CN101592660A - Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit - Google Patents
Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit Download PDFInfo
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Abstract
The object of the present invention is to provide a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit China's animal epidemic prevention demand, quick, responsive, special, stable that satisfies.It is to utilize the purifying lipopolysaccharides potpourri of smooth type Brucella and rough type Brucella to make antigen coated ELISA Plate, prepare positive control milk liquid with the immune health ox, prepare negative control milk liquid with the non-immune cattle of health, with enzyme labeling combine 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 is formed.Brucellosis indirect enzyme-linked immunosorbent assay of the present invention milk humoral antibody detection kit is set up quick, responsive, special, the method for inspection accurately that are fit to brucellosis diagnosis and epidemiology survey, satisfies the demand of China's animal epidemic prevention.
Description
(1) technical field
The present invention relates to the immunological test method, is exactly Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection 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 Design Theory.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 is also enlarging day by day, not only becomes the important method of various clinical medical diagnosis on disease, and also the research for numerous subjects provides convenience.
Antigen and corresponding antibodies meet and specificity can take place combine, and present certain reacting phenomenon under extraneous condition effect, and as aggegation or precipitation, available by this known antigens (or antibody) detects unknown antibody (or antigen).The antibody that test is adopted often is present in the serum, therefore is referred to as serological reaction (serological reaction) again.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, do following processing 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 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 (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 and the carrier coupling can become comlete antigen.
Brucellosis (Brucellosis) claim the Mediterranean remittent fever again, 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 heating, 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.Wright in 1897 and its colleague find that agglutinating reaction can take place for patients serum and brucellar culture, are called the Wright agglutinating reaction, thereby have set up so far the still serological diagnostic method of usefulness.Though in the Ancient Times in China doctor nationality this disease is had description, in Chongqing this disease is made formal report up to Boone in 1905.Should disease distribute in the world at present, have only several countries to eliminate this disease, and in the northeast of China, North China, northwest one have popular and distributes, 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.Under the nature situation, have more than 60 kind of animal can infect brucella, it mainly is a goat, sheep, and ox and pig, based on miscarriage, the pregnancy period animal sense of being advisable most.Human to the brucella susceptible, after bacterium enters human body, protracted course of disease, outbreak repeatedly, heating is wave, and as not treating, consequence is serious.
According to clinical symptoms, epidemiology characteristics and characteristic pathology, be not difficult to make the tentative diagnosis of brucellosis, but, must could make a definite diagnosis at last brucellosis by the laboratory diagnosis technology because there are similar symptom in enterocolitis yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157 infection clinically.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 big quantity research, and have obtained significant achievement.Successively set up in cause of disease evaluation, the fluorescence antibody and detection method such as detection.Yet 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 higher relatively.
(3) summary of the invention
The object of the present invention is to provide a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection 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 positive control milk liquid with S1119.3 immune health ox, prepare negative control milk liquid with the non-immune cattle of health, with enzyme labeling combine 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 is formed.
