CN101936998A

CN101936998A - Antirabies virus IgG (Immunoglobulin G) antibody ELISA (Enzyme Linked Immunosorbent Assay) detection kit for dogs

Info

Publication number: CN101936998A
Application number: CN2010102575274A
Authority: CN
Inventors: 廖园园; 刘汉平; 刘洁; 王威; 彭杏; 漆世华; 温文生; 王小红
Original assignee: WUHAN CHOPPER BIOLOGY CO Ltd
Current assignee: WUHAN CHOPPER BIOLOGY CO Ltd
Priority date: 2010-08-19
Filing date: 2010-08-19
Publication date: 2011-01-05
Anticipated expiration: 2030-08-19
Also published as: CN101936998B

Abstract

The invention relates to an antirabies virus IgG (Immunoglobulin G) antibody kit for dogs. In the kit, an enzyme label plate is coated with anti-rabies virus monoclonal antibodies in advance, wherein a coating buffer solution is 0.05M of carbonate buffer solution with pH of 9.6, and the coating amount is 0.1-1ug per hole; 1-10% of BSA (Bull Serum Albumin) or skimmed milk in percentage by mass concentration is used as a blocking buffer; rabies virus purified antigens are coated after blocking, wherein the coating amount is 0.1-1ug per hole; 0.01mol/L PBS (Phosphate-Buffered Saline) with pH 7.2-7.4 containing 0.1-10% of BSA and 0.01-0.05% of NaN3 in percentage by mass concentration is used as a sample diluent; a horse radish peroxidase-rabbit anti-rabies IgG enzyme conjugate is used as an enzyme conjugate; 0.01mol/L PBS with pH 7.2-7.4 containing tween-20 with volume concentration of 0.05% is used as a washing concentrate; an enzyme substrate A solution is a 3,3'-5,5'-tetramethyl benzidine solution, and an enzyme substrate B solution is a hydrogen peroxide solution; a 1 mol/L H2SO4 solution is adopted as a stop solution; and a positive control and a negative control are arranged in the kit. With 100% of specificity and 1:640 of sensitivity, the kit is used for the immune effect evaluation of experimental dogs, pet dogs and common dogs which are inoculated with rabies vaccines.

Description

Dog is used anti-rabies virus IgG antibody ELISA detection kit

Technical field

The present invention relates to a kind of detection kit, be specifically related to a kind of dog and use anti-rabies virus IgG antibody ELISA detection kit.

Background technology

Rabies are that (its main host is animals such as dog, cat for Rabies virus, the infectious diseases common to human beings and animals that RV) causes by rabies viruses.Classical symptom is a hydrophobia, claims again: hydrophobia.This disease is very dangerous, and case fatality rate is almost 100%.Dog not only is used for hunting, track down, guard the gate, eat meat and as raising pets, dog still carries out one of life science, medical science, pharmacy, chemical industry, agricultural, military affairs, environmental protection, space flight and the widely used important animal used as test of bioengineering field.Along with the raising of expanding economy and living standards of the people, the quantity of pet dog is not only increasing, and the zone of action of dog is also in continuous expansion.From the information that the pet medical market obtains, whether dog only obtained protection after people urgently wished to understand pet dog inoculation rabies vaccine, and the testing of rabies virus antibodies at present is not widely used because of the restriction of examined condition and expense.Rabies viruses is rabic pathogen, and China is rabic country occurred frequently, occupies the second place of the world, is only second to India.Because rabies are infecting both domestic animals and human diseases, most human rabies is that it endangers and has caused showing great attention to of the government and the people by the band poison dog propagation that contact closely with the mankind.

