CN103901213A - Detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus - Google Patents

Abstract

The invention provides an ELISA (Enzyme-Linked Immunosorbent Assay) detection method for distinguishing generation of antibody through activated vaccine and inactivated vaccine of tembusu virus. The method comprises the following steps: separating and purifying E protein and NS5 protein of the tembusu virus which is expressed by using escherichia coli pronucleus; adding a sealing solution, duck serum to be detected and a PBST (Plumbous Stearate) dilution solution into a 96-hole ELISA plate, diluting duck-resisting rabbit (or sheep) secondary antibodies which are marked by HRP (Horse Reddish Peroxidase) by using the PBST dilution solution, and reading OD450nm value after the reaction is ended, wherein the antibody is proved to be generated by the inactivated vaccine if the OD450nm of the E protein is positive and an OD450nm value of the NS protein is negative in the result, and the antibody is proved to be generated by infection of the activated vaccine if both the OD450nm of the E protein and the OD450nm value of the NS protein are positive. The ELISA detection method of the tembusu virus is capable of distinguishing generation of antibody through the activated vaccine and the inactivated vaccine of the tembusu virus, plays an important role of monitoring duck tembusu virus antibody level and identifying infection of the tembusu virus, and is simple to operate, low in cost and applicable to detection of samples in large scales.

Description

A kind of detection method of distinguishing tembusu virus poison alive and inactivated vaccine generation antibody

(1) technical field

The present invention relates to a kind of detection method of distinguishing tembusu virus poison alive and inactivated vaccine generation antibody, belong to cause of disease detection technique field.

(2) background technology

Flavivirus (Flavivirus) is the tunicary single positive chain RNA virus of large numbers of tools, flaviviridae (Flaviviridae) comprises 3 Tobamovirus, be Flavivirus (flavivirus), pestivirus (pestivirus) and Hepacivirus (hepacivirus), have the virus of kind more than 60.At present, research shows, cause duck infection morbidity in flaviviridae, Flavivirus tembusu virus (Tembusu virus, TMUV).TMUV belongs to mosquito and passes arboviruse, may be through mosquitoes spread; In dead sparrow body in duck field, detect TMUV, prompting virus can be through bird; Can cause the propagation of cause of disease between different ducks field with malicious mosquito and sparrow.In sick duck tissue sample, the recall rate of membrana follicularis is the highest, reaches 93%, and whether prompting TMUV can, through ovum vertical transmission, should study and draw attention.With malicious duck the allocation and transportation of different regions (or the means of transport polluting) very easily become infectious disease on a large scale with the channel of fast propagation.Therefore the prevention of qualification in advance that, tembusu virus is lived malicious can reduce the generation of extensive infectious disease.

Tembusu virus be 2010 in East China emerging cause of disease.Inactivated vaccine inoculation is one of these sick effective measures of prevention, easily in tembusu virus epidemiology survey, produces and obscures but the antibody producing after inactivated vaccine inoculation infects with the poison of living the antibody producing.The antibody detecting that is not easily distinguishable is live poison infection or inactivated vaccine generation, easily causes larger economic loss to production.At present, the competitive ELISA of having reported and indirect ELISA only can detect tembusu virus antibody, and cannot distinguish antibody is to be produced by natural infection or inactivated vaccine immunity.Therefore, set up a kind of can detect and distinguish tembusu virus infect with the method for inactivated vaccine immunity generation antibody significant.

(3) summary of the invention

In order to address the above problem, the invention provides a kind of detection method of distinguishing tembusu virus poison alive and inactivated vaccine generation antibody.

The present invention can detect and distinguish tembusu virus and infect and inactivated vaccine immunity generation antibody, contributes to evaluate immune effect of vaccine, whether understands animal by situations such as virus infectionses.