The present invention also has following technical characterictic:
1. the preparation method of described antigen coated ELISA Plate is as follows:
(1) bag quilt: under the aseptic condition, the freeze-dried antigen of two kinds of purifying is suspended to volume before the freeze-drying with distilled water, mix smooth type and rough in 1: 5 ratio, carbonic acid buffer with 0.05MpH9.6 diluted by 1: 1000 again, with 100 μ l/ holes, bag is by polystyrene 96 orifice plates (NUNC620 or equivalent material), adds to be placed on 4 ℃ of temperature and to incubate 18 hours;
(2) washing: with 0.01mol/l pH value is that 7.2 PBST makes cleansing solution, 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: with containing the albuminous 0.01mol/l pH of 0.1% milk liquid value is that 6.3 PBST is by the 100ul/ hole, 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 air dry;
(4) packing: will seal good antigen coated ELISA Plate and be put in the aluminium foil bag, add the drying agent of 1g packing, antigen coated ELISA Plate be packaged with vacuum packing machine, 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 liquid need detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, the healthy ox that no enterocolitis yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157 infect and do not have negative antibodies such as propagating system infection.Observe a week;
(2) immunogen preparing: 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, aggegation 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 drug allusion quotation ", centrifugal 10 minutes in 4 ℃ with 20000 rev/mins, get precipitation, do 10 times of dilutions, as immunogene with physiological saline; Preserve standby in 2 ℃～8 ℃ refrigerators;
(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 liquid, detect with ELISA; Milk liquid OD
450nmWith feminine gender milk liquid OD
450nmRatio (P/N)＞1 can adopt milk liquid in a large number; As disqualified upon inspection, booster immunization once again;
(4) positive milk liquid manufacturing: with the conventional method blood sampling, separate its milk liquid, it as milk liquid to be checked, is done 1: 2 with milk liquid dilution, 1: 4......1: 128 dilutions are done to get milk liquid OD to be checked with reference to carrying out the ELISA test with the positive milk of Quality Control liquid
450nmThe positive milk of/Quality Control liquid OD
450nmThe milk liquid dilutability to be checked that is worth at=1 o'clock dilutes milk liquid with milk liquid dilution as milk liquid extension rate;
(5) degerming and packing: the liquid of will suckling carries out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion, and quantitative packing, freeze-drying under the aseptic condition then is labelled.
3. the preparation method of described negative control milk liquid is as follows:
(1) gathers healthy animal milk liquid with conventional method, separate its milk liquid;
(2) milk liquid is handled and packing: the liquid of will suckle carries out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition descends adding thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion, and quantitative packing under the aseptic condition then is labelled.
4. described enzyme labeling bond is acceptor--the a-protein G that combines 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, and aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion; Packing freeze-drying aseptic condition is pressed 0.2ml/ bottle packing enzyme labeling bond down.
Brucellosis indirect enzyme-linked immunosorbent assay of the present invention milk humoral antibody detection kit is set up quick, responsive, special, the method for inspection accurately that are fit to 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 humoral antibody detection 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 positive control milk liquid with the healthy ox of S19 vaccine immunity, prepare negative control milk liquid with the non-immune cattle of health, with enzyme labeling combine 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 is formed.The present invention also has following technical characterictic:
1. the preparation method of described antigen coated ELISA Plate is as follows:
(1) bag quilt: under the aseptic condition, the freeze-dried antigen of two kinds of purifying is suspended to volume before the freeze-drying with distilled water, mix smooth type and rough in 1: 5 ratio, carbonic acid buffer with 0.05M pH9.6 diluted by 1: 1000 again, with 100 μ l/ holes, bag is by polystyrene 96 orifice plates (NUNC620 or equivalent material), adds to be placed on 4 ℃ of temperature and to incubate 18 hours;
(2) washing: with 0.01mol/l pH value is that 7.2 PBST makes cleansing solution, 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: with containing the albuminous 0.01mol/l pH of 0.1% milk liquid value is that 6.3 PBST is by the 100ul/ hole, 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 air dry;
(4) packing: will seal good antigen coated ELISA Plate and be put in the aluminium foil bag, add the drying agent of 1g packing, antigen coated ELISA Plate be packaged with vacuum packing machine, 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 liquid need detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, the healthy ox that no enterocolitis yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157 infect and do not have negative antibodies such as propagating system infection.Observe a week;
(2) immunogen preparing: 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, aggegation 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 drug allusion quotation ", centrifugal 10 minutes in 4 ℃ with 20000 rev/mins, get precipitation, do 10 times of dilutions, as immunogene with physiological saline; Preserve standby in 2 ℃～8 ℃ refrigerators;
(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 liquid, detect with ELISA; Milk liquid OD
450nmWith feminine gender milk liquid OD
450nmRatio (P/N)＞1 can adopt milk liquid in a large number; As disqualified upon inspection, booster immunization once again;
(4) positive milk liquid manufacturing: with the conventional method blood sampling, separate its milk liquid, it as milk liquid to be checked, is done 1: 2 with milk liquid dilution, 1: 4......1: 128 dilutions are done to get milk liquid OD to be checked with reference to carrying out the ELISA test with the positive milk of Quality Control liquid
450nmThe positive milk of/Quality Control liquid OD
450nmThe milk liquid dilutability to be checked that is worth at=1 o'clock dilutes milk liquid with milk liquid dilution as milk liquid extension rate;
(5) degerming and packing: the liquid of will suckling carries out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion, and quantitative packing, freeze-drying under the aseptic condition then is labelled.