At present, be used for method and reagent corresponding box that animal used as test comprises that rabies virus antibodies such as dog, monkey, mouse detect, certain research and report are arranged.But the existing method that detects rabies virus antibodies comprises that all there are corresponding weakness in common ELISA method, fluorescent immune method, radioimmunology and chemoluminescence method etc.How common ELISA kit does envelope antigen with the totivirus of prokaryotic expression recombinant albumen or cultivation.There is the renaturation difficulty in recombinant protein, lacks the shortcoming of space conformation epi-position, and there is the shortcoming that is difficult for purifying in totivirus, and these all can cause detection sensitivity low.Fluorescent immune method, radioimmunology and chemoluminescence method need certain experimental apparatus, and also there is the problem of radioisotope pollution in radioimmunology.Though and colloidal gold immunity chromatography has advantage simple to operate, remolding sensitivity is lower.The Chinese patent publication number is that the patented claim of CN1547027A discloses the indirect enzyme immunosorbent adsorption test detection of a kind of usefulness rabies poison IgG kit and preparation method, the Chinese patent publication number is that the patented claim of CN1547028A discloses and a kind ofly detects total antibody kit of rabies poison and preparation method with competition enzyme-linked immunosorbent adsorption test, the Chinese patent publication number is that the patented claim of CN101413948 discloses a kind of rabies virus NP-ELISA antibody detection reagent kit, and the used check-out console method for coating of above-mentioned patent is antigen direct coated method.

Summary of the invention

The purpose of this invention is to provide a kind of dog and use anti-rabies virus IgG antibody ELISA detection kit.The present invention adopts the monoclonal antibody bag by the indirect envelope antigen preparation feedback of method plate, and then form with indirect immunoadsorption and test the kit that detects dog anti rabies virus IgG antibody, this kit is except the advantage with common ELISA kit, also remedy the low shortcoming of sensitivity that causes owing to the antigen problem, thereby improved sensitivity and the specificity that detects.

Technical scheme of the present invention is:

A kind of dog is used anti-rabies virus IgG antibody ELISA detection kit, described detection kit includes: wrap in advance by rabies virus antigen ELISA Plate, confining liquid, sample diluting liquid, positive control, negative control, enzyme conjugates, concentrated cleaning solution, enzyme substrate solution and stop buffer, it is characterized in that: ELISA Plate is wrapped in advance by anti-rabies monoclonal antibodies, bag is cushioned the carbonate buffer solution that liquid is 0.05M pH9.6, promptly contains 1.59g Na in 1 liter of solution ₂CO ₃, 2.93g NaHCO ₃, package amount is every hole 0.1-1ug; Confining liquid is that mass concentration is BSA or the skim milk of 1-10%; Sealed and wrapped by the rabies viruses purifying antigen again, package amount is every hole 0.1-1ug; Sample diluting liquid is for containing mass concentration 0.1-10% bovine serum albumin(BSA) BSA and containing mass concentration 0.01-0.05%NaN ₃0.01mol/L and the phosphate buffer (PBS) of pH7.2-7.4; Enzyme conjugates is the anti-dog IgG of a horseradish peroxidase-rabbit enzyme conjugates; Concentrated cleaning solution is to contain the 0.01mol/L of volumetric concentration 0.05% Tween-20 and the PBS of pH7.2-7.4; Zymolyte A solution is 3,3 '-5,5 '-tetramethyl biphenyl amine aqueous solution, and zymolyte B solution is hydrogen peroxide solution; Stop buffer is 1mol/L H ₂SO ₄Solution, positive control, negative control are placed in the box.Horseradish peroxidase-labeled be the anti-dog IgG of rabbit antibody.

