CN203011927U

CN203011927U - Detection kit of avian leukemia virus ELISA (Enzyme-Linked Immune Sorbent Assay) antigen

Info

Publication number: CN203011927U
Application number: CN 201220730978
Authority: CN
Inventors: 何丹婷; 徐飞; 吴小平; 苏丽芳; 杨铮
Original assignee: BEIJING WDWK BIOTECHNOLOGY Co Ltd
Current assignee: BEIJING WDWK BIOTECHNOLOGY Co Ltd
Priority date: 2012-12-27
Filing date: 2012-12-27
Publication date: 2013-06-19
Anticipated expiration: 2022-12-27

Abstract

The utility model relates to a detection kit of avian leukemia virus ELISA (Enzyme-Linked Immune Sorbent Assay) antigen, which comprises a kit body, wherein five cover board films, a self-sealed bag, a specification, a 96-hole enzyme labelled plate coated by an avian leukemia virus monoclonal antibody, a downwardly concave bottle position formed by a fixed foam mould for placing reagents, and seven reagent bottles on the downwardly concave bottle position, namely a bottle of negative contrast, a bottle of positive contrast, a bottle of enzyme labelled antibody, a bottle of substrate A solution, a bottle of substrate B solution, a bottle of 25* concentrated washing solution and a bottle of stopping solution, are contained in the kit body. P27 antigen of avian leukosis is detected by adopting a double-antibody sandwich enzyme linked immunosorbent assay principle; in the event of detecting, if containing the P27 antigen, the sample is combined with the monoclonal antibody on the surface of an adsorption plate hole to form an antigen antibody compound; after the antigen antibody compound is washed, the enzyme labelled antibody is added; a substrate is catalyzed by enzyme to react, thereby displaying blue; and the shade is positively correlated with the content of the P27 antigen in the sample. The kit disclosed by the utility model has the advantages of being simple, convenient and rapid for detection, high in sensitivity, strong in specificity, low in detection cost and the like, is capable of satisfying self-quality control of poultry breeding production enterprises, and can also be used for epidemiological surveillance of avian leukemia virus and early diagnosis of diseases.

Description

Avian leukosis virus ELISA antigen detection kit

Technical field

The invention belongs to biological technical field, particularly, the present invention relates to a kind of fast detecting tool that detects animal virus, specifically the detection kit of avian leukosis virus.

Background technology

Avian leukosis (Avian Leukosis) is the general designation of the bird kinds of tumors disease that caused by the virus in avian leukosis virus (Avian Leukosis Virus, ALV) and fowl sarcosis virus (Avian Sarcoma Virus, ASV) group.This disease can cause that to chicken many kinds have communicable optimum and malignant tumour.

Since Roloff in 1868 reported the chicken lymphosarcoma, avian leukosis had been distributed in all over the world, and China also has natural cases to occur.This disease can cause chicken weightening finish slowly, the sexal maturity delay, egg is little, eggshell is thin, egg production descends, fertility rate and hatchability is low, the discarded rate of trunk increases, mortality ratio increases etc., causes direct economic loss.Can cause also that simultaneously the body nonspecific resistance descends and immunosupress, causes indirect economic loss.Due to this disease energy vertical transmission, therefore very large to aviculture harm, be one of principal disease of harm poultry husbandry.

At present, there is no the effectively preventing measure for this disease, also without available vaccine, various countries mainly by regularly breeder flock being quarantined, eliminate positive chicken, thereby by constantly purifying the chicken group who selects without avian leukosis.In numerous detection methods, the ELISA detection method is with its sensitivity, special, easy to operate and be widely used in clinical detection.This method is suitable for animal doctor department of basic unit and chicken house diagnosis and the serology of this disease is detected.

Summary of the invention

The object of the present invention is to provide low, quick, easy and simple to handle, the highly sensitive avian leukosis virus ELISA antigen detection kit of a kind of cost.

