CN101629956A - ELISA kit for detecting porcine circovirus antibody II - Google Patents

ELISA kit for detecting porcine circovirus antibody II Download PDF

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CN101629956A
CN101629956A CN200910043875A CN200910043875A CN101629956A CN 101629956 A CN101629956 A CN 101629956A CN 200910043875 A CN200910043875 A CN 200910043875A CN 200910043875 A CN200910043875 A CN 200910043875A CN 101629956 A CN101629956 A CN 101629956A
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elisa
kit
antigen
cap
enzyme
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CN101629956B (en
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余兴龙
葛猛
李润成
蒋大良
罗维
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Beijing golden Nuobaitai Biological Technology Co. Ltd.
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Hunan Agricultural University
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Abstract

The invention relates to an ELISA kit for detecting porcine circovirus antibodies II, wherein, the kit is developed according to the double antigen sandwich ELISA principle. The ELISA kit comprises a prepacked ELISA antigen plate bar, a cleaning solution, 100 times concentrated enzyme labeled antigen, an enzyme labeled dilution, a zymolyte coloration, a stopping solution, standard PCV2 negative sera, and standard PCV2 positive sera. The kit replaces enzyme labeled anti-antibodies with the enzyme labeled antigen. The enzyme labeled antigen can not combine with antibodies absorbed with ELSA plate without specificity. Meanwhile, the serum specimen to be detected does not need to be pre-diluted, and the serum specimen can be directly mixed with the enzyme labeled antigen with working concentration to be directly used for determination. The ELISA kit has the advantages of simple operation and shorter detecting time.

Description

Detect the ELISA kit of porcine circovirus 2 type antibody
Technical field:
The present invention relates to the ELISA kit of a kind of detection porcine circovirus 2 type (PCV2) antibody, belong to the Vet Biotechnology field.Be used for the antibody test of 2 porcine circovirus, judge in the swinery circovurus type 2 infection conditions and this sick popularity and immune effect are monitored with this.
Background technology:
Porcine circovirus 2 type (porcine circovirus 2, PCV2) with swinery in the numerous disease that takes place closely related, such as weanling pig multisystemic wasting syndrome (Post weaning multisystemwasting syndrome, PMWS) the scorching ephritis syndrome of pigskin (Porcine dermatitis andnephropathy syndrome, PDNS), the sow breeding difficulty, the hyperplasia enteritis, piglet is congenital trembles, the sick syndrome of porcine respiratory etc., we will be referred to as PCVD (porcinecircovirus disease by the various disease conditions that PCV2 causes, PCVD), in many PCVD, the most common with PMWS, PMWS causes mainly that pig is gradual and becomes thin, diarrhoea, respiratory symptom, ochrodermia or jaundice occurs, production performance reduces.PCV2 is except itself causes swine disease, immune system because of the infringement pig, the immunologic function that suppresses pig can also interact with common schweineseuche disease pathogen such as pig blue-ear disease, swine flu, porcine mycoplasmal pneumonia, haemophilus parasuis and Streptococcus suis, increases the weight of the harm of these diseases.At present PCV2 wide-scale distribution has worldwide caused enormous economic loss to pig industry.
The Histopathology that mainly comprises the laboratory diagnostic method of PCV2 detects, the separation of virus and evaluation, PCR, indirect immunofluorescence assay (IFA), in situ hybridization (ISH), SABC method (IHC) etc., but these methods are mainly used in the detection cause of disease, and higher to test condition and technical requirement.
In recent years, many both at home and abroad R﹠D institutions have set up the enzyme linked immunosorbent assay (ELISA) that detects PCV2 antibody, and advantages such as that this method has is simple to operate, susceptibility is high, quick, easy standardization are particularly suitable for the detection of extensive blood serum sample.And be used at present check that the ELISA method of PCV2 antibody mainly is an indirect method, the shortcoming of this method is that the non-specific antibody of high concentration contained in the serum can directly be adsorbed onto on the solid phase carrier, sometimes also adsorbable surface to envelope antigen, thus cause background higher or false-positive result may occur.
