CN101592660B - Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit - Google Patents
Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit Download PDFInfo
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Abstract
The invention aims to provide a brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit which meets the demand of epidemic prevention of animals in our country and is quick, sensitive, specific and steady. The reagent kit is assembled by utilizing a purified lipopolysaccharide mixture of smooth bacterium burgeri and rough bacterium burgeri as an antigen coated ELIAS plate, using an immune healthy cow to prepare a positive control milk liquid, using a healthy non-immune cow to prepare a negative control milk liquid, taking a receptor-protein AG which is labeled by enzyme and is combined with Fc segments of mammals as an enzyme labeled conjugate, and is matched with a diluent, a washing liquid, a substrate solution A, a substrate solution B, H2O2, and a stop buffer. The brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit establishes a quick, sensitive, specific and accurate inspection method which is suitable for brucellosis diagnosis and epidemiological investigation, and meets the demand of the epidemic prevention of the animals in our country.
Description
(1) technical field
The present invention relates to immunological detection, is exactly Brucellosis indirect enzyme-linked immunosorbent assay milk antibody assay kit specifically.
(2) background technology
Immunological detection method is the experimental technique of cell factor of a series of mensuration antigens, antibody, immunocyte and the secretion thereof of applied immunology Theoretical Design.Interpenetrate along with interdisciplinary, the scope that immunology relates to constantly enlarges, and new immunological detection method emerges in an endless stream.The range of application of immunological method not only becomes the important method of various clinical medical diagnosis on disease also at expanding day, also provides convenience for the multi-disciplinary research of crowd.
Antigen and corresponding antibodies meet specific binding can occur, and present certain reacting phenomenon under the impact of external condition, and such as aggegation or precipitation, available known antigens (or antibody) detects unknown antibody (or antigen) by this.The antibody that test is adopted often is present in the serum, therefore is referred to as again serological reaction (serological reaction).Antigen type is various, can divide graininess and solubility two classes by its physical behavior.The former phalangeal cell antigen (comprising bacterial antigens), its preparation is comparatively easy, generally is made into finite concentration with new fresh cell after with stroke-physiological saline solution or phosphate buffer washing.If be bacterial antigens, then get the fresh cultured thing, be handled as follows through the collection bacterium, H antigen is because of thermo-labile usefulness 0.3%～0.5% formaldehyde treated, and O antigen is used after 100 ℃ of 2h remove H antigen by heat-resisting the heating.Soluble antigen can be the cell ingredients such as cell membrane, cytoplasm, nucleus and nuclear membrane, also may be through emiocytosis some soluble factors to the body fluid.The cell ingredient often need pass through fragmentation, the rough antigens of centrifugal acquisition such as machinery or enzymatic isolation method, and is further purified by methods such as selective precipitation or chromatographies.The soluble antigen of (such as serum etc.) then can directly obtain required composition with biochemical means in the body fluid.Some soluble antigen only has immunoreactivity, and non-immunogenicity, this type of antigen is still needed can become comlete antigen with the carrier coupling.
Brucellosis (Brucellosis) claim again the Mediterranean remittent fever, Malta fever, and undulant fever or undulant fever are the infecting both domestic animals and human whole body infectious diseases that is caused by brucella, its clinical characters is long-term fever, hidrosis, arthralgia and hepatosplenomegaly etc.Britain medical officer Bruce isolated " brucella " on the island, Malta from soldier's spleen of dying from " Malta fever " in 1886, clear and definite first pathogen that should disease.Agglutinating reaction can occur in Wright in 1897 and its colleague's Discover the patients serum and brucellar culture, is called the Wright agglutinating reaction, thereby has set up so far the still serological diagnostic method of usefulness.In the Ancient Times in China doctor nationality though this disease is had description, until Boone in 1905 makes formal report in Chongqing to this disease.Should disease distribute in the world at present, only have several countries to eliminate this disease, and in the northeast of China, North China, northwest one are with popular and distribution, distribute in other area, and increasingly extensive serious with harm.To animal husbandry and human next serious economic loss.
Brucella is the Gram negative bacillus pumilis of a class, and endotoxin is important morbid substance.Brucella has strong invasiveness, and bacterium can enter the host by intact skin and mucous membrane.Brucella has 6 bions, and what China was popular is Brucella melitensis, and three kinds of Brucella abortus and Brucella suis are wherein the most common with Brucella melitensis.In the nature situation, have 60 many animals can infect brucella, it mainly is goat, sheep, and ox and pig, take miscarriage as main, the pregnancy period animal sense of being advisable most.Human to the brucella susceptible, after bacterium enters human body, protracted course of disease, repeatedly outbreak, heating is wave, and as not treating, consequence is serious.