3. the preparation method of described negative control milk liquid is as follows:
(1) gathers healthy animal milk liquid with conventional method, separate its milk liquid;
(2) milk liquid is handled and packing: the liquid of will suckle carries out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition descends adding thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion, and quantitative packing under the aseptic condition then is labelled.
4. described enzyme labeling bond is acceptor--the a-protein G that combines 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, and aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion; Packing freeze-drying aseptic condition is pressed 0.2ml/ bottle packing enzyme labeling bond down.
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 compound 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 0.3%, 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.
Embodiment 2, and the relevant issues of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit just 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 of 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 Detection of antigen brucellosis antibody with the mixture of two kinds of bacteria lipopolysaccharides, purpose is in order to detect all serotype Brucella antibodies, to improve the recall rate of detection kit, avoiding omission.
2 antigen preparation
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, 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 that the viable bacteria operation, other work can be carried out in P2 level laboratory.The deactivation test effect shows that the 0.5% phenol physiological saline washing lotion of brucella RB51 and S1119.3 was kept 90 minutes for 80 ℃, effective killing bacteria, and lipopolysaccharides extracted not influence.
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 the characteristic of organic phase phenol specifically, adopt sodium acetate formalin precipitation boivin antigen, water dialysis again, thus obtain purifying, concentrated antigen.
2.4 the titration purified antigen is for being used for coated elisa plate, need monoclonal antibody measuring antigen valence with the lipotropism polysaccharide, according to experimental study, because monoclonal antibody is discerned the selectivity of single 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 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 changes slightly, its OD promptly 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 enzymes mark bond
Anti-mammal Fc acceptor--a-protein G is the unicity problem that overcomes traditional indirect ELISA method ELIAS secondary antibody host, adopts the acceptor-a-protein G that combines with mammal Fc section of enzyme labeling to combine as the enzyme labeling thing of homogeneous and with the Fc section of all mammal antibody.
4 negative control animal milk liquid and positive control animal milk liquid
Whether special positive control milk liquid be whether correct, the reaction of checking ELISA test operation a important component in the kit, also is the index whether reaction kit works.For reducing the non-specific of contrast milk liquid, the animal of preparation negative control milk liquid and positive control milk liquid all need carry out Brucella antibody, enterocolitis yersinia enterocolitica antibody, Bacillus paratyphosus B antibody, Escherichia coli O:157 antibody test, and detect negative.
With the repeatedly immune positive milk of the method preparation liquid of heavy dose of deactivation brucella.The animal first immunisation begins to produce antibody after 10 days, along with immunostimulation repeatedly, its antibody horizontal peaked in immunity for the second time in back about 20 days, and the duration reached about half a year;
The milk liquid 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 liquid brucellosis antibody indirect ELISA test, positive control milk liquid to the positive rate of monoclonal antibody near 100%P (positive percentage), colour developing fully basically.Negative control milk liquid is not competed inhibition to monoclonal antibody, because the influence of milk liquid, 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 the very important index of decision testing result accuracy.Detectability should be and detects a large amount of negative serum samples in theory, carry out positive percentage distribution research, influence 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 detectable.