Summary of the invention:
Technical matters to be solved by this invention is: at above-mentioned the deficiencies in the prior art, provide a kind of ELISA kit of the detection porcine circovirus 2 type antibody of developing according to the principle of double antigens sandwich ELISA, be used for the detection of porcine circovirus 2 type antibody.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of ELISA kit that detects porcine circovirus 2 type antibody, and described kit develops according to double antigens sandwich ELISA principle, comprising:
The a.ELISA lath: each kit comprises 4 vacuum-packed elisa plates, and every block of plate contains dismountable ELISA micropore lath that has wrapped detected antigen, and specification is 8 holes * 12; This envelope antigen is that (accession number: AY943819), design two primers, upstream primer is: GC according to the gene order of PCV2 among the GenBank GAATTCATGGGCATCTTCAACACCCGCCTCTC; Downstream primer is: GC GTCGACTTACTTAGGGTTAAGTGGGGGGTC; Method by PCR increases from pig PCV2 genome and does not contain the Cap gene of nuclear localization signal, and it is cloned in pET32-a (+) prokaryotic expression carrier, makes up the pET32a-Cap recombinant expression carrier; Be converted in the e. coli bl21 (DE3), with reference to the pET Syetem Manual of Novagen company express recombinant Cap fusion, the fusion of expressing with the affinity chromatography purifying makes;
B. cleansing solution: 10 times of concentrated pH are 7.4 the 1M PBS solution 200ml that contains 0.5% (percent by volume) Tween-20;
C.100 doubly concentrated enzyme-labelled antigen 500 μ l: the Cap fusion of above-mentioned purifying is cut the fusion part with pig source enterokinase enzyme, and reuse the Cap albumen that the affinity column purifying obtains not contain nuclear internalization signal peptide, and with the ultrafiltration pipe it is concentrated into 2.0mg/ml, and adopt improvement sodium periodate method with horseradish peroxidase on Cap albumen;
D. enzyme mark dilution: pH is 7.4 0.1M PBS solution 50ml;
E. substrate colour developing liquid 50ml: take by weighing the 200mg tetramethyl benzidine, after 100ml absolute ethyl alcohol or DMSO dissolving, be settled to 1000ml, configuration colour developing liquid A with distilled water; Take by weighing the 9.33g citric acid, the 14.6g sodium hydrogen phosphate adds 0.75% (mass volume ratio) hydrogen peroxide urea 6.4ml, adjust pH to 5.0～5.4, and distilled water is settled to 1000ml, preparation colour developing liquid B; Colour developing liquid A, B equal-volume mix, and are developer; Measuring the 111.2ml18M diluting concentrated sulfuric acid is settled to 1000ml and promptly uses;
F. stop buffer: 2M sulfuric acid solution 50ml;
G. each 1.0ml of Standard PC V2 positive and negative serum.
Above-mentioned employing improvement sodium periodate method on Cap albumen, specifically is with horseradish peroxidase in 5mg/ml HRP solution, adds the 0.1M NaIO that 0.2ml newly joins 4Solution, lucifuge stirred 20 minutes under the room temperature, 4 ℃ of dialysed overnight of 1mM sodium-acetate buffer to pH4.4, add 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH of the HRP of above hydroformylation be elevated to 9.0～9.5, add 2ml Cap albumen to be marked then immediately to 1ml 0.01M carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently, added the 4mg/ml NaBH that 0.1ml newly joins 4Liquid, mixing was placed 2 hours for 4 ℃, and the 0.15M PBS4 ℃ dialysed overnight to pH7.4 under agitation dropwise adds the equal-volume saturated ammonium sulfate, places centrifugal half an hour of 3000rpm after 1 hour, abandons supernatant for 4 ℃; Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of 0.15M of a small amount of PH7.4, to the 0.15MPBS buffer saline dialysis of pH7.4, remove ammonium ion after, 10000rpm removed precipitation in centrifugal 30 minutes, supernatant is enzyme-labelled antigen.
Double antigens sandwich ELISA is owing to having higher susceptibility and specificity, and is widely-used on people doctor.Be to replace enzyme mark antiantibody with the main difference part of indirect ELISA with enzyme-labelled antigen, enzyme-labelled antigen not can with the antibodies of non-specific adsorption, detect sample simultaneously and need not dilute, can be directly used in mensuration, so its susceptibility and specificity are relatively higher than indirect method.On this basis, the present invention joins enzyme-labelled antigen and serum to be checked in the detection hole simultaneously, and one step of reaction is finished.
Use this kit the pig breeding is detected with breathing syndrome virus standard positive serum, CSFV standard positive serum, Pseudorabies virus standard positive serum, foot and mouth disease virus standard positive serum, pig parvoviral standard positive serum, Escherichia coli standard positive serum, the result is all negative.No cross reaction between the used diagnostic antigen of this kit and other pathogenic autoantibody is described.