According to clinical symptoms, epidemic and characteristic pathology, be not difficult to make the tentative diagnosis of brucellosis, but because there are similar symptom in Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157 infection clinically, must could make a definite diagnosis at last brucellosis by the laboratory diagnosis technology.Be fit to that this disease diagnosed and quick, responsive, special, the method accurately of epidemiology survey in order to set up, domestic and international many scholars have carried out large quantity research, and have obtained significant achievement.Successively set up in cause of disease evaluation, the fluorescence antibody and the detection method such as detection.Yet the brave red flat board that China uses at present and tube agglutination method are that method is eliminated in international trade, and backward in technique, false positive is also relatively high.
(3) summary of the invention
The object of the present invention is to provide a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk antibody assay kit China's animal epidemic prevention demand, quick, responsive, special, stable that satisfies.
The object of the present invention is achieved like this: it is to utilize smooth type Brucella (B.abortus S1119.3) and the purifying lipopolysaccharides potpourri of rough type Brucella (B.abortus RB51) to make antigen coated ELISA Plate, prepare the positive control milk with S1119.3 immune health ox, prepare the negative control milk with the nonimmune ox of health, with enzyme labeling be combined with mammal Fc section acceptor--a-protein G makes the enzyme labeling bond, be equipped with dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling forms.
The present invention also has following technical characterictic:
1. the preparation method of described antigen coated ELISA Plate is as follows:
(1) coated: under the aseptic condition, the freeze-dried antigen of two kinds of purifying is suspended to volume before the freeze-drying with distilled water, ratio function with mixed smoothness type and rough in 1: 5, carbonic acid buffer with 0.05MpH9.6 diluted by 1: 1000 again, with 100 μ l/ holes, coated polystyrene 96 orifice plates (NUNC620 or equivalent material) add and are placed on 4 ℃ of temperature and incubated 18 hours;
(2) washing: the PBST as 7.2 makes cleansing solution take 0.01mol/l pH value, and the effect of adding capacity room temperature was got rid of after 3 minutes, as above repeated 3 times, or repeated to wash 3 times with washing the plate machine, pats dry till the no-watermark;
(3) sealing: be that 6.3 PBST is by the 100ul/ hole with containing the albuminous 0.01mol/l pH of 0.1% milk liquid value, be added in the ELISA Plate, 37 ℃ act on 1 hour, get rid of, add the capacity cleansing solution, the room temperature effect was got rid of after 3 minutes, as above repeat 3 times, or repeat to wash 3 times with washing the plate machine, pat dry till the no-watermark, natural drying;
(4) packing: will seal good antigen coated ELISA Plate and be put in the aluminium foil bag, add the drying agent of 1g packing, with vacuum packing machine antigen coated ELISA Plate be packaged, labelled.
2. described positive control method for preparing milk liquor is as follows:
(1) animal is used in manufacturing: the ox of preparation positive control milk need to detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, infect and without the healthy ox of the negative antibodies such as propagating system infection without Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157.Observe a week;
(2) immunogene preparation: with the flat bottle of Brucella S1119.3 inoculation potato immersion liquid agar, putting 37 ℃ cultivated 48 hours, during one deck bacterium colony to be formed, choose pure flat bottle, agglutinating water is abandoned in suction, with the sterile saline that contains 0.5% phenol lawn is washed, in the neutral glass bottle of impouring, adding formalin to final concentration is 0.2%, puts 37 ℃ of vibration deactivations 48 hours, is up to the standards by " Chinese veterinary pharmacopoeia ", centrifugal 10 minutes with 20000 rev/mins in 4 ℃, get precipitation, do 10 times of dilutions with physiological saline, as immunogene; For subsequent use in 2 ℃～8 ℃ Refrigerator stores;
(3) immune programme for children: press minute 4 intramuscular injection of 5ml/ point with immunogene, immunity animal in conceived late period, after 14 days, with the method immunity once, and 14 days again, wait for childbirth, collect milk, detect with ELISA; Milk OD
450nmWith negative milk OD
450nmRatio (P/N)＞1 can adopt in a large number milk; Such as disqualified upon inspection, booster immunization once again;
(4) positive milk manufacturing: with the conventional method blood sampling, separate its milk, it as milk to be checked, is done 1: 2 with the milk dilution, 1: 4......1: 128 dilutions are done to get milk OD to be checked with reference to carrying out the ELISA test with the positive milk of Quality Control
450nmThe positive milk OD of/Quality Control
450nmBe worth=1 o'clock milk dilutability to be checked as the milk extension rate, with the milk dilution milk diluted;
(5) degerming and packing: milk is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal under the aseptic condition and gentamicin makes its final concentration be 0.02% with anticorrosion, then quantitative separating, freeze-drying under the aseptic condition, labelled.