In the research experiment of the present invention, the animal milk liquid brucellosis antibody indirect ELISA kit of using development detects the negative milk of 30 parts of brucellosis liquid, 53 parts of immune animal brucella infect milk liquid and immunity milk liquid, use in the world that OIE has confirmed that the animal milk liquid 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 liquid, standard female milk liquid, negatively detect with reference to suckle liquid, immunity milk liquid of milk liquid, artificial challenge, negative recall rate is 100%.Detect with Bacillus paratyphosus B, enterocolitis yersinia enterocolitica, Escherichia coli O:157 positive and negative milk liquid, the specificity that overcomes cross reaction reaches 96.5%.Test findings shows to have higher specificity according to animal milk liquid 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 to institute is ill, so when working out specific criteria only to the positive milk of enterocolitis yersinia enterocolitica liquid, the positive milk of Bacillus paratyphosus B liquid, the positive milk of Escherichia coli O:157 liquid, the checking of testing of vaccine type antibody, OIE standard positive and standard female milk liquid.
The susceptibility of 7 kits
Because there is immune antiboidy in the extensive compulsory immunization animal of China in the drove, thereby make general serology detection method be 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 work such as actual detected generaI investigation, sensitivity, the little screening detection method of as far as possible avoiding immune antiboidy to disturb again of cross reaction.
Select for use artificial brucella to attack 37 parts of poison milk liquid in the research process, 16 parts of OIE standard positive milk liquid, 8 parts of OIE standard female milk liquid, the positive milk of immune vaccine liquid detects with animal milk liquid brucella indirect ELISA reagent kit respectively for 5 parts, 51 parts of sick positive milk liquid 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 unusual sensitive detecting 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 of 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, influence 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 calculation method in the world.
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 influence the detection effect of kit thereby at room temperature reach frequent heating and cooling, so must deposit use on request; In addition, developer in the kit and stop buffer all contain the chemical substance that is pernicious to people, and should avoid the skin contact in the experiment.
10 about being placed on 2～8 ℃ and room temperature respectively after stability, storage and the term of validity brucellosis antibody I-ELISA detection kit, getting several bags every 1 month is tested by good ELIAS strip, 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 diagnosis 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 height of FPA 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 antibodies 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 (micromolecule rotation fast, bigger molecule is slower) in solution.If a micromolecule makes it become big by being attached on the bigger molecule, just can measure micromolecular rotational speed and change.FPA uses is by the hydrolysis specific polysaccharide antigen of mark fluorescent preparation again.The average molecular mass of this molecule is 22kD, and making it is the antibody molecule (as IgG antibody) of 160kD much smaller than molecular weight.In the method for operating of FPA, can use the FPA analyser to remove the fluorescence reading of background in glass tube or 96 orifice plates after dilute serum, blood or the milk.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.Add fluorescently-labeled specific polysaccharide antigen then, 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, with antibodies on solid phase, with a kind of can with the reaction of the enzyme labeling reagent of the antibodies 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, short relatively proving time, adaptability such as relatively cheap.Indirect enzyme-linked immunosorbent assay (IELISA) is used to detect antibody level and smooth or that the rough type brucella produces, can be used for most of mammal.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) is used to detect antibody level and smooth or that the rough type brucella produces fast, can be used for most of mammal.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 liquid indirect enzyme-linked immunosorbent assay (MIELISA) is used to detect the Brucella antibody in the ruminant milk, and this conventional sense method can be measured the antibody of smooth type brucella in milk from cows and goats.90 minutes consuming time, be shaker test as the mixed milk sample.Because the milk liquid that detects animal can be used for the monitoring of brucellosis, also reduced stimulation simultaneously to dairy animal.
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) is used to detect the antibody that the smooth type brucella produces, and is applicable to people and all animals almost.This detection method is the test of making a definite diagnosis of well last brucellosis based on high sensitive monoclonal antibody, also is the unique method that is used to distinguish vaccine immunity and wild virus infection.