Technical scheme of the present invention is: comprise a kit box body, five cover plate films, valve bag, a instructions are housed in box body, the recessed bottle position of the coated 96 hole ELISA Plate of avian leukosis virus monoclonal antibody, placement reagent that the fixed foam mould forms, be positioned at the reagent bottle on recessed bottle position, it is characterized in that: 12 dismountable enzyme mark bars are housed on ELISA Plate, each enzyme mark bar has 8 reacting holes, anti-avian leukosis virus monoclonal antibody that each enzyme mark hole is pre-coated, put into aluminium foil bag, the drying agent final vacuum of packing into; Described reagent bottle comprises that 2 mL negative controls, 2 mL positive controls, 50mL enzyme labelled antibody, 25 mL substrate A liquid, 25 mL substrate B liquid, 50 mL 25 * concentrated cleaning solutions, 50 mL stop buffers are fixed in the polyfoam mould with recessed bottle position, and cover plate film, valve bag and instructions together are contained in the top that is placed in ELISA Plate in a valve bag.Kit can one-time continuous be measured multiple sample, also can take plate hole apart repeated detection.

The principle that this kit detects is: this kit is to adopt DASELISA immunosorbent adsorption test principle to detect the P27 antigen of avian leukosis.During detection, add negative and positive contrast and testing sample in corresponding reacting hole, if contain P27 antigen in sample, be combined with the monoclonal antibody of adsorption plate hole surface and form antigen antibody complex, on the contrary can not in conjunction with.After washing, add enzyme labelled antibody, substrate for enzymatic activity reaction presents blueness, and in shade and sample, the content of P27 antigen becomes positive correlation.

This kit has very high sensitivity and very high specificity, and detectable sample comprises egg white, cloaca swab, vaccine, meconium.Be limited to 5.7ng/ml under the detection of this kit to ALV P27 protein solution, common disease poultry and livestock poison, Escherichia coli, white diarrhea salmonella, negative swab, SPF egg white and the SPF chicken plasmas such as NDV, IBDV, REV, IBV, AEV, EDSV, MDV, FPV, CIAV, ILTV are not produced specific reaction.

This kit detects easy, and required instrument is less, only needs the simple Laboratory Instruments such as microplate reader, hydro-extractor, and required testing cost is low.The self-quality control of fowl breeding production enterprise be can satisfy, also the epidemiological surveillance of avian leukosis virus and the early diagnosis of disease can be used as.

Description of drawings

Fig. 1 is the novel schematic diagram of this use

Fig. 2 is the schematic diagram of the utility model foam mold

Specific embodiments

Referring to accompanying drawing 1, represented the described avian leukosis virus ELISA of the present embodiment antigen detection kit, described 96 hole ELISA Plate 2 are housed in described box body 1, and described cover plate film, valve bag, instructions jointly are contained in and are positioned over above described ELISA Plate 2 in a valve bag; Have the recessed bottle position that fixed foam mould 3 forms in described kit lower floor, have 7 recessed bottle positions: 2 mL negative controls 4,2 mL positive controls 5,50mL enzyme labelled antibody 6,25 mL substrate A liquid 7,25 mL substrate B liquid 8,50 mL 25 * concentrated cleaning solutions 9,50 mL stop buffers 10.Described recessed bottle position is used for placing the reagent bottle (7 bottles) that corresponding solution is housed.

Embodiment of the present utility model

One, the preparation of working fluid

Wash operating solution: press 1:25(1 part concentrate+24 parts of deionized waters with deionized water) dilution.

PBS solution: 3.58 g disodium hydrogen phosphates, 0.24 g potassium dihydrogen phosphate, 8 g sodium chloride, 0.2 g potassium chloride adds deionized water and is settled to 1L.