3. the preparation method of described negative control milk is as follows:
(1) gathers the healthy animal milk with conventional method, separate its milk;
(2) milk is processed and packing: milk is carried out the suction filtration degerming with the filter of 0.45 μ m, and adding thimerosal and gentamicin make its final concentration be 0.02% with anticorrosion under the aseptic condition, and then quantitative separating under the aseptic condition is labelled.
4. described enzyme labeling bond is acceptor--the a-protein G of being combined with mammal Fc section of enzyme labeling, and enzyme labeling a-protein G uses pH7.2, and PBS solution is pressed 0.5mg/ml dilution enzyme labeling bond as enzyme labeling bond dilution; Filtration sterilization; The enzyme labeling bond is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal under the aseptic condition and gentamicin makes its final concentration be 0.02% with anticorrosion; Press 0.2ml/ bottle packing enzyme labeling bond under the packing freeze-drying aseptic condition.
Brucellosis indirect enzyme-linked immunosorbent assay milk antibody assay kit of the present invention set up to be fit to quick, responsive, special, the method for inspection accurately of brucellosis diagnosis and epidemiology survey, satisfies the demand of China's animal epidemic prevention.
(4) embodiment
Below the invention will be further described.
Embodiment 1, Brucellosis indirect enzyme-linked immunosorbent assay milk antibody assay kit of the present invention, it is to utilize smooth type Brucella (B.abortus S1119.3) and the purifying lipopolysaccharides potpourri of rough type Brucella (B.abortus RB51) to make antigen coated ELISA Plate, prepare the positive control milk with the healthy ox of S19 vaccine immunity, prepare the negative control milk with the nonimmune ox of health, with enzyme labeling be combined with mammal Fc section acceptor--a-protein G makes the enzyme labeling bond, is equipped with dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling forms.The present invention also has following technical characterictic:
1. the preparation method of described antigen coated ELISA Plate is as follows:
(1) coated: under the aseptic condition, the freeze-dried antigen of two kinds of purifying is suspended to volume before the freeze-drying with distilled water, ratio function with mixed smoothness type and rough in 1: 5, carbonic acid buffer with 0.05M pH9.6 diluted by 1: 1000 again, with 100 μ l/ holes, coated polystyrene 96 orifice plates (NUNC620 or equivalent material) add and are placed on 4 ℃ of temperature and incubated 18 hours;
(2) washing: the PBST as 7.2 makes cleansing solution take 0.01mol/l pH value, and the effect of adding capacity room temperature was got rid of after 3 minutes, as above repeated 3 times, or repeated to wash 3 times with washing the plate machine, pats dry till the no-watermark;
(3) sealing: be that 6.3 PBST is by the 100ul/ hole with containing the albuminous 0.01mol/l pH of 0.1% milk liquid value, be added in the ELISA Plate, 37 ℃ act on 1 hour, get rid of, add the capacity cleansing solution, the room temperature effect was got rid of after 3 minutes, as above repeat 3 times, or repeat to wash 3 times with washing the plate machine, pat dry till the no-watermark, natural drying;
(4) packing: will seal good antigen coated ELISA Plate and be put in the aluminium foil bag, add the drying agent of 1g packing, with vacuum packing machine antigen coated ELISA Plate be packaged, labelled.
2. described positive control method for preparing milk liquor is as follows:
(1) animal is used in manufacturing: the ox of preparation positive control milk need to detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, infect and without the healthy ox of the negative antibodies such as propagating system infection without Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157.Observe a week;
(2) immunogene preparation: with the flat bottle of Brucella S1119.3 inoculation potato immersion liquid agar, putting 37 ℃ cultivated 48 hours, during one deck bacterium colony to be formed, choose pure flat bottle, agglutinating water is abandoned in suction, with the sterile saline that contains 0.5% phenol lawn is washed, in the neutral glass bottle of impouring, adding formalin to final concentration is 0.2%, puts 37 ℃ of vibration deactivations 48 hours, is up to the standards by " Chinese veterinary pharmacopoeia ", centrifugal 10 minutes with 20000 rev/mins in 4 ℃, get precipitation, do 10 times of dilutions with physiological saline, as immunogene; For subsequent use in 2 ℃～8 ℃ Refrigerator stores;
(3) immune programme for children: press minute 4 intramuscular injection of 5ml/ point with immunogene, immunity animal in conceived late period, after 14 days, with the method immunity once, and 14 days again, wait for childbirth, collect milk, detect with ELISA; Milk OD
450nmWith negative milk OD
450nmRatio (P/N)＞1 can adopt in a large number milk; Such as disqualified upon inspection, booster immunization once again;
(4) positive milk manufacturing: with the conventional method blood sampling, separate its milk, it as milk to be checked, is done 1: 2 with the milk dilution, 1: 4......1: 128 dilutions are done to get milk OD to be checked with reference to carrying out the ELISA test with the positive milk of Quality Control
450nmThe positive milk OD of/Quality Control
450nmBe worth=1 o'clock milk dilutability to be checked as the milk extension rate, with the milk dilution milk diluted;
(5) degerming and packing: milk is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal under the aseptic condition and gentamicin makes its final concentration be 0.02% with anticorrosion, then quantitative separating, freeze-drying under the aseptic condition, labelled.
