CN101799470A - Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit - Google Patents
Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit Download PDFInfo
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Abstract
The invention aims to provide a brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit which has moderate cost and short reaction time and is closely combined with the requirements of actual detection and general survey operations in China. The brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit is assembled by using a purified lipopolysaccharide mixture of smooth type brucella and rough type brucella as antigen coated wells, using S1119.3 immune and healthy cattle to prepare positive control serum and standard weak positive serum, using healthy and non-immune cattle serum as negative control serum, using protein AG which is labeled by enzyme and is a receptor in combination with mammal Fc segment as enzyme labeling combo, and together taking serum diluent, a cleaning solution, a substrate solution A, a substrate solution B, H2O2 and a stopping solution as coordination. The invention establishes a rapid, sensitive, special and accurate method suitable for brucellosis diagnosis and epidemiological survey, has objective result judgment, simple and convenient operation, and is suitable for both large-scale screening tests and final confirmed diagnosis of the brucellosis.
Description
(1) technical field
The present invention relates to medical biotechnology goods technology, is exactly the Brucellosis indirect enzyme-linked immunosorbent assay 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 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 inspection.China uses the method for the red and tube agglutination of tiger at present, and their susceptibility and poor specificity be the technology that international trade is eliminated, and the reaction time are longer, has certain contradiction with China actual detected census operations demand.
(3) summary of the invention
The object of the present invention is to provide a kind of moderate cost, the reaction time is short, with the married Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit of China's actual detected census operations demand.
The object of the present invention is achieved like this: 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 serum, standard weak positive serum with S1119.3 immune health ox, make negative control sera with the non-immune cattle serum of health, with enzyme labeling combine with mammal Fc section acceptor--a-protein G makes the enzyme labeling bond, be equipped with serum 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:
Described antigen coated ELISA Plate preparation method is as follows:
(1) bacterial classification
Make antigen and be respectively B.abortus S1119.3 and B.abortus RB51 with bacterial classification; B.abortus S1119.3 strain was hatched the beginning for 37 ℃ and can be grown transparent, colourless or light yellow, entire bacterium colony, colony diameter 1-2mm on potato immersion liquid agar through 48 hours; B.abortus RB51 strain was hatched the beginning for 37 ℃ and can be grown that transparency is low, the edge is rough, the bacterium colony of dry tack free on potato immersion liquid agar through 48 hours, be dark white or yellow under refract light, colony diameter 1-2mm;
(2) antigen
First order seed preparation and evaluation: freeze-drying B.abortus S1119.3 strain and B.abortusRB51 strain are dissolved with the equivalent sterile saline, inoculate potato immersion liquid agar slant respectively, cultivated 48 hours at 37 ℃; Observe colonial morphology, choose the bacterium colony of smooth type and rough type cultural character respectively, transplant in the flat bottle of the potato immersion liquid agar that contains 5% calf serum and 0.1% yeast extract, inoculating surfaces was cultivated 72 hours in 37 ℃ of incubators downwards, with 0.5% phenol physiological saline results bacterial cultures, carry out biological purity check, qualified in first order seed, put-20 ℃ of preservations;
The secondary seed breeding:
Get two kinds of bacterial strain first order seeds and be inoculated in 37 ℃ of cultivations of potato immersion liquid agar 72 hours, results bacterial cultures respectively; Carry out checks such as form and biochemical characteristic, cultural character, serological characteristic, variation inspection, pure check respectively; Satisfactory bacterial classification is put 2 ℃～8 ℃ preservations;
(3) antigen preparation
The bacteria suspension preparation:
B.abortus S1119.3 strain and B.abortus RB51 strain secondary seed with accreditation is inoculated in the flat bottle of the potato immersion liquid agar that contains 5% calf serum and 0.1% yeast extract respectively, cultivates 72 hours for 37 ℃; Check living contaminants will be arranged or grow atypical flat bottle to discard through variation; Aggegation water in the flat bottle is abandoned in suction, and every bottle of adding contains the physiological saline 20ml of 0.5% phenol, washes culture, is collected in the good glass bottle of autoclaving; Be heated to 80 ℃ with collecting good bacteria suspension, kept 90 minutes; Be stored in 4 ℃; Should carry out activity check to inactivated bacterial liquid, 37 ℃ of incubations bacterial growth that do not have after 10 days;
(4) antigen extracts
Respectively with two kinds of inactivated bacterial liquid centrifugal 75 minutes with 3000g, collecting precipitation; 50g weight in wet base bacterium is added the 170ml distilled water, be heated to 66 ℃, add 66 ℃ 90% phenol solution then; 66 ℃ continue to be stirred 15 minutes, in 4 ℃ with 10, centrifugal 15 minutes of 000g; Abandon the henna phenol phase of lower floor with long tube suction, in case of necessity, remove big bacterial chip with No. 1 filter paper filtering of Whatman; Add 500ml then and contain the cold methanol of 1% saturated acetic acid sodium, hatched 2 hours for 4 ℃, centrifugal 10 minutes of 10000g abandons supernatant, will precipitate with the 80ml distilled water resuspended, after 18 hours, 4 ℃ 10, centrifugal 10 minutes of 000g; Supernatant is in 4 ℃ of preservations; Precipitation is suspended in the 80ml sterile purified water, stirred again 2 hours in 4 ℃; Comply with the centrifugal supernatant that obtains of last method, and mix with aforementioned supernatant; Subsequently, add the 8g trichloroacetic acid in supernatant, stirring at room is after 15 minutes, and by 10, centrifugal 15 minutes of 000g discards precipitation, and supernatant spends the night with distill water dialysis, changes liquid 2 times (at least 4000 milliliters each time), then with bag filter content-crude antigen freeze-drying;
(5) antigen coated microplate
The preparation of antigen coated microplate:
Bag is by 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;
Washing is that 7.2 PBST (seeing 5 cleansing solutions) makes cleansing solution with 0.01mol/l pH value, adds the effect of capacity room temperature and gets rid of after 3 minutes, as above repeats 3 times, or repeats to wash 3 times with washing the plate machine, pats dry till the no watermark;
Sealing is that 6.3 PBST (seeing 4 serum dilutions) presses the 100ul/ hole with the 0.01mol/l pH value that contains 0.1% bovine serum albumin(BSA), 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.
Described positive control serum, weak positive serum preparