goat-anti pig IgG of 10mg purifying is added in the enzyme of above-mentioned activation, be transferred to bag filter, in the carbonic acid buffer of 0.05mM PH9.6, dialyse, 4 ℃ of stirrings spend the night (changing liquid twice).Dislysate is drawn in the centrifuge tube, adds the NaBH of 5mg/ml
4Liquid 0.4ml, 4 ℃ were reacted 2 hours.The saturated ammonium sulfate solution that adds equivalent, 4 ℃ 30 minutes, 4 ℃ 4000 left the heart 20 minutes, abandoned supernatant, drained.Precipitation is dissolved in a small amount of PBS, and in the bag filter of packing into, to 0.02M PBS (PH7.4) dialysis, 4 ℃ are spent the night.Liquid in the dislysate is drawn in the centrifuge tube, centrifugal, with the supernatant sucking-off, add equivalent glycerine, mixing ,-20 ℃ of preservations.Again containing 0.1%BSA, 0.05% Tween-20, the phosphate buffer of 0.01% merthiolate (pH7.4) is by 1: the frozen marker enzyme of 200-5000 dilution is as the enzyme conjugates working fluid.
(5) preparation of sample diluting liquid, cleansing solution, stop buffer
Sample diluting liquid is the 0.01M phosphate buffer (KH that contains 0.05% Tween-20
2PO
40.2g, Na
2HPO
412H
2O 2.9g, NaCl8g is settled to 1000ml, pH7.4 adds the 0.5ml Tween-20 again); 10 * concentrated cleaning solution is the 0.1M phosphate buffer (KH that contains 0.5% Tween-20
2PO
42g, Na
2HPO
412H
2O 29g, NaCl80g is settled to 1000ml, pH7.4 adds the 5ml Tween-20 again); Stop buffer is the 2M sulfuric acid solution, promptly measures the 111.2ml concentrated sulphuric acid (18M) dilution and is settled to 1000ml.
(6) preparation of positive control and negative control
The pig II type PCV-II standard positive serum that screening is obtained dilutes (OD with sample diluting liquid at 1: 100
450nm〉=1.00) add a small amount of red pigment and also press 1000U/ml adding penicillin and streptomysin, aseptic filtration is as PCV2 antibody indirect ELISA diagnostic kit positive control; The pig standard female serum that screening is obtained dilutes (OD with sample diluting liquid at 1: 100
450nm≤ 0.150), press 1000U/ml and add penicillin and streptomysin, aseptic filtration is as PCV2 antibody indirect ELISA diagnostic kit negative control.
(7) preparation of colour developing liquid
Take by weighing 200mg tetramethyl benzidine (TMB), after 100ml absolute ethyl alcohol or DMSO dissolving, be settled to 1000ml, preparation colour developing liquid A with distilled water; Take by weighing the 21g Citric Acid Mono, 28.2g ADSP (Na
2HPO
4), 6.4ml 0.75% hydrogen peroxide urea, distilled water is settled to 1000ml, and adjust pH is prepared colour developing liquid B to 4.5-5.0.
(8) kit detecting operation program
Its trace routine is: 1) dilution of 10 * concentrated cleaning solution is cleansing solution for 10 times; 2) serum to be checked is done dilution in 1: 100 with sample diluting liquid, add in the antibody test plate by 100 μ l/ holes, establish blank (only adding 100 μ l sample diluting liquids), negative control (PCV2 negative serum), positive control (PCV2 standard positive serum) simultaneously, hatched 20-45 minute for 37 ℃, dry; 3) every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; 4) every hole adds 100 μ l enzyme conjugates working fluids (blank does not add), hatches 20-45 minute for 37 ℃, dries; 5) every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; 6) add 50 μ l colour developing liquid A and 50 μ l colour developing liquid B successively, mixing, 37 ℃ of lucifuges were hatched 5-15 minute; 7) add 50 μ l stop buffers, under the 450nm wavelength, read every hole absorbance value (OD with microplate reader
450nmValue).
(9) criterion as a result
Criterion is with sample OD to be checked
450nmValue and standard female OD
450nmValue ratio (P/N) is judged to the positive more than or equal to 2.2, and is negative smaller or equal to 1.7.Be suspicious, must heavily examine that the P/N value is judged to the positive more than or equal to 2.0 when resurveying, and is negative less than 2.0 between 1.7-2.2.If positive control does not have obvious chromogenic reaction, or negative control appearance colour developing, show that kit lost efficacy or detecting operation is wrong, need to detect again.
Embodiment PCV2 antibody indirect ELISA diagnostic kit
Principle is referring to Fig. 1, press (1) method step preparation reorganization PCV2 dCap albumen among the embodiment, prepare PCV2 antibody test plate by (2) method step among the embodiment, prepare PCV2 antibody assay kit enzyme conjugates working fluid by (3) (4) method step among the embodiment, prepare sample diluting liquid by (5) method step among the embodiment, cleansing solution, stop buffer, prepare antibody assay kit positive control and negative control by (6) method step among the embodiment, press (7) method step preparation colour developing liquid A among the embodiment, colour developing liquid B, at last, various compositions are assembled into the PCV2 antibody assay kit.
When using PCV2 antibody indirect ELISA diagnostic kit to detect pig PCV2 antibody or antibody horizontal, the porcine blood serum that separates will be gathered, make 1: 100 or gradient dilution with sample diluting liquid, prepare sample solution to be checked or gradient dilution sample solution, with sample to be checked, positive and negative control adds the Detection of antigen plate respectively, press (8) middle running program among the embodiment, room temperature effect 30 minutes, cleansing solution washing, add the enzyme conjugates working fluid, room temperature effect 30 minutes, the same washing adds colour developing liquid A, each 50 μ l of colour developing liquid B, room temperature lucifuge colour developing 5min drips the reaction of stop buffer 50 μ l color development stopping, and microplate reader detects OD
450nmValue.Press the criterion result of determination in (9) among the embodiment.Detect hole OD
450nmValue and standard negative control OD
450nmValue ratio (P/N) is judged to the positive more than or equal to 2.2.Then detect the PCV2 antibody titer of the high dilution of the positive sample in hole for the gradient dilution sample for this serum to be checked.If the PCV2 positive control does not have obvious chromogenic reaction, or negative control appearance colour developing, show that kit lost efficacy or detecting operation is wrong, need to detect again.
Claims (8)
1. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit, it is characterized in that: be provided with the antibody test plate in the kit, the enzyme conjugates working fluid, positive control, negative control, sample diluting liquid, 10 * concentrated cleaning solution, colour developing liquid A, colour developing liquid B and stop buffer, the antibody test plate is for wrapping by the removable 96 hole ELISA Plate of pig II type PCV-II reorganization nls-defected capsid protein, and the enzyme conjugates working fluid is the anti-pig IgG polyclonal antibody of horseradish peroxidase-labeled goat, positive control is a pig II type PCV-II standard positive serum, and negative control is a pig standard female serum.
2. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1, it is characterized in that: said pig II type PCV-II reorganization nls-defected capsid protein be prepared as the capsid protein gene sequence (fragment of capsid protein gene 3 ' end 579bp) of utilizing round pcr amplification pig II type PCV-II stoning positioning signal, glutathione-S-transferase gene (GST) downstream that is inserted into prokaryotic expression carrier pGEX-4T-1 makes up the pGEX-PCV2-dCap expression vector, and transformed into escherichia coli BL21, obtain pig II type PCV-II reorganization nls-defected capsid protein through 0.1mM IPTG abduction delivering and through commercial GST affinity column purifying, this albumen has kept the antigenicity of complete capsid protein, can with pig II type PCV-II positive serum specific bond.
3. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1; it is characterized in that: the optimum preparating condition of said antibody test plate is; 0.05M Tris-HCl damping fluid with pH8.5 is made coating buffer; the dilution of pig II type PCV-II reorganization nls-defected capsid protein is 0.1-10 μ g/ml; add in the polystyrene micropore plate by 100 μ l/ holes; 37 ℃ 2 hours; 4 ℃ of bags are spent the night again; dry; contain 1% bovine serum albumin(BSA) (BSA) by the adding of 100 μ l/ holes; 37 ℃ of sealings of the phosphate buffer of PH7.4 2 hours; washing dries; added 20% sucrose phosphate buffer room temperature protection again 3 hours, put the hothouse drying after, preserve in the packaging bag that contains drying agent of packing into.
4. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1, it is characterized in that: being prepared as of said enzyme conjugates working fluid: prepare horseradish peroxidase (HRP with ultrapure water, RZ 〉=3.0) solution, added 4 ℃ of lucifuges of 0.06M sodium periodate solution 1 hour, add 160mM ethylene glycol, room temperature reaction 30 minutes, 1 hour 2-8 ℃ of dialysed overnight of acetate buffer solution of adding PH4.4 is changed liquid twice.The goat-anti pig IgG of purifying is added in the enzyme of above-mentioned activation, be transferred to bag filter, in the carbonic acid buffer of 0.05mMPH9.6, dialyse, 4 ℃ of stirrings spend the night (changing liquid twice).Dislysate is drawn in the centrifuge tube, adds the NaBH that newly joins
4Liquid, 4 ℃ were reacted 2 hours.4 ℃ of the saturated ammonium sulfate solution 30 minutes that add equivalent, 4 ℃ centrifugal 20 minutes, abandon supernatant, drain.Precipitation is dissolved in a small amount of PBS, and in the bag filter of packing into, to 0.02MPBS (PH7.4) dialysis, 4 ℃ are spent the night.Liquid in the dislysate is drawn in the centrifuge tube, centrifugal, with the supernatant sucking-off, add equivalent glycerine, mixing ,-20 ℃ of preservations.Again containing 0.1%BSA, 0.05% Tween-20,0.01% merthiolate, the phosphate buffer of pH7.4 is by 1: the anti-pig IgG polyclonal antibody of goat of the horseradish peroxidase-labeled that the 200-5000 dilution is frozen is as the enzyme conjugates working fluid.
5. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1, it is characterized in that: said sample diluting liquid is the 0.01M phosphate buffer (pH7.4) that contains 0.05% Tween-20; 10 * concentrated cleaning solution is the 0.1M phosphate buffer (pH7.4) that contains 0.5% Tween-20; Colour developing liquid A is tetramethyl benzidine (TMB) solution of 0.2mg/ml, and colour developing liquid B is the citric acid-phosphate buffer that contains 0.5 ‰ hydrogen peroxide ureas; Stop buffer is the 2M sulfuric acid solution.
6. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1 is characterized in that: the pig II type PCV-II standard positive serum (OD of said positive control for obtaining through screening
450nm〉=1.00) add a small amount of red pigment and also press 1000U/ml adding penicillin and streptomysin, aseptic filtration; Negative control is the pig standard female serum (OD that obtains through screening
450nm≤ 0.150), presses 1000U/ml and add penicillin and streptomysin, aseptic filtration.
7. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1, its trace routine is: 1) dilution of 10 * concentrated cleaning solution is cleansing solution for 10 times; 2) serum to be checked is done dilution in 1: 100 with sample diluting liquid, add in the antibody test plate by 100 μ l/ holes, establish blank (only adding 100 μ l sample diluting liquids), negative control (PCV2 negative serum), positive control (PCV2 standard positive serum) simultaneously, hatched 20-45 minute for 37 ℃, dry; 3) every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; 4) every hole adds the enzyme conjugates working fluid (blank does not add) of 100 μ l, hatches 20-45 minute for 37 ℃, dries; 5) every hole adds cleansing solution 200 μ l, washs 6 times, each 1 minute at interval, pats dry; 6) add 50 μ l colour developing liquid A and 50 μ l colour developing liquid B successively, 37 ℃ of lucifuges were hatched 5-15 minute; 7) add 50 μ l stop buffers, under the 450nm wavelength, read every hole absorbance value (OD with microplate reader
450nmValue).
8. a boar II type circovirus antibody indirect ELISA diagnostic reagent kit according to claim 1, its criterion that detects sample is: with sample OD to be checked
450nmValue and standard female OD
450nmThe ratio (P/N) of value is judged to the positive more than or equal to 2.2, and is negative smaller or equal to 1.7.Be suspicious, must heavily examine that the P/N value is judged to the positive more than or equal to 2.0 when resurveying, and is negative less than 2.0 between 1.7-2.2.
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|---|---|---|---|
| CN 200410066998 CN1588076A (en) | 2004-09-28 | 2004-09-28 | Indirect ELISA diagnostic reagent kit for pig B type circular virus antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410066998 CN1588076A (en) | 2004-09-28 | 2004-09-28 | Indirect ELISA diagnostic reagent kit for pig B type circular virus antibody |
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