3. the preparation method of described negative control milk is as follows:
(1) gathers the healthy animal milk with conventional method, separate its milk;
(2) milk is processed and packing: milk is carried out the suction filtration degerming with the filter of 0.45 μ m, and adding thimerosal and gentamicin make its final concentration be 0.02% with anticorrosion under the aseptic condition, and then quantitative separating under the aseptic condition is labelled.
4. described enzyme labeling bond is acceptor--the a-protein G of being combined with mammal Fc section of enzyme labeling; Enzyme labeling a-protein G uses pH7.2, and PBS solution is pressed 0.5mg/ml dilution enzyme labeling bond as enzyme labeling bond dilution; Filtration sterilization; The enzyme labeling bond is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal under the aseptic condition and gentamicin makes its final concentration be 0.02% with anticorrosion; Press 0.2ml/ bottle packing enzyme labeling bond under the packing freeze-drying aseptic condition.
5. described dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer is 0.05M carbon acid solution, 1% BSA solution, 10 times of washing lotions (PBST), 10 times of sample diluting liquids, 10 times of substrate solution A, substrate solution B, H
2O
2Liquid and 10 times of stop buffers;
Described 0.05M carbon acid solution is prepared as follows: sodium carbonate, 1.93 grams; Sodium bicarbonate, 3.80 grams; Adding distil water to 1000 milliliter, 9.6,10 pounds of high pressure of pH 15 minutes;
Described 1% BSA solution preparation method is: get 1000 milliliters of PBST solution, add BSA 10 grams, adjust pH 7.0, filtration sterilization;
Described 10 times of washing lotions (PBST) preparation: sodium chloride, 8 grams; Potassium dihydrogen phosphate, 0.2 gram; Sodium hydrogen phosphate (12H
2O), 6.29 grams; Adding distil water to 100 milliliter adds 0.5 milliliter of polysorbas20; 7.2,10 pounds of high pressure of adjust pH 15 minutes;
The preparation of described 10 times of sample diluting liquids: sodium chloride, 8.5 grams; Potassium dihydrogen phosphate, 0.2 gram; Sodium hydrogen phosphate (12H
2O), 6.29 grams; 0.5 the milliliter polysorbas20, EDTA, 5.7 grams, adding distil water to 100 milliliter, pH6.3,10 pounds of high pressure 15 minutes;
The preparation of described 10 times of substrate solution A: get citric acid 4.2g, sodium acetate 13.5g adds deionized water 100ml, adjust pH 4.0, filtration sterilization; 1.2ml/ the bottle packing, sealing bottleneck, adhesive label;
Described substrate solution B preparation: get TMB 0.2192g, dimethyl sulfoxide (DMSO) 20ml dissolves under the room temperature; 0.5ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
Described H
2O
2Liquid is that 0.3%, 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
The preparation of described 10 times of stop buffers: the concentrated sulphuric acid of getting 55.6 milliliter 98% adds in 80 ml waters, transfers to 100 milliliters of cumulative volumes; 1.2 milliliter/bottle is sub-packed in bottle, the sealing bottleneck, adhesive label, batch.
Embodiment 2, and just the relevant issues of Brucellosis indirect enzyme-linked immunosorbent assay milk antibody assay kit of the present invention are explained as follows:
1 bacterial classification is selected
Brucella ox type S1119.3 is the bacterial strain of traditional extraction antigen.It derives from U.S. USDA, because S1119.3 is difficult for changing into rough type, is the bacterial strain of the extraction smooth type bacteria lipopolysaccharide of International Animal Health tissue (OIE) approval.RB51 is the dissociant of Brucella abortus rough type, as a kind of Brucella abortus vaccine strain, also is the bacterial strain of international extraction rough type bacteria lipopolysaccharide.Make antigen with the mixture of two kinds of bacteria lipopolysaccharides and detect brucellosis antibody, purpose is in order to detect all serotype Brucella antibodies, to improve the recall rate of detection kit, avoiding undetected.
The preparation of 2 antigens
Although 2.1 the bacterial reproduction brucella divides two class cause of diseases in China, owing to be the infecting both domestic animals and human cause of disease, the breeding of all viable bacterias and calibration operation are all carried out in the P3 laboratory.
