CN204008662U - Detect the ELISA kit of PRRS virus antibody - Google Patents
Detect the ELISA kit of PRRS virus antibody Download PDFInfo
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- CN204008662U CN204008662U CN201420201362.2U CN201420201362U CN204008662U CN 204008662 U CN204008662 U CN 204008662U CN 201420201362 U CN201420201362 U CN 201420201362U CN 204008662 U CN204008662 U CN 204008662U
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- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 title claims abstract description 28
- 241000700605 Viruses Species 0.000 title claims abstract description 15
- 238000008157 ELISA kit Methods 0.000 title claims abstract description 10
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- 238000002965 ELISA Methods 0.000 claims abstract description 26
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- 101100256910 Drosophila melanogaster sick gene Proteins 0.000 description 4
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model relates to a kind of ELISA kit that detects PRRS virus antibody.This kit comprises: the anti-pig antibody of elisa plate bar, sample diluting liquid, enzyme mark rabbit, substrate A liquid, substrate B liquid, stop buffer, PRRS virus positive serum, PRRS virus negative serum, concentrated cleaning solution using the recombinant N protein of purifying as envelope antigen.The utility model kit adopts the principle of indirect enzyme-linked immunosorbent assay to detect the antibody of PRRS virus.When detection, in corresponding reacting hole, add negative and positive contrast and testing sample, if contain specific antibody in sample, be combined with the antigen of adsorption plate hole surface and form antigen antibody complex, otherwise can not be in conjunction with, add enzyme labelled antibody, substrate for enzymatic activity reaction presents blueness, and shade becomes positive correlation with the content of antibody in sample.
Description
Technical field
The utility model belongs to zoosis toxicity disease surveillance and diagnostic reagent development field, is specifically related to a kind of ELISA kit that detects PRRS virus antibody.
Background technology
Pig blue-ear disease is again porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS), is a kind of infectious disease of the serious harm pig industry of the appearance 1980s.This sick principal character is the weak piglet of premature labor, miscarriage, stillborn foetus, mummy tire and product that causes in-pig, and piglet main manifestations is for to cough, to have difficulty in breathing, be short of breath.First the U.S. in 1987 have reported the generation of this disease, and this disease has also been broken out in the country such as Canadian, German, Dutch and area in succession subsequently.
This sick pathogen is porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus; PRRSV), PRRSV is separated first and identifies in 1991 by Dutch scholar Wensvoort, called after Lelystad virus (LV).Within 1992, the U.S. is separated to a strain PRRSV first, and called after VR2332.Now, there is generation in respectively raise pigs in world country, area of this disease, causes more and more people's concern.There is the generation of this disease in China in reported first in 1996.China is the big country of raising pigs of the world, and this disease occurs in China, popular present situation allows of no optimist.
Porcine reproductive and respiratory syndrome, from occurring so far, continues to bring out about this sick diagnostic method and achievement in research.What be most widely used is the ELISA method of setting up using totivirus as envelope antigen, i.e. the ELISA of American I DEXX company kit.Because virus need be by propagation in a large number in cell culture, and purified virus is truly difficult to obtain, and the testing cost of this kit is too high is restricted its promotion and application.The N gene of coding nucleocapsid protein is quite conservative in two genotype, and when pig is subject to after PRRSV invasion and attack, the antibody that body produces is at first for virus nucleocapsid albumen.Our company has been expressed and has been obtained purification of Recombinant N albumen by early-stage Study, detects the ELISA kit of porcine reproductive and respiratory syndrome antibody and the technology of detection method thereof but also do not utilize albumen to set up.
Utility model content
The purpose of this utility model is to provide a kind of ELISA kit that detects porcine reproductive and respiratory syndrome antibody, and for the detection of porcine reproductive and respiratory syndrome antibody, kit specificity is good, susceptibility is high, and testing cost is cheap, is suitable for promoting.Differentiate and take to provide important method aspect effective anti-measure processed this sick antibody horizontal detection, epidemiology survey, class disease.
The technical solution of the utility model is: this kit is to adopt the principle of indirect enzyme-linked immunosorbent assay (Indirect-ELISA) to detect the antibody of PRRSV.When detection, in corresponding reacting hole, add negative and positive contrast and testing sample, if contain specific antibody in sample, be combined with the antigen of adsorption plate hole surface and form antigen antibody complex, on the contrary can not be in conjunction with.After washing, add enzyme labelled antibody, substrate for enzymatic activity reaction presents blueness, and shade becomes positive correlation with the content of antibody in sample.