Claims (5)
1. Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit, it is characterized in that: 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 positive control milk liquid with the healthy ox of S1119.3 vaccine immunity, prepare negative control milk liquid with the non-immune cattle of health, with enzyme labeling combine 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 is formed.
2. a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit according to claim 1, it is characterized in that: the preparation method of described antigen coated ELISA Plate is as follows:
(1) bag quilt: under the aseptic condition, the freeze-dried antigen of two kinds of purifying is suspended to volume before the freeze-drying with distilled water, mix smooth type and rough in 1: 5 ratio, carbonic acid buffer with 0.05MpH9.6 diluted by 1: 1000 again, with 100 μ l/ holes, bag is by polystyrene 96 orifice plates (NUNC620 or equivalent material), adds to be placed on 4 ℃ of temperature and to incubate 18 hours;
(2) washing: with 0.01mol/l pH value is that 7.2 PBST makes cleansing solution, 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: with containing the albuminous 0.01mol/l pH of 0.1% milk liquid value is that 6.3 PBST is by the 100ul/ hole, 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 air dry;
(4) packing: will seal good antigen coated ELISA Plate and be put in the aluminium foil bag, add the drying agent of 1g packing, antigen coated ELISA Plate be packaged with vacuum packing machine, labelled.
3. a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit according to claim 1, it is characterized in that: described positive control method for preparing milk liquor is as follows:
(1) animal is used in manufacturing: the ox of preparation positive control milk liquid need detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, the healthy ox that no enterocolitis yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157 infect and do not have negative antibodies such as propagating system infection is observed a week;
(2) immunogen preparing: 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, aggegation 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, be up to the standards by " Chinese veterinary drug allusion quotation ", in 4 ℃ with 20000 rev/mins centrifugal 10 minutes, get precipitation, do 10 times of dilutions with physiological saline, as immunogene, preserve standby in 2 ℃～8 ℃ refrigerators;
(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 liquid, detect milk liquid OD with ELISA
450nmWith feminine gender milk liquid OD
450nmRatio (P/N)＞1 can adopt milk liquid in a large number, as disqualified upon inspection, booster immunization is once again;
(4) positive milk liquid manufacturing: with the conventional method blood sampling, separate its milk liquid, it as milk liquid to be checked, is done 1: 2 with milk liquid dilution, 1: 4......1: 128 dilutions are done to get milk liquid OD to be checked with reference to carrying out the ELISA test with the positive milk of Quality Control liquid
450nmThe positive milk of/Quality Control liquid OD
450nmThe milk liquid dilutability to be checked that is worth at=1 o'clock dilutes milk liquid with milk liquid dilution as milk liquid extension rate;
(5) degerming and packing: the liquid of will suckling carries out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion, and quantitative packing, freeze-drying under the aseptic condition then is labelled.
4. a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit according to claim 1, it is characterized in that: the preparation method of described negative control milk liquid is as follows:
(1) gathers healthy animal milk liquid with conventional method, separate its milk liquid;
(2) milk liquid is handled and packing: the liquid of will suckle carries out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition descends adding thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion, and quantitative packing under the aseptic condition then is labelled.
5. a kind of Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit according to claim 1, it is characterized in that: described enzyme labeling bond is acceptor--the a-protein G that combines 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, and aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion; Packing freeze-drying aseptic condition is pressed 0.2ml/ bottle packing enzyme labeling bond down.
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| 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 |
| CN103344770A (en) * | 2013-07-15 | 2013-10-09 | 山东省农业科学院奶牛研究中心 | Brucella abortus indirect enzyme linked immunosorbent assay (ELISA) detection kit |
| 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 |
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| 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 |
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| 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 |
| RU2792079C1 (en) * | 2022-03-09 | 2023-03-16 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кубанский государственный аграрный университет имени И.Т. Трубилина" | Method for rapid diagnosis of brucellosis in sheep and goat milk intended for sale on the agricultural market |
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