2.2 the bacteria inactivation mode considers that for biological safety except the viable bacteria operation, other work can be carried out in P2 level laboratory.The deactivation test effect shows, 80 ℃ of the 0.5% phenol physiological saline washing lotions of brucella RB51 and S1119.3 were kept 90 minutes, effective killing bacteria, and lipopolysaccharides extracted not impact.
2.3 the purity of purifying, concentrated antigen is the specific key index of guarantee reagent box.Be to improve the purity of lipopolysaccharides, utilize the brucella lipopolysaccharides can be present in specifically the characteristic of organic phase phenol, adopt sodium acetate formalin precipitation boivin antigen, again water dialysis, thus obtain purifying, concentrated antigen.
2.4 the titration purified antigen is for being used for coated elisa plate, need the monoclonal antibody measuring antigen valence with the lipotropism polysaccharide, according to experimental study, because the selectivity of monoclonal antibody identification form one antigen site, antigen purity by the producting rule preparation meets the kit requirement substantially, but the amount of LPS is not definite constant in the manufacture process of every batch of antigen, so every batch of antigen all need carry out titration.Use the monoclonal antibody through demarcating, when the antigen amount is enough, its OD should be 1.5～2.0, and this colour developing interval is that ELISA is the most responsive, and the antigen amount slightly changes, its OD namely changes rapidly, the antigen amount is during less than standard, and the OD value may be on the low side, tests insensitive, the sensing range of microplate reader may appear exceeding in OD value higher (greater than 3.0).
3 enzyme mark bonds
Anti-mammal Fc acceptor--a-protein G is the unicity problem that overcomes traditional indirect ELISA method ELIAS secondary antibody host, and the acceptor of being combined with mammal Fc section of employing enzyme labeling-a-protein G is combined as the enzyme labeling thing of homogeneous and with the Fc of all mammal antibody section.
4 negative control animal milks and positive control animal milk
The positive control milk is whether correct, the reaction of a checking ELISA test operation special important component whether in the kit, also is the index whether reaction kit works.For reducing the non-specific of contrast milk, the animal of preparation negative control milk and positive control milk all need carry out Brucella antibody, Caused by Yersinia enterocolitica antibody, Bacillus paratyphosus B antibody, Escherichia coli O:157 antibody test, and detect negative.
With heavy dose of deactivation brucella repeatedly the immunity method prepare positive milk.The animal first immunisation begins to produce antibody after 10 days, along with immunostimulation repeatedly, its antibody horizontal peaked in for the second time immunity in rear about 20 days, and the duration reached about half a year;
The milk that is diagnosed as natural infection brucella animal also can be used as positive control serum after aseptic filtration, but must demarcate through quality controlled serum.
According to the sensitivity experimental study of animal milk brucellosis antibody indirect ELISA test, the positive control milk to the positive rate of monoclonal antibody near 100%P (positive percentage), basically fully colour developing.The negative control milk is not competed inhibition to monoclonal antibody, because the impact of milk, its OD value just is slightly larger than blank in the indirect ELISA experiment.
5 determine that about critical value (cut off) value the yin and yang attribute limit value is to determine the very important index of testing result accuracy.Detectability should be and detects a large amount of negative serum samples in theory, carry out positive percentage distribution research, affect specificity to get rid of nonspecific reaction, but in real work, be difficult to obtain a large amount of definite negative samples, can only be by comparing to determine with other detection reagent.
In the research experiment of the present invention, the animal milk brucellosis antibody indirect ELISA kit of using development detects the negative milk of 30 parts of brucellosis, 53 parts of immune animal Infected with Brucella milks and immune milk, use in the world that OIE has confirmed that the animal milk result of accuracy compares, 30 parts of its positive percentage rates of definite negative sample are all below 20%.Therefore, determine that detection is limited to 20%P (positive percentage).
The specificity specificity of 6 kits is to determine one of important indicator that whether accurate diagnostic result is.For verifying its specificity, with OIE standard positive animal milk, standard female milk, negative with reference to milk, artificial challenge's milk, the detection of immune milk, negative recall rate is 100%.Detect with Bacillus paratyphosus B, Caused by Yersinia enterocolitica, Escherichia coli O:157 positive and negative milk, the specificity that overcomes cross reaction reaches 96.5%.Test findings shows to have higher specificity according to animal milk brucellosis antibody indirect ELISA kit.
Can not also there is no need when when practical application and to every batch of product, detecting to detect one by one institute is ill, so when working out specific criteria only to the positive milk of Caused by Yersinia enterocolitica, the positive milk of Bacillus paratyphosus B, the positive milk of Escherichia coli O:157, the checking of testing of vaccine type antibody, OIE standard positive and standard female milk.