Dog is used the preparation method of anti-rabies virus IgG antibody ELISA detection kit, carry out according to the following steps: the preparation of (1) anti-rabies monoclonal antibodies: with the rabies virus antigen immunity BALB/c mouse of purifying, obtain the spleen cell of antigenic stimulus, merge the myeloma cell line of this spleen cell and mouse, utilize HAT selective medium screening hybridoma, identify the hybridoma cell strain of rabies poison with ELISA method and IFA (indirect immunofluorescence) method, carry out obtaining behind three subclonings secreting the cell line of the monoclonal antibody of high specific, the monoclonal antibody of producing the rabies poison by mouse ascites; (2) preparation of the anti-dog IgG of rabbit and horseradish peroxidase mark: extract purifying dog 1gG according to a conventional method; Immunizing rabbit reaches 1: 32 and gets serum when above when rabbit anti-serum ELISA tires; The ammonium sulfate precipitation purifying; Periodate oxidation method with improvement is carried out mark; Last enzyme labeling thing adds the neutral glycerine of 0.1-10% bovine serum albumin(BSA), 0.1-10% casein and 50%, measure working concentration after ,-20 ℃ of preservations are standby; (3) preparation of above-mentioned various solution: 1. sample diluting liquid: 0.1-10%BSA, 0.01-0.05%NaN ₃0.01mol/L, the phosphate buffer of pH7.2-7.4 (PBS); 2. cleansing solution: in the 0.01M of 1000ml PBS solution, add 0.5ml Tween-20 (Tween-20); 3. zymolyte 3, and 3 '-5,5 '-tetramethyl benzidine (TMB) solution: i) substrate A liquid: take by weighing TMB100mg and add in the 10ml dimethyl sulfoxide (DMSO) (DMSO), promptly get 100 times tmb substrate concentrate after making it to dissolve fully; Ii) substrate B liquid: take by weighing sodium acetate 10g and be dissolved in the 1L purified water, transferring pH value with acetate is 5.0, and adding concentration is 30%H ₂O ₂400ul promptly; 4. stop buffer: get 54.3ml concentration and be 95-98% concentrated sulphuric acid adding distil water and get final product to 1000ml.

Described rabies virus antigen is cultivated on the Vero cell, increase with the hydrophobia strain, and through freeze thawing, ultrasonic disruption, differential centrifugation extract and Sepharose 4FF post filters purifying, and-20 ℃ of preservations are as wrapping quilt with rabies viruses antigen.

Experimental result of the present invention and analysis:

1. specific detection: the kit with manufacturing of the present invention detects rabies viruses immunity dog serum respectively, the dog negative serum, dog hexavaccine (canine distemper disease, canine parvovirus disease, canicola fever, catarrhal jaundice, infective bronchitis and parainfluenza virus) immune dog serum, operation and judgement are carried out with the operation steps of anti-rabies virus IgG antibody ELISA detection kit according to dog in the embodiment, the result shows, rabies viruses immunity dog serum test positive, other sample detection are negative, and specificity is 100%.

2. sensitivity detects: the kit made from the present invention detects different dilution positive reference serums, operation and judgement are carried out with the operation steps of anti-rabies virus IgG antibody ELISA detection kit according to dog in the embodiment, and testing result is: highest detection is 1: 640 to the dilutability of positive reference sample.

Description of drawings

Fig. 1 is a process chart of the present invention.

Embodiment

One. dog is used the preparation method of anti-rabies virus IgG antibody ELISA detection kit

1. the preparation of rabies monoclonal antibodies: with the rabies virus antigen immunity BALB/c mouse of purifying, obtain the spleen cell of antigenic stimulus, merge the myeloma cell line of this spleen cell and mouse, utilize HAT selective medium screening hybridoma, identify the hybridoma cell strain of rabies poison with ELISA method and IFA method, carry out obtaining behind three subclonings secreting the cell line of the monoclonal antibody of high specific, the monoclonal antibody of producing the rabies poison by mouse ascites;

2. the anti-preparation of the anti-dog IgG of horseradish peroxidase-labeled rabbit two: extract purifying dog 1gG according to a conventional method; Immunizing rabbit reaches 1: 32 and gets serum when above when rabbit anti-serum ELISA tires; The ammonium sulfate precipitation purifying; Periodate oxidation method with improvement is carried out mark; Last enzyme labeling thing adds the neutral glycerine of 1% bovine serum albumin(BSA), 1% casein and 50%, measure working concentration after ,-20 ℃ of preservations are standby;

3. described rabies virus antigen is cultivated on the Vero cell, increase with the hydrophobia strain, and through freeze thawing, ultrasonic disruption, differential centrifugation extract and Sepharose 4FF post filters purifying, and-20 ℃ of preservations are as wrapping quilt with rabies viruses antigen;