Distinguish the detection method that tembusu virus poison alive and inactivated vaccine produce antibody, realize by following steps:

1) separation and purification utilizes tembusu virus E albumen and the NS5 albumen of escherichia coli prokaryotic expression; Dilute respectively E albumen and NS5 albumen with 1 × carbonate buffer solution (pH is between 9.5-9.7), making E protein concentration is 0.5ng/ μ L, and NS5 protein concentration is 1.0ng/ μ L; The E protein solution of dilution and NS5 protein solution are all joined respectively in two 96 hole elisa plates by 100 μ L/ holes, hatch 12 hours for 4 DEG C, wash 5 times with PBS cleansing solution, dry residual liquid on plate;

2) every hole in two 96 hole elisa plates after step 1) drying is added to 200 μ L confining liquids, incubated at room 1h, washes 5 times with PBS cleansing solution, dries residual liquid on plate;

3) by step 2) every hole first adds 10 μ L duck serum to be checked to add 90 μ LPBST dilutions again in two 96 hole elisa plates after drying, mixes, and hatches 1h for 37 DEG C, washes 5 times residual liquid on drying plate with PBS cleansing solution;

4) two anti-with the anti-duck of rabbit (or sheep) of PBST diluted HRP mark, by two anti-two 96 hole elisa plates that add after step 3) dries of dilution (every hole 100 μ L), hatches 1h for 37 DEG C, washes 5 times residual liquid on drying plate with PBS cleansing solution;

5) TMB nitrite ion is joined in two 96 hole elisa plates after step 4) dries by 100 μ L/ holes, 37 DEG C of lucifuges are hatched 15min;

6) every hole adds 50 μ L3M sulfuric acid stop buffers, and vibration is gently read OD450nm value by microplate reader:

E albumen OD450nm value result of determination in sample:

OD450 >=0.6897, serum to be checked is positive;

OD450<0.5841, serum to be checked is negative;

0.6897 > OD450 >=0.5841, be judged to suspicious, according to step 1)-6) again detect;

NS5 albumen OD450nm value result of determination in sample:

OD450 >=0.5017, serum to be checked is positive;

OD450<0.4223, serum to be checked is negative;

0.5017 > OD450 >=0.4223, be judged to suspicious, according to step 1)-6) again detect;

In result, the positive and negative explanation antibody of NS5 albumen OD450nm value of E albumen OD450nm is that inactivated vaccine produces; If both all positive explanation antibody by live poison infect produce.

Beneficial effect of the present invention:

The tembusu virus ELISA detection method that the present invention sets up has fast, stablizes, specificity is high, sensitivity is higher, can distinguish tembusu virus poison alive and produce antibody with inactivated vaccine, be applicable to the operation that robotization and batch sample detect, duck tembusu virus antibody surveillance and tembusu virus infection qualification are had to important effect.The present invention can use in basic unit, simple to operate, and cost is lower, is applicable to large-scale sample detection.

(4) brief description of the drawings

Fig. 1 is western bloting figure after E and NS5 fusion protein purification.

Wherein M swimming lane is molecular weight of albumen standard; Swimming lane 1 is the western bloting analysis result of E fusion; Swimming lane 2 is the western bloting analysis result of NS5 fusion.Illustrate that E and NS5 fusion have good specificity.

(5) embodiment

Various reagent component of the present invention is:

1 × carbonate buffer solution (coated damping fluid) Na ₂cO ₃0.2756g, NaHCO ₃0.6216g, dissolves and is settled to 100mL, pH value 9.5-9.7,4 DEG C of preservations with distilled water;

PBS cleansing solution: NaCl4.25g, NaH ₂pO ₄2H ₂o0.178g, Na ₂hPO ₄12H ₂o1.386g, dissolves and is settled to 500mL with distilled water, and pH value is 7.1-7.3;

PBST dilution: every 1L PBS cleansing solution adds Tween-200.5mL, fully mixes;

Confining liquid: 5g drymilk is dissolved in 100mL PBST dilution, and short-term preservation, in 4 DEG C, is stored in-20 DEG C for a long time.