The susceptibility of 7 kits
Because there is immune antiboidy in the extensive compulsory immunization animal of China in the drove, thereby so that general Serology test is difficult to distinguish diagnosis.ELISA detects one of the most responsive method of anti-brucellosis antibody at present, but is badly in need of a kind of can being applied to fast in the work such as reality detection generaI investigation, sensitivity, the little screening detection method of as far as possible avoiding again immune antiboidy to disturb of cross reaction.
Select artificial brucella to attack 37 parts of malicious milks in the research process, 16 parts of OIE standard positive milks, 8 parts of OIE standard female milks, 5 parts of positive milks of immune vaccine detect with animal milk brucella indirect ELISA reagent kit respectively, 51 parts of sick positive milks of Shandong Salmonella are positive as a result, and susceptibility is 96.23% (51/53).
8 is closely related with judgement test method and test findings about usage, correct operation and appropriate judgement just can draw correct result, ELISA is highstrung detection method, often because experiment for improper causes testing result widely different, therefore to usage and as a result decision method also make concrete regulation: kit should return to room temperature before use, because joining, ice-cold diagnostic reagent need the time just can make reaction conditions reach 18～28 ℃ on the ELISA Plate, so just cause the real reaction deficiency of time, affect the combination of antigen antigen.Washing under the plate machine operation condition, it is just enough to wash plate for 4 times.The result has designed two yin and yang attributes and blank with calculating mean value in calculating, and detects the accuracy of test.The computing method of positive percentage are general in the world computing method.
9 about points for attention because the ELISA detection kit in composition be bioactive ingredients, antibody wherein etc. all is again protein ingredient, can cause albuminous degeneration to affect the detection effect of kit thereby at room temperature reach frequent heating and cooling, so must deposit on request use; In addition, the developer in the kit and stop buffer all contain the chemical substance that is pernicious to people, and should avoid skin contact in the experiment.
10 about being placed on respectively 2～8 ℃ and room temperature after stability, storage and the term of validity brucellosis antibody I-ELISA detection kit, getting several coated good ELIAS strip every 1 month tests, the result shows that detection kit was 2～8 ℃ of preservations 12 months, room temperature preservation at least 4 months, its physical behavior, detection specificity and sensitivity are all unaffected.Therefore storage life is fixed tentatively and be: 2～8 ℃ of preservations, the term of validity 1 year.
Embodiment 3, and the elementary combination test of Test in Diagnosis of Human Brucellosis comprises that fluorescence polarization detects and enzyme linked immunosorbent assay.Fluorescence polarization method is a kind of homogeneous phase method, does not need to remove not binding reagents, and operating speed is fast, just obtains the result, and can eliminate some vaccine reactions in two minutes.It both can be used for the laboratory detects, and also can apply to pasture and field, is fit to the detection of wild animal brucellosis.The susceptibility of FPA is high, is outstanding shaker test.Relative low price, accuracy is elementary the same in conjunction with test with other.Such mensuration also is fit to robotization.Its principle is to measure the molecular reaction rate to change, this change be since in test specimen antibody react with soluble antigen and cause.If the antibody conjugated antigen, the specific rotation of antigen will descend, and then can measure this reaction.The ultimate principle of FPA is that molecule rotates Stochastic Interest Rates and its size be inversely proportional to (rotation of little molecule fast, larger molecule is slower) in solution.If a little molecule makes it become large by being attached on the larger molecule, just can measure micromolecular rotational speed and change.What FPA used is by being hydrolyzed the again antigen of mark fluorescent preparation of specific polysaccharide.The average molecular mass of this molecule is 22kD, and making it is the antibody molecule (such as IgG antibody) of 160kD much smaller than molecular weight.In the method for operating of FPA, can in glass tube or 96 orifice plates after dilute serum, blood or the milk, use the FPA analyser to remove the fluorescence reading of background.This reading be stored and the value of reading afterwards in deduct it.The FPA analyser is by the velocity of rotation of polarized light by specific measurement of angle molecule.Then add fluorescently-labeled specific polysaccharide antigen, after the mixing, hatch 2 minutes (serum or milk; Whole blood was hatched 15 seconds), read final numerical value with analyser at last.If tested sample at quilt antibody is arranged, the rotational time of antigen molecule increases, and causes higher milli polarization unit (mP) reading.The quantity that the size of milli polarization unit (mP) reading and antibody exist is proportional.