For this reason, the utility model provides a kind of ELISA kit that detects porcine reproductive and respiratory syndrome antibody, comprises recessed bottle position, 96 hole ELISA Plate, 8 bottles of reagent and a cover plate film of box body, placement reagent.Wherein, ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the check-out console that coated recombinant N protein is made in ELISA Plate micropore, 8 bottles of reagent are respectively concentrated cleaning solution, sample diluting liquid, substrate A liquid, substrate B liquid, the anti-pig antibody of enzyme mark rabbit, stop buffer, porcine reproductive and respiratory syndrome positive serum, porcine reproductive and respiratory syndrome negative serum, and cover plate film size is in the same size with ELISA Plate square section.Box body is carton box, and be made up of plastic foam recessed bottle position, and cover plate film is plastic hard membrane.ELISA Plate is made up of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, and each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, and each reacting hole is coated with recombinant N protein; 1 bottle of concentrated cleaning solution, 70mL; 1 bottle of sample diluting liquid, 120mL; 1 bottle of the anti-pig antibody of enzyme mark rabbit, 60mL; 1 bottle of substrate A liquid, 30mL; 1 bottle of substrate B liquid, 30mL; 1 bottle of stop buffer, 60mL; 1 bottle of porcine reproductive and respiratory syndrome positive serum, 2mL; 1 bottle of porcine reproductive and respiratory syndrome negative serum, 2mL.
Our experiments show that this kit has very high sensitivity, with respect to other porcine reproductive and respiratory syndrome antibody detection methods, the required instrument of this kit is less, and generally all there is outfit in the similar laboratory of required instrument, and required cost is lower.
The beneficial effects of the utility model are: can be quick, easy, sensitive, exactly for the detection of porcine reproductive and respiratory syndrome antibody.
Brief description of the drawings
Fig. 1 is the lateral longitudinal sectional view (long is 8.55cm) of the utility model ELISA Plate;
Fig. 2 is the side drawing in side sectional elevation (long is 12.8cm) of the utility model ELISA Plate;
Fig. 3 is the vertical view of the utility model ELISA Plate;
Fig. 4 is the utility model cover plate membrane plane figure;
Fig. 5 is the vertical view of the utility model fixed foam mould;
Fig. 6 is the side view of the utility model box body and fixed foam mould.
Embodiment
embodiment 1: kit assembling
Referring to accompanying drawing: have the reaction micropore (3) of coated recombinant N protein on enzyme mark reaction capillary strip (2), the outer frame support (1) that is fixed on ELISA Plate is upper, enzyme mark reaction capillary strip (2) can be with requiring dismounting; Cover plate film (4) is put capping enzyme mark reaction capillary strip (2) while reacting in constant temperature oven for ELISA Plate; Plastic foaming mold (5) has 8 bottle positions, placement location is followed successively by: 2mL porcine reproductive and respiratory syndrome positive serum bottle position (6), 2mL porcine reproductive and respiratory syndrome negative serum bottle position (7), 70mL concentrated cleaning solution bottle position (8), 30mL substrate A liquid bottle position (9), 30mL substrate B liquid bottle position (10), 60mL enzyme mark rabbit anti-pig antibody working fluid bottle position (11), 60mL stop buffer bottle position (12), 120mL sample diluting liquid bottle position (13), box body (14) is for being carton box.
embodiment 2: the each material preparation of kit
1, the preparation of enzyme labelled antibody
(1) take horseradish peroxidase (HRP) 2 mg and be dissolved in 0.5 mL water, add 0.5 mL 0.06 mol/L NaIO
4solution, 4 DEG C of lucifuge effect 30 min;
(2) add the ethylene glycol 0.5mL of 160 mmol/L, room temperature effect 30 min;
(3) add anti-pig antibody 2 mg of rabbit, after mixing, pack in the bag filter of processing, put in the 0.05 mmol/L sodium carbonate buffer of 1000 mL and dialyse, 4 DEG C are spent the night;
(4) dislysate is drawn in the centrifuge tube of 10 mL, adds the NaBH of 0.25mL 5g/L
4solution, mixes rearmounted 4 DEG C of 2 h;
(5) add isopyknic saturated ammonium sulfate solution, after 4 DEG C of effect 30 min, centrifugal 25 min of 3000 r/min at 4 DEG C, abandon supernatant;
(6) precipitation is dissolved in the PBS of 1.5 mL0.02 mol/L pH 7.4, sucks in bag filter, 0.02mol/L pH 7.4 PBS dialysis, 4 DEG C spend the night (changing PBS midway 3 times);
(7) liquid in bag filter is drawn in microcentrifugal tube, the centrifugal 30min of 10000r/min at 4 DEG C, by supernatant sucking-off, adds equivalent glycerine, mixes, and-20 DEG C save backup.
