CN101936998B

CN101936998B - Antirabies virus IgG (Immunoglobulin G) antibody ELISA (Enzyme Linked Immunosorbent Assay) detection kit for dogs

Info

Publication number: CN101936998B
Application number: CN 201010257527
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: 2013-04-10
Anticipated expiration: 2030-08-19
Also published as: CN101936998A

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 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 anti-rabies virus IgG antibody ELISA detection kit.

Background technology

Rabies are the infectious diseases common to human beings and animals that caused by rabies viruses (Rabies virus, RV), and its main host is the animals such as dog, cat.Classical symptom is 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 Medical pet market obtains, whether dog only obtained protection after people urgently wished to understand pet dog inoculation rabies vaccine, and the at present testing of rabies virus antibodies 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 by propagating with the band poison dog of human close contact, and its harm has caused showing great attention to of government and the people.

At present, be used for method and corresponding kit that animal used as test comprises that the 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.The recombinant protein of the multiplex prokaryotic expression of common ELISA kit or the totivirus of cultivation are done envelope antigen.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.Although and colloidal gold immunity chromatography has advantage simple to operate, remolding sensitivity is lower.China Patent Publication No. be CN1547027A Patent Application Publication a kind ofly detect rabies poison IgG kit and preparation method with the indirect enzyme immunosorbent adsorption test, China Patent Publication No. be CN1547028A Patent Application Publication a kind ofly detect the total antibody kit of rabies poison and preparation method with competition enzyme-linked immunosorbent adsorption test, China Patent Publication No. be CN101413948 Patent Application Publication a kind of rabies virus NP-ELISA antibody detection reagent kit, 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 anti-rabies virus IgG antibody ELISA detection kit.The present invention adopts monoclonal antibody to be coated with the indirect envelope antigen preparation feedback of method plate, and then composition detects the kit of dog anti rabies virus IgG antibody with indirect immune adsorption experiment, 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 anti-rabies virus IgG antibody ELISA detection kit, described detection kit includes: pre-coated 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 coated anti-rabies monoclonal antibodies in advance, coated damping fluid is the carbonate buffer solution of 0.05M pH9.6, namely 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 coated rabies viruses purifying antigen, package amount is every hole 0.1-1ug again; 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 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.

The dog 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 according to a conventional method purifying dog 1gG; 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 ℃ save backup; (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), namely 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 acetic acid is 5.0, and adding concentration is 30%H ₂O ₂400ul and get final product; 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 uses the hydrophobia strain to cultivate, increase at the Vero cell, and through freeze thawing, ultrasonic disruption, differential centrifugation extract and Sepharose 4FF post filters purifying, and-20 ℃ of preservations are as being coated with rabies viruses antigen.

Experimental result of the present invention and analysis:

1. specific detection: the kit with manufacturing of the present invention detects respectively rabies viruses immunity dog serum, 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 process chart of the present invention.

Embodiment

One. the dog 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 according to a conventional method purifying dog 1gG; 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 ℃ save backup;

3. described rabies virus antigen uses the hydrophobia strain to cultivate, increase at the Vero cell, and through freeze thawing, ultrasonic disruption, differential centrifugation extract and Sepharose 4FF post filters purifying, and-20 ℃ of preservations are as being coated with rabies viruses antigen;

4. rabies monoclonal antibodies is coated on the ELISA Plate hole uniformly, coated damping fluid is the carbonate buffer solution of 0.05M pH9.6, namely 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 coated is the rabies viruses purifying antigen again, and package amount is every hole 0.1-1ug;

5. other solution preparations of kit: 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), namely 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 acetic acid is 5.0, and adding concentration is 30%H ₂O ₂400ul and get final product; 4. stop buffer: get 54.3ml concentration and be 95% concentrated sulphuric acid adding distil water and get final product to 1000ml.

Two, the dog 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 simultaneously the positive and negative controls, 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 with anti-rabies virus IgG antibody ELISA detection kit, described detection kit includes: pre-coated 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 coated anti-rabies monoclonal antibodies in advance, coated damping fluid is the carbonate buffer solution of 0.05M pH9.6, namely contains 1.59g Na in 1 liter of solution ₂CO ₃, 2.93g NaHCO ₃, package amount is every hole 0.1-1 μ g; Confining liquid is that mass concentration is BSA or the skim milk of 1-10%; Sealed coated rabies viruses purifying antigen, package amount is every hole 0.1-1 μ g again; 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 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 anti-rabies virus IgG antibody ELISA detection kit, it is characterized in that the strain of described rabies virus antigen usefulness hydrophobia is cultivated at the Vero cell, amplification, through freeze thawing, ultrasonic disruption, differential centrifugation extract and Sepharose 4FF post filters purifying,-20 ℃ of preservations are as being coated with rabies viruses antigen.