Two types enzyme linked immunosorbent assay is used for the detection of antagonist.They are indirect enzyme-linked immunosorbent assay and competition enzyme-linked immunosorbent adsorption test.Indirect enzyme-linked immunosorbent assay is combined on the solid phase with antibody, with a kind of reaction of the enzyme labeling reagent that can be combined with the antibody of selected or all isotypes.Because vaccine antibody also may detect with this method, it mainly is intended for shaker test.It has several advantages, comprises robotization, the commercial supply of material, accuracy, relatively short proving time, the adaptability such as relatively cheap.Indirect enzyme-linked immunosorbent assay (IELISA) can be used for most of mammal for detection of antibody level and smooth or that Rough Anti-Brucella produces.This method of inspection can be finished in 90 minutes and also can be used as outstanding shaker test, was used for the laboratory and detected.Its susceptibility is the highest, mainly contains in the elimination plan of brucella bacterium disease.
Indirect enzyme-linked immunosorbent assay (RIELISA) for detection of antibody level and smooth or that Rough Anti-Brucella produces, can be used for most of mammal fast.This method of inspection can be finished in 15 minutes and can be used as outstanding shaker test, and great advantage is can be in the pasture, field operation, quick.
Milk indirect enzyme-linked immunosorbent assay (MIELISA), for detection of the Brucella antibody in the ruminant milk, this conventional sense method can be measured the antibody of smooth type brucella in milk from cows and goats.90 minutes consuming time, be the shaker test as the mixed milk sample.Because the milk that detects animal can be used for the monitoring of brucellosis, also reduced the stimulation to dairy animal simultaneously.
Competition enzyme-linked immunosorbent adsorption test uses the antibody competition in monoclonal antibody and the check sample.It not only has the indirect enzyme-linked immunosorbent assay attribute, and have select suitable affinity competition antibody in order to eliminate because the serological reaction that the antibody of vaccine and cross reaction causes.Competition enzyme-linked immunosorbent adsorption test (CELISA) for detection of the antibody that the smooth type brucella produces, is applicable to people and all animals almost.This detection method is the test of making a definite diagnosis of well last brucellosis take high sensitive monoclonal antibody as the basis, also is for the unique method of distinguishing vaccine immunity and wild virus infection.
Claims (1)
1. Brucellosis indirect enzyme-linked immunosorbent assay milk antibody assay kit, it is characterized in that: it is to utilize the purifying lipopolysaccharides potpourri of smooth type Brucella B.abortus S1119.3 and rough type Brucella B.abortus RB51 to make antigen coated ELISA Plate, prepare the positive control milk with the healthy ox of S19 vaccine immunity, prepare the negative control milk with the nonimmune ox of health, so that enzyme labeling resisting mammal Fc section--a-protein G makes the enzyme labeling bond, be equipped with dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling forms;
The preparation method of described antigen coated ELISA Plate is as follows:
(1) coated: under the aseptic condition, the freeze-dried antigen of two kinds of purifying is suspended to volume before the freeze-drying with distilled water, ratio function with mixed smoothness type and rough in 1: 5, carbonic acid buffer with 0.05M pH9.6 diluted by 1: 1000 again, with 100 μ l/ holes, coated polystyrene 96 orifice plates add and are placed on 4 ℃ of temperature and incubated 18 hours;
(2) washing: the PBST as 7.2 makes cleansing solution take 0.01mol/l pH value, and the effect of adding capacity room temperature was got rid of after 3 minutes, as above repeated 3 times, or repeated to wash 3 times with washing the plate machine, pats dry till the no-watermark;
(3) sealing: be that 6.3 PBST is by the 100ul/ hole with containing the albuminous 0.01mol/l pH of 0.1% milk liquid value, be added in the ELISA Plate, 37 ℃ act on 1 hour, get rid of, add the capacity cleansing solution, the room temperature effect was got rid of after 3 minutes, as above repeat 3 times, or repeat to wash 3 times with washing the plate machine, pat dry till the no-watermark, natural drying;
(4) packing: will seal good antigen coated ELISA Plate and be put in the aluminium foil bag, add the drying agent of 1g packing, with vacuum packing machine antigen coated ELISA Plate be packaged, labelled;
The preparation method of described negative control milk is as follows:
(1) gathers the healthy animal milk with conventional method, separate its milk;