Two, sample pre-treatments

Egg white: get shell egg, draw egg white with micropipettor after fragmentation and directly detect;

The cloaca swab: get cotton swab after chicken cloaca to be checked sampling, put in 1ml PBS solution (0.01 mol/L, pH value 7.4), in freeze thawing below-20 ℃ once, after the recovery room temperature for detection of;

Vaccine: with being used for measuring after PBS solution (0.01 mol/L, pH value 7.4) dissolving;

Meconium: need not dilution, can be directly used in detection.If contain precipitation or impurity in sample, need carry out being used further to detect after low-speed centrifugal.

Three, operation steps

The sample of handling well is added coated plate, and 100 μ l/ holes select 4 holes to do positive and negative contrast (each 2 holes) simultaneously; Enzyme-linked reaction plate is added a cover or sealer, at room temperature hatched 60 minutes under (25 ± 2 ℃); Get rid of liquid in the hole, wash enzyme-linked reaction plate with wash operating solution, wash 3 times in 260 μ l/ holes, pats dry; Add enzyme labelled antibody, 100 μ l/ holes; Put the cover plate film, at room temperature hatched 60 minutes; Get rid of liquid in the hole, wash enzyme-linked reaction plate with wash operating solution, wash 3 times in 260 μ l/ holes, pats dry; Substrate A liquid is mixed by 1:1 with substrate B liquid, add in hand-hole, 100 μ l/ holes; Put the cover plate film, at room temperature hatched 15 minutes; Add stop buffer, 100 μ l/ holes, and the mixing that vibrates gently; Reading OD650 value (should complete reading in 15 minutes after adding stop buffer) on microplate reader.

Four, judge

NC value＜0.200 and 0.500＜PC value≤2.000, test findings are just effective.Otherwise, should carry out revision test; If S/P＜0.17, in interpret sample without avian leukosis virus P27 albumen; If there is avian leukosis virus P27 albumen S/P 〉=0.17 in interpret sample.

S/P=(SC-NC)/(PC-NC), wherein: the negative control wells OD value of NC average; The positive control wells OD value of PC average; SC is test sample hole OD value average.

Five, measure points for attention

1. please don't use expired product, the reagent of different lot number products must not be used with.

2. do not use expired kit, the component that has broken a seal should be used before the deadline.

3. when each the use, all should set up respectively 2 hole positive controls and 2 hole negative controls.

4. must use water (as distilled water, deionized water or pure water) the dilution cleansing solution that meets the pharmacopeia regulation.

5. each component of kit answers pre-balance to room temperature before use.

6. untapped enzyme-linked reaction plate should be put back to aluminium foil bag and sealing, 2～8 ℃ of preservations.

7. should avoid using metal species material splendid attire and stir reagent.

8. concentrated cleaning solution crystallization may occur when low temperature (2～8 ℃) stores, and must at room temperature dissolve fully also to exercise use after mixing again.

9. substrate solution is not exposed under high light.

10. this product stop buffer is sulfuric acid, avoids with eye, skin contact.

11. liquor-transferring system should guarantee accurate cleaning, should not cause cross pollution when accurate liquor capacity is provided.

12. the transportation of sample must be undertaken by the relevant regulations of country.

Claims

1. avian leukosis virus ELISA antigen detection kit, it is characterized in that: comprise a kit box body, five cover plate films, valve bag, a instructions are housed in box body, the recessed bottle position of the coated 96 hole ELISA Plate of avian leukosis virus monoclonal antibody, placement reagent that the fixed foam mould forms, be positioned at the reagent bottle on recessed bottle position, described reagent bottle comprises 2 mL negative controls, 2 mL positive controls, 50mL enzyme labelled antibody, 25 mL substrate A liquid, 25 mL substrate B liquid, 50 mL 25 * concentrated cleaning solutions, 50 mL stop buffers.

2. avian leukosis virus ELISA antigen detection kit according to claim 1, its described ELISA Plate is characterised in that: 12 dismountable enzyme mark bars are housed on ELISA Plate, each enzyme mark bar has 8 reacting holes, anti-avian leukosis virus monoclonal antibody that each enzyme mark hole is pre-coated, put into aluminium foil bag, the drying agent final vacuum of packing into.