And ELISA kit of the present invention and Spain INGENASA company kit are relatively: the indirect ELISA reagent kit (INGEZIM CIRCO IgG 11.PCV.K1) that PCV2 antibody detects in INGENASA company is widely used in pig industry developed countries such as America and Europes, is the high-quality diagnostic kit of the present a kind of PCV2 of detection antibody of generally acknowledging in the world.With this kit 94 parts of clinical censorship serum of indirect ELISA reagent kit (INGEZIM CIRCO IgG 11.PCV.K1) detection with the detection PCV2 of INGENASA company antibody, relatively its result's consistance the results are shown in Table 1 respectively.
The comparison test of this kit of table 1 and INGENASA company kit
Figure G2009100438759D00041
From the contrast test of ELISA kit of the present invention and INGENASA company kit, the final decision in most of test sample is unanimity as a result, has only minority weak positive serum and OD 450Value is inconsistent near the testing result of the negative serum of criterion, and this may be relevant with the selected diagnostic antigen errors different and two systems itself of two kinds of ELISA detection systems.
In addition, detect the indirect ELISA reagent kit of PCV2 antibody compares with INGENASA company, ELISA kit of the present invention only needs a step to hatch, and saved the washing process that enzyme labelled antibody is hatched process (30 minutes) and 6 times, thereby saved 1 hour detection time nearly.
ELISA kit of the present invention is compared with the indirect ELISA method of traditional detection porcine circovirus 2 type antibody, has close susceptibility and better specificity, and operating process simpler, required detection time still less.
Description of drawings:
Fig. 1 is recombinant C ap Expression of Fusion Protein and purifying rear electrophoresis figure.
Wherein: the 1st swimming lane: recombinant expressed bacterium cracking postprecipitation; The 2nd swimming lane: supernatant after the recombinant expressed bacterium cracking; The 3rd swimming lane: the full bacterium of negative control; The 4th swimming lane: the protein standard is respectively 94KD, 66KD, 45KD, 30KD, 26KD, 20KD, 14KD from top to bottom; The 5th swimming lane: the recombinant C ap fusion of purifying.Fig. 2 is that the enzyme of recombinant C ap fusion is cut and the purifying and the concentrated rear electrophoresis figure of Cap albumen.
Wherein: the 1st swimming lane: Cap albumen behind the ultrafiltration and concentration; The 2nd swimming lane: enzyme is cut Cap albumen behind the purifying; The 3rd swimming lane: recombinant C ap fusion protease is cut; The 4th swimming lane: the recombinant C ap fusion of purifying; The 5th swimming lane: the protein standard is respectively 97KD, 66KD, 44KD, 29KD, 22KD, 14KD from top to bottom.
Embodiment:
The preparation of envelope antigen:
According to the gene order of PCV2 among the GenBank (accession number: AY943819), design two primers, on have primer to be: GC GAATTCATGGGCATCTTCAACACCCGCCTCTC; Downstream primer is: GC GTCGACTTACTTAGGGTTAAGTGGGGGGTC.Method by PCR increases from pig PCV2 genome and does not contain the Cap gene of nuclear localization signal, with its be cloned into pET32-a (+) prokaryotic expression carrier (available from Novagen company, production code member: 69015-3), make up the pET32a-Cap recombinant expression carrier.Be converted in the e. coli bl21 (DE3), with reference to the pET Syetem Manual of Novagen company express recombinant Cap fusion, this albumen is solubility expression.With affinity chromatography (His-BindKit of Novagen company, production code member: 70239-3) the purifying fusion of expressing, see Fig. 1.
The preparation of PCV2 antibody test plate:
Cap fusion with 0.05M carbonate buffer solution (pH9.6) dilution purifying, determine that with the square formation method best bag is 5 μ g/ml by concentration, get removable 96 hole ELISA Plate, every hole adds 100 μ l, 4 ℃ of bags washed 2 times with PBS (pH7.4) cleansing solution (PBST) that contains 0.05% Tween-20 (volume ratio) after 24 hours, with 37 ℃ of sealings of 5% skimmed milk power (mass volume ratio) 1 hour, fully washed with PBST, room temperature is air-dry, preparation antibody test plate.