4. rabies monoclonal antibodies is coated on the ELISA Plate hole uniformly, bag is cushioned the carbonate buffer solution that liquid is 0.05M pH9.6, promptly contains 1.59g Na in 1 liter of solution ₂CO ₃, 2.93g NaHCO ₃, package amount is every hole 0.1-1ug; Confining liquid is BSA or skim milk, and concentration is 1-10%; Having sealed what wrap quilt again is the rabies viruses purifying antigen, and package amount is every hole 0.1-1ug;

5. other solution of kit preparation: 1. sample diluting liquid: 1%BSA, 0.05%NaN ₃0.01mol/L, the phosphate buffer of pH7.2 (PBS); 2. cleansing solution: in the 0.01M of 1000ml PBS solution, add 0.5ml Tween-20 (Tween-20); 3. zymolyte 3, and 3 '-5,5 '-tetramethyl benzidine (TMB) solution: i) substrate A liquid: take by weighing TMB100mg and add in the 10ml dimethyl sulfoxide (DMSO) (DMSO), promptly get 100 times tmb substrate concentrate after making it to dissolve fully; Ii) substrate B liquid: take by weighing sodium acetate 10g and be dissolved in the 1L purified water, transferring pH value with acetate is 5.0, and adding concentration is 30%H ₂O ₂400ul promptly; 4. stop buffer: get 54.3ml concentration and be 95% concentrated sulphuric acid adding distil water and get final product to 1000ml.

Two, dog is used the operation steps of anti-rabies virus IgG antibody ELISA detection kit:

1. with sample to be checked 10 times of dilutions of sample diluting liquid, every hole adds 100ul, adds the positive and negative controls simultaneously, establishes blank.Put 37 ℃ and hatched 1 hour, cleansing solution is washed plate 5 times, dries, and every hole adds enzyme labelled antibody 100ul, hatches 1 hour for 37 ℃; Cleansing solution is washed plate 5 times, dries, and adds enzyme substrate solution, every hole 100 μ l, and lucifuge colour developing 5-10 minute adds stop buffer, every hole 50 μ l.Utilize microplate reader to measure absorbance value A450nm. at 450nm

2. the result judges: Cut off (CO value)=negative control absorbance value A450nm * 2.1 times, and sample value=sample absorbance value A450nm/CO value, sample value is judged to the positive greater than 1; Sample value is judged to feminine gender less than 1.

Claims

1. a dog is used anti-rabies virus IgG antibody ELISA detection kit, described detection kit includes: wrap in advance by rabies virus antigen ELISA Plate, confining liquid, sample diluting liquid, positive control, negative control, enzyme conjugates, concentrated cleaning solution, enzyme substrate solution and stop buffer, it is characterized in that: ELISA Plate is wrapped in advance by anti-rabies monoclonal antibodies, bag is cushioned the carbonate buffer solution that liquid is 0.05M pH9.6, promptly contains 1.59g Na in 1 liter of solution ₂CO ₃, 2.93g NaHCO ₃, package amount is every hole 0.1-1ug; Confining liquid is that mass concentration is BSA or the skim milk of 1-10%; Sealed and wrapped by the rabies viruses purifying antigen again, package amount is every hole 0.1-1ug; Sample diluting liquid is for containing mass concentration 0.1-10% bovine serum albumin(BSA) BSA and containing mass concentration 0.01-0.05%NaN ₃0.01mol/L and the phosphate buffer (PBS) of pH7.2-7.4; Enzyme conjugates is the anti-dog IgG of a horseradish peroxidase-rabbit enzyme conjugates; Concentrated cleaning solution is to contain the 0.01mol/L of volumetric concentration 0.05% Tween-20 and the PBS of pH7.2-7.4; Zymolyte A solution is 3,3 '-5,5 '-tetramethyl biphenyl amine aqueous solution, and zymolyte B solution is hydrogen peroxide solution; Stop buffer is 1mol/L H ₂SO ₄Solution, positive control, negative control are placed in the box.

2. dog according to claim 1 is used anti-rabies virus IgG antibody ELISA detection kit, it is characterized in that described rabies virus antigen cultivates, increases with the hydrophobia strain on the Vero cell, through freeze thawing, ultrasonic disruption, differential centrifugation extract and Sepharose 4FF post filters purifying,-20 ℃ of preservations, as bag by with rabies viruses antigen.