(2) milk is processed and packing: milk is carried out the suction filtration degerming with the filter of 0.45 μ m, and adding thimerosal and gentamicin make its final concentration be 0.02% with anticorrosion under the aseptic condition, and then quantitative separating under the aseptic condition is labelled;
Described enzyme labeling bond is enzyme labeling resisting mammal Fc section--a-protein G; Commercialization enzyme labeling a-protein G uses pH7.2, and PBS solution is pressed 0.5mg/ml dilution enzyme labeling bond as enzyme labeling bond dilution; Filtration sterilization; The enzyme labeling bond is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal under the aseptic condition and gentamicin makes its final concentration be 0.02% with anticorrosion; Press 0.2ml/ bottle packing enzyme labeling bond under the packing freeze-drying aseptic condition;
Described dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer is 0.05M carbon acid solution, 1% BSA solution, 10 times of washing lotion PBST, 10 times of sample diluting liquids, 10 times of substrate solution A, substrate solution B, H
2O
2Liquid and 10 times of stop buffers; It is characterized in that:
Described 0.05M carbon acid solution is prepared as follows: sodium carbonate, 1.93 grams; Sodium bicarbonate, 3.80 grams; Adding distil water to 1000 milliliter, 9.6,10 pounds of high pressure of pH 15 minutes;
Described 1% BSA solution preparation method is: get 1000 milliliters of PBST solution, add BSA 10 grams, adjust pH 7.0, filtration sterilization;
Described 10 times of washing lotion PBST preparation: sodium chloride, 8 grams; Potassium dihydrogen phosphate, 0.2 gram; Sodium hydrogen phosphate, 6.29 grams; Adding distil water to 100 milliliter adds 0.5 milliliter of polysorbas20; 7.2,10 pounds of high pressure of adjust pH 15 minutes;
The preparation of described 10 times of sample diluting liquids: sodium chloride, 8.5 grams; Potassium dihydrogen phosphate, 0.2 gram; Sodium hydrogen phosphate, 6.29 grams; 0.5 the milliliter polysorbas20, EDTA, 5.7 grams, adding distil water to 100 milliliter, pH6.3,10 pounds of high pressure 15 minutes;
The preparation of described 10 times of substrate solution A: get citric acid 4.2g, sodium acetate 13.5g, urea peroxide 0.5g adds deionized water 100ml, adjust pH 4.0, filtration sterilization; 1.2ml/ the bottle packing, sealing bottleneck, adhesive label;
Described substrate solution B preparation: get TMB 0.2192g, dimethyl sulfoxide (DMSO) 20ml dissolves under the room temperature; 0.5ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
Described H
2O
2Liquid is to be produced by U.S. VWR company, and the 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
The preparation of described 10 times of stop buffers: the concentrated sulphuric acid of getting 55.6 milliliter 98% adds in 80 ml waters, transfers to 100 milliliters of cumulative volumes; 1.2 milliliter/bottle is sub-packed in bottle, the sealing bottleneck, adhesive label, batch.
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| CN102707055A (en) * | 2012-06-11 | 2012-10-03 | 郑州安图绿科生物工程有限公司 | Kit for joint or single detection of autoimmune liver disease related antibody and detection method of kit |
| CN102768276A (en) * | 2012-07-23 | 2012-11-07 | 甘肃农业大学 | Indirect ELISA (enzyme linked immunosorbent assay) kit for alicyclobacillus and application thereof |
| CN103344770B (en) * | 2013-07-15 | 2015-09-23 | 山东省农业科学院奶牛研究中心 | Brucella abortus indirect ELISA testing kit |
| CN103543261B (en) * | 2013-09-13 | 2015-11-18 | 中国农业科学院特产研究所 | Cattle and sheep Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit and preparation method thereof |
| CN104698182A (en) * | 2013-12-04 | 2015-06-10 | 中国检验检疫科学研究院 | Brucella outer membrane protein antibody detection ELISA (enzyme-linked immuno sorbent assay) kit |
| CN106198973A (en) * | 2016-09-07 | 2016-12-07 | 扬州大学 | What a kind of simplicity quickly detected B rucella melitensis antibody directly blocks ELISA method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (6)
| Title |
|---|
| Anastasios Minas, et al..Validation of a competitive ELSIA for diagnosis of Brucella melitensis infection in sleep an goats..《The Veterinary Journal》.2007,第177卷(第2008期),411-417. |
| OIE标准全乳iELISA与国标全乳环状试验在奶牛布鲁氏菌病检测中的研究;赵智香等;《中国动物检疫》;20071101;第24卷(第11期);24-25 * |
| Validation of a competitive ELSIA for diagnosis of Brucella melitensis infection in sleep an goats.;Anastasios Minas, et al.;《The Veterinary Journal》;20070615;第177卷(第2008期);411-417 * |
| 张东林等.蛋白AG检测多种动物弓形虫抗体AG-ELISA的效果评价.《湖北农业科学》.2008,第47卷(第06期),692-695. |
| 蛋白AG检测多种动物弓形虫抗体AG-ELISA的效果评价;张东林等;《湖北农业科学》;20080605;第47卷(第06期);692-695 * |
| 赵智香等.OIE标准全乳iELISA与国标全乳环状试验在奶牛布鲁氏菌病检测中的研究.《中国动物检疫》.2007,第24卷(第11期),24-25. |
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