The preparation of enzyme-labelled antigen:
Use pig source enterokinase enzyme (available from Nanjing Jin Site company the Cap fusion of purifying, production code member: Z01003) cut the fusion part, and reuse the Cap albumen that the affinity column purifying obtains not contain nuclear internalization signal peptide, and with the ultrafiltration pipe (available from Millipore company, filter core 10KDa, production code member: U650886) it is concentrated into 2.0mg/ml and is used for mark, see Fig. 2.
Adopt improvement sodium periodate method that horseradish peroxidase (HRP) is coupled on the Cap albumen.Detailed process is as follows: in 5mg/ml HRP solution, add the 0.1M NaIO that 0.2ml newly joins 4Solution, lucifuge stirred 20 minutes under the room temperature, to 4 ℃ of dialysed overnight of 1mM sodium-acetate buffer (PH4.4), add 20 μ l 0.2MPH9.5 carbonate buffer solutions, make the pH of the HRP of above hydroformylation be elevated to 9.0～9.5, add 2ml Cap albumen to be marked then immediately to 1ml 0.01M carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently, added the 4mg/ml NaBH that 0.1ml newly joins 4Liquid, mixing was placed 2 hours for 4 ℃, to 4 ℃ of dialysed overnight of 0.15M PBS (PH7.4), under agitation dropwise added the equal-volume saturated ammonium sulfate, placed centrifugal half an hour of 3000rpm after 1 hour, abandoned supernatant for 4 ℃.Sediment is washed secondary with semi-saturation ammonium sulfate, last sediment is dissolved among the PBS (PH7.4) of a small amount of 0.15M, and PBS (PH7.4) the buffer saline dialysis to 0.15M (detects with Nai Shi reagent) behind the removal ammonium ion, 10000rpm removed precipitation in centrifugal 30 minutes, and supernatant is enzyme-labelled antigen.Determine with the individual event titrimetry that the ELISA of enzyme-labelled antigen tires and reach 1: 6000.
The preparation of damping fluid:
Preparation 0.1M PBS (PH7.4) is as the enzyme-labelled antigen dilution of PCV2 antibody assay kit, and the 1M PBS (PH7.4) that contains 0.5% Tween-20 is 10 * concentrated cleaning solution.
The preparation of developer:
Take by weighing 200mg tetramethyl benzidine (TMB), after 100ml absolute ethyl alcohol or DMSO dissolving, be settled to 1000ml, configuration colour developing liquid A with distilled water.Take by weighing the 9.33g citric acid, 14.6g sodium hydrogen phosphate (Na 2HPO 412H 2O), add 0.75% (mass volume ratio) hydrogen peroxide urea 6.4ml, adjust pH to 5.0～5.4, distilled water is settled to 1000ml, preparation colour developing liquid B.Colour developing liquid A, B equal-volume mix, and are developer.Measure the 111.2ml concentrated sulphuric acid (18M) dilution and be settled to 1000ml, standby.
Positive and negative contrast and criterion:
In the ELISA process, can there be certain error between different operating personnel and the different detection batch, such as, the error in application of sample and reaction time etc., these errors can cause the error of test sample OD value.But test sample OD value/standard model OD value (S/P value) can be eliminated error, and the accurate response testing result is the criterion of optimizing.The NaN3 that adds 0.2mg/ml in PCV2 standard positive and negative serum respectively makes them can be 4 ℃ of long preservation and antibody titer changes little.
Criterion: when standard positive serum OD value 〉=0.8; Standard female serum OD value＜0.3 illustrates that test effectively.When S/P 〉=0.3, sample is anti-PCV2 antibody positive, and when S/P＜0.3, sample is anti-PCV2 negative antibody.
The assembling of kit:
The a.ELISA lath: each kit comprises 4 vacuum-packed elisa plates, and every block of plate contains dismountable elisa plate bar that has wrapped detected antigen, and specification is 8 holes * 12.
B. cleansing solution: 10 times of concentrated 1M PBS (PH7.4) 200ml that contain 0.5% (volume) Tween-20.
C.100 doubly concentrated enzyme-labelled antigen 500 μ l.
D. enzyme is marked dilution 50ml.
E. substrate colour developing liquid 50ml.
F. stop buffer: 2M sulfuric acid solution 50ml.
G. each 1.0ml of Standard PC V2 positive and negative serum.
The basic operational steps of sample detection is:
A. the enzyme-labelled antigen that concentrates is diluted to working concentration with enzyme mark dilution 100, joins in the reacting hole of elisa plate, every hole adds 80 μ l, and every then hole adds serum 20 μ l, the concussion mixing, and shrouding is put 37 ℃ and was hatched 1 hour.