2, be coated with ELISA Plate and the preparation thereof of recombinant N protein
(1) with the carbonate solution of 0.05M, recombinant N protein is diluted to 2.5ng/mL, coated 96 hole polystyrene ELISA Plate, every hole 100 μ L, 37 DEG C of incubation 2h, coating buffer inclines, with cleansing solution washing 3 times, each 10s, pats dry, then in every hole, add 150 μ L confining liquids, 37 DEG C of incubation 2h, liquid in the hole of inclining, dry rear with aluminium film vacuum seal preservation.
(2) coated damping fluid: pH9.6, the sodium carbonate buffer of 0.05mo1/L;
(3) confining liquid: every 1 liter of confining liquid is prepared as follows: 5mL horse serum, 1g sodium azide, 30g casein are mixed, dissolve and be settled to 1000mL with phosphate buffer, obtain confining liquid; Wherein, the concentration of phosphate buffer is 0.02M, and pH value is 7.2.
embodiment 3: the pre-treatment of sample
1, dosing:
Wash operating solution:, for the washing of ELISA Plate, wash operating solution can be preserved one month at 4 DEG C of environment with deionized water, 20 × concentrated cleaning solution to be diluted to (1 part of 20 × concentrated cleaning solution+19 part deionized water) by 1:19 volume ratio.
2, pig serum or plasma sample processing:
(1) sample preparation: get pig blood, prepare according to a conventional method serum or blood plasma, require limpid without haemolysis;
(2) pig serum or blood plasma press to 1:40 (1 part pig serum or blood plasma+39 duplicate samples dilution) in serum dilution plate or centrifuge tube with sample diluting liquid and diluted, mix;
(3) getting 100 μ L detects.
Note: dilution factor: 40; Negative control and positive control need not dilute.
embodiment 4: the operation steps that uses kit
1, in ELISA Plate, contrast and testing sample add pattern as shown in drawings;
In accompanying drawing ELISA Plate, contrast and testing sample add pattern
Note: N-represents to add negative control; P-represents to add positive control; S1, S2, S3, the expressions such as S4 add various test samples.
2, in A1 and the coated hole of B1 antibody, add negative control 100 μ L; In C1 and the coated hole of D1 antibody, add positive control 100 μ L;
3, in each sample well, add the test sample 100 μ L that handle well;
4, cover cover plate film, under 37 DEG C of constant incubators, hatch 30 min;
5, the plate film of uncapping, gets rid of liquid in hole, and with wash operating solution detersive enzyme target, 300 μ L/ holes, wash 4 times, pat dry;
6, in each hole, add enzyme labelled antibody immediately, 100 μ L/ holes;
7, cover cover plate film, under 37 DEG C of constant incubators, hatch 30 min;
8, open cover plate film, get rid of liquid in hole, with wash operating solution detersive enzyme target, 300 μ L/ holes, wash 4 times, pat dry;
9, substrate A liquid is mixed by 1:1 with substrate B liquid, add in hand-hole, 100 μ L/ holes, cover cover plate film, and under room temperature (25 scholar 2 0C), lucifuge is reacted 15 min;
10, open cover plate film, in every hole, add 100 μ L stop buffers;
11, in microplate reader, read OD
650value (should add after stop buffer complete reading in 5 min).
embodiment 5: result is judged
1, NC value <0.200 and 0.500 < PC value≤2.000, test findings is just effective.Otherwise, should carry out revision test.
If 2 test sample hole OD value <0.45, in interpret sample without porcine reproductive and respiratory syndrome virus antibody; If test sample hole OD value >0.45, has porcine reproductive and respiratory syndrome virus antibody in interpret sample.
embodiment 6: points for attention
1, this product is only for vitro detection.
2, the component in different lot number kits can not be used with.
3, do not use expired kit, the component having broken a seal should use before the deadline.
4, in the time of each use, in ELISA Plate, all should set up respectively positive control and each 2 holes of negative control.
5, must use water (as distilled water, deionized water or pure water) the dilution cleansing solution that meets pharmacopeia and specify.