TMB nitrite ion: formed with the ratio mixed preparing of volume ratio 39:1 by Buffer A and Buffer B;

Buffer A: take 66.5063g potassium citrate (potassium citrate), steam water-soluble solution and regulate pH value to 4.0 with concentrated hydrochloric acid with 800mL tetra-, add 314 μ L H ₂o ₂, be settled to 1L with distilled water; 4 DEG C of preservations;

Buffer B: take 0.2956g tetramethyl benzidine (TMB), 0.0633g tetrabutyl ammonium borohydride (TBABH) is dissolved in 30mL dimethyl acetamide (DMA), and 4 DEG C keep in Dark Place;

When use, Buffer A and Buffer B are mixed with the ratio of volume ratio 39:1, now with the current;

Stop buffer 3M H ₂sO ₄: by dense H ₂sO ₄add in distilled water with the ratio of volume ratio 1:5, mix for subsequent use.

Embodiment 1

1, the expression of E albumen and NS5 albumen adopts duck embryo allantoic cavity inocalation method to separate tembusu virus from sick duck brain tissue (or membrana follicularis) with purifying, by these two albumen of escherichia coli prokaryotic expression, adopt E albumen and the NS5 albumen of Ni-NTA separating column purifying prokaryotic expression.

2, trace routine:

2.1 use 1 × carbonate buffer solutions (pH=9.6) join the dilution of E protein concentration in 96 hole elisa plates by 100 μ L/ holes for 0.5ng/ μ L; Be 1.0ng/ μ L by the dilution of NS5 protein concentration again, join in another 96 hole elisa plate by 100 μ L/ holes; After 4 DEG C of overnight incubation, use and wash plate machine cleansing solution and wash 5 times, dry residual liquid on plate.

2.2 sealing: every hole adds 200 μ L confining liquids, and incubated at room 1h, washes plate according to method in step 2.1.

2.3 add primary antibodie: the ratio of 1:9 first adds 10 μ L duck serum to be checked to add 90 μ LPBST dilutions in every hole again by volume, mixes, and hatches 1h for 37 DEG C, washes plate according to the method in step 2.1.

2.4 add two resists: the IgG bis-anti-(Sigma) of the anti-duck of rabbit (or goat) of HRP mark uses PBST diluted to specifications, resist and add (every hole 100 μ L) in two 96 hole elisa plates that dry in 2.3 steps two of dilution, hatch 1h for 37 DEG C, wash plate according to method in step 2.1.

2.5 colour developings: the TMB nitrite ion newly preparing is joined in two 96 hole elisa plates according to 100 μ L/ holes, and 37 DEG C of lucifuges are hatched 15min.

2.6 cessation reactions: in two 96 hole elisa plates, every hole adds 50 μ L3M sulfuric acid stop buffers, vibration gently, reads OD450nm value by microplate reader.

2.7 results are judged

The criterion of E antigen indirect ELISA result:

Sample OD450 >=0.6897, serum to be checked is positive;

Sample OD450<0.5841, serum to be checked is negative;

0.6897 > OD450 >=0.5841, is judged to suspiciously, again detects according to step 2.1～2.6; .

The criterion of NS5 antigen indirect ELISA result:

Sample OD450 >=0.5017, serum to be checked is positive;

Sample OD450<0.4223, serum to be checked is negative;

0.5017 > OD450 >=0.4223, is judged to suspiciously, again detects according to 2.1～2.6 steps.

In result, the positive and negative explanation antibody of NS5 albumen of E albumen is that inactivated vaccine produces; If both all positive explanation antibody by live poison infect produce.

Embodiment 2

A, respectively duck serum, 45 parts of healthy duck serum and 30 parts of healthy meat duck serum of planting of 48 parts of doubtful infection tembusu viruses of random acquisition, according in summary of the invention 1)～6) step carries out respectively E albumen and NS5 protein antibodies horizontal detection.

B, respectively organize duck serum E albumen to be checked and NS5 protein antibodies ELISA testing result, according to summary of the invention 6) carry out the judgement of sample yin and yang attribute to be checked

B1,48 parts of doubtful infection tembusu virus duck serum ELISA testing results

48 parts of doubtful Tan Busu infected duck antibody level of serum entirety are all very high, and E albumen and NS5 protein antibodies level basically identical (27 plumage), show to infect in sample to be checked the individual ratio of tembusu virus higher.Testing result and clinical symptoms are diagnosed, RT-PCR testing result is basically identical, illustrate that this inventive method credibility is higher.

B2,45 parts of healthy duck serum ELISA testing results of planting

Health kind duck antibody level of serum entirety after 45 parts of immunity is higher, and E protein antibodies level is apparently higher than NS5 albumen, shows that vaccine immunization has higher immune protective efficiency to duck, but does not get rid of the possibility of subclinical infection (infect and do not fall ill).

B3,30 parts of healthy meat duck serum ELISA testing results

30 parts of healthy meat duck antibody level of serums are a little more than yin and yang attribute decision content, and E albumen is more consistent with NS5 protein antibodies level, possible cause: contain a certain amount of maternal antibody at meat duck body; Another part derives from tembusu virus and infects resistance to mistake.Illustrate that in healthy meat duck cultivation, Tan Busu infection conditions is comparatively serious, but maternal antibody and the resistance to source antibody of crossing can provide certain protection.

Claims (1)

1. distinguish the detection method that tembusu virus poison alive and inactivated vaccine produce antibody, it is characterized in that realizing by following steps:

1) separation and purification utilizes tembusu virus E albumen and the NS5 albumen of escherichia coli prokaryotic expression; 1 × the carbonate buffer solution that is 9.5-9.7 with pH dilutes respectively E albumen and NS5 albumen, and making E protein concentration is 0.5ng/ μ L, and NS5 protein concentration is 1.0ng/ μ L; The E protein solution of dilution and NS5 protein solution are all joined respectively in two 96 hole elisa plates by 100 μ L/ holes, hatch 12 hours for 4 DEG C, wash 5 times with PBS cleansing solution, dry residual liquid on plate;

2) every hole in two 96 hole elisa plates after step 1) drying is added to 200 μ L confining liquids, incubated at room 1h, washes 5 times with PBS cleansing solution, dries residual liquid on plate;

3) by step 2) every hole first adds 10 μ L duck serum to be checked to add 90 μ LPBST dilutions again in two 96 hole elisa plates after drying, mixes, and hatches 1h for 37 DEG C, washes 5 times residual liquid on drying plate with PBS cleansing solution;

4) two anti-with the rabbit of PBST diluted HRP mark or goat-anti duck, by two anti-two 96 hole elisa plates that add after step 3) dries of dilution, every hole 100 μ L, hatch 1h for 37 DEG C, wash 5 times residual liquid on drying plate with PBS cleansing solution;

5) TMB nitrite ion is joined in two 96 hole elisa plates after step 4) dries by 100 μ L/ holes, 37 DEG C of lucifuges are hatched 15min;

6) every hole adds 50 μ L3M sulfuric acid stop buffers, and vibration is gently read OD450nm value by microplate reader:

E albumen OD450nm value result of determination in sample:

OD450 >=0.6897, serum to be checked is positive;

OD450<0.5841, serum to be checked is negative;

0.6897 > OD450 >=0.5841, be judged to suspicious, according to step 1)-6) again detect;

NS5 albumen OD450nm value result of determination in sample:

OD450 >=0.5017, serum to be checked is positive;

OD450<0.4223, serum to be checked is negative;

0.5017 > OD450 >=0.4223, be judged to suspicious, according to step 1)-6) again detect;

In result, the positive and negative explanation antibody of NS5 albumen OD450nm value of E albumen OD450nm is that inactivated vaccine produces; If both all positive explanation antibody by live poison infect produce.

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