B. every hole adds 300 μ l cleansing solutions and washes plate 5 times, blot empty in behind the residual liquid every hole add 100 μ l substrate TMB, 37 ℃ of lucifuges colour developings 15 minutes.
C. every hole adds stop buffer 2M sulfuric acid 100 μ l color development stopping, uses microplate reader reading under the 450nm wavelength immediately.
Annotate: except that the enzyme-labelled antigen that concentrates, other reagent constituents and serum to be checked are with before returning to room temperature.

Claims (2)

1, a kind of ELISA kit that detects porcine circovirus 2 type antibody, it is characterized in that: described kit develops according to double antigens sandwich ELISA principle, comprising:
The a.ELISA lath: each kit comprises 4 vacuum-packed elisa plates, and every block of plate contains dismountable ELISA micropore lath that has wrapped detected antigen, and specification is 8 holes * 12; This envelope antigen is that (accession number: AY943819), design two primers, upstream primer is: GC according to the gene order of PCV2 among the GenBank GAATTCATGGGCATCTTCAACACCCGCCTCTC; Downstream primer is: GC GTCGACTTACTTAGGGTTAAGTGGGGGGTC; Method by PCR increases from pig PCV2 genome and does not contain the Cap gene of nuclear localization signal, and it is cloned in pET32-a (+) prokaryotic expression carrier, makes up the pET32a-Cap recombinant expression carrier; Be converted in the e. coli bl21 (DE3), with reference to the pET Syetem Manual of Novagen company express recombinant Cap fusion, the fusion of expressing with the affinity chromatography purifying makes;
B. cleansing solution: 10 times of concentrated pH are 7.4 the 1M PBS solution 200ml that contains 0.5% (percent by volume) Tween-20;
C.100 doubly concentrated enzyme-labelled antigen 500 μ l: the Cap fusion of above-mentioned purifying is cut the fusion part with pig source enterokinase enzyme, and reuse the Cap albumen that the affinity column purifying obtains not contain nuclear internalization signal peptide, and with the ultrafiltration pipe it is concentrated into 2.0mg/ml, and adopt improvement sodium periodate method with horseradish peroxidase on Cap albumen;
D. enzyme mark dilution: pH is 7.4 0.1M PBS solution 50ml;
E. substrate colour developing liquid 50ml: take by weighing the 200mg tetramethyl benzidine, after 100ml absolute ethyl alcohol or DMSO dissolving, be settled to 1000ml, configuration colour developing liquid A with distilled water; Take by weighing the 9.33g citric acid, the 14.6g sodium hydrogen phosphate, the hydrogen peroxide urea 6.4ml of adding 0.75% (mass volume ratio), adjust pH to 5.0～5.4, distilled water is settled to 1000ml, preparation colour developing liquid B; Colour developing liquid A, B equal-volume mix, and are developer; Measuring the 111.2ml18M diluting concentrated sulfuric acid is settled to 1000ml and promptly uses;
F. stop buffer: 2M sulfuric acid solution 50ml;
G. each 1.0ml of Standard PC V2 positive and negative serum.
2, the ELISA kit of detection porcine circovirus 2 type antibody as claimed in claim 1, it is characterized in that: described employing improvement sodium periodate method with horseradish peroxidase on Cap albumen, specifically be in 5mg/ml HRP solution, add the 0.1M NaIO that 0.2ml newly joins 4Solution, lucifuge stirred 20 minutes under the room temperature, 4 ℃ of dialysed overnight of 1mM sodium-acetate buffer to pH4.4, add 20 μ l 0.2M pH9.5 carbonate buffer solutions, make the pH of the HRP of above hydroformylation be elevated to 9.0～9.5, add 2ml Cap albumen to be marked then immediately to 1ml 0.01M carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently, added the 4mg/ml NaBH that 0.1ml newly joins 4Liquid, mixing was placed 2 hours for 4 ℃, and 0.15MPBS4 ℃ of dialysed overnight to pH7.4 under agitation dropwise adds the equal-volume saturated ammonium sulfate, places centrifugal half an hour of 3000rpm after 1 hour, abandons supernatant for 4 ℃; Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of 0.15M of a small amount of PH7.4, to the PBS buffer saline dialysis of the 0.15M of pH7.4, remove ammonium ion after, 10000rpm removed precipitation in centrifugal 30 minutes, supernatant is enzyme-labelled antigen.
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