6, kit each component answers pre-balance to room temperature (25 ± 2 DEG C) before use, otherwise will directly affect experimental result.
7, the ELISA Plate that end is used should be put back to aluminium foil bag sealing, 2 ~ 8 DEG C of preservations.
8, should avoid using metal species material splendid attire and stir reagent.
9, may there is crystallization in concentrated cleaning solution and stop buffer in the time that low temperature (2 ~ 8 DEG C) stores, and palpus room temperature is dissolved mix rear use completely.
10, substrate solution is not exposed under high light.
11, this product stop buffer is sulfuric acid, avoids stop buffer with eye, skin contact.
12, liquor-transferring system should ensure accurately to clean, and does not cause cross pollution when should providing accurate liquor capacity.
13, the transport of sample must be undertaken by national relevant regulations.
Claims (3)
1. one kind is detected the ELISA kit of PRRS virus antibody, comprise box body, place the recessed bottle position of reagent, 96 hole ELISA Plate, 8 bottles of reagent and a cover plate film, it is characterized in that: ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the check-out console that coated recombinant N protein is made in ELISA Plate micropore, 8 bottles of reagent are respectively concentrated cleaning solution, sample diluting liquid, substrate A liquid, substrate B liquid, the anti-pig antibody of enzyme mark rabbit, stop buffer, PRRS virus positive serum, PRRS virus negative serum, cover plate film size is in the same size with ELISA Plate square section.
2. ELISA kit according to claim 1, is characterized in that: affiliated box body is carton box, and be made up of plastic foam recessed bottle position, and cover plate film is plastic hard membrane.
3. ELISA kit according to claim 1, it is characterized in that: ELISA Plate is made up of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, and each reacting hole is coated with recombinant N protein; 1 bottle of concentrated cleaning solution, 70mL; 1 bottle of sample diluting liquid, 120mL; 1 bottle of the anti-pig antibody of enzyme mark rabbit, 60mL; 1 bottle of substrate A liquid, 30mL; 1 bottle of substrate B liquid, 30mL; 1 bottle of stop buffer, 60mL; 1 bottle of PRRS virus positive serum, 2mL; 1 bottle of PRRS virus negative serum, 2mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420201362.2U CN204008662U (en) | 2014-04-24 | 2014-04-24 | Detect the ELISA kit of PRRS virus antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420201362.2U CN204008662U (en) | 2014-04-24 | 2014-04-24 | Detect the ELISA kit of PRRS virus antibody |
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| Publication Number | Publication Date |
|---|---|
| CN204008662U true CN204008662U (en) | 2014-12-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201420201362.2U Expired - Lifetime CN204008662U (en) | 2014-04-24 | 2014-04-24 | Detect the ELISA kit of PRRS virus antibody |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104744573A (en) * | 2015-03-27 | 2015-07-01 | 中华人民共和国吉林出入境检验检疫局 | Liquid-phase-chip detection kit for detecting blue-eared pig disease virus |
| CN110894243A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Porcine reproductive and respiratory syndrome virus chimeric antigen and colloidal gold immunochromatographic test strip for detecting porcine reproductive and respiratory syndrome virus antibody |
| CN113588948A (en) * | 2021-08-03 | 2021-11-02 | 江苏量界生物技术有限公司 | Kit for quantitatively detecting novel coronavirus N protein based on ELISA method |
-
2014
- 2014-04-24 CN CN201420201362.2U patent/CN204008662U/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104744573A (en) * | 2015-03-27 | 2015-07-01 | 中华人民共和国吉林出入境检验检疫局 | Liquid-phase-chip detection kit for detecting blue-eared pig disease virus |
| CN104744573B (en) * | 2015-03-27 | 2020-05-29 | 中华人民共和国吉林出入境检验检疫局 | Liquid chip detection kit for detecting porcine reproductive and respiratory syndrome virus |
| CN110894243A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Porcine reproductive and respiratory syndrome virus chimeric antigen and colloidal gold immunochromatographic test strip for detecting porcine reproductive and respiratory syndrome virus antibody |
| CN110894243B (en) * | 2019-12-16 | 2021-07-13 | 中国农业大学 | Porcine reproductive and respiratory syndrome virus chimeric antigen and colloidal gold immunochromatographic test strip for detecting porcine reproductive and respiratory syndrome virus antibody |
| CN113588948A (en) * | 2021-08-03 | 2021-11-02 | 江苏量界生物技术有限公司 | Kit for quantitatively detecting novel coronavirus N protein based on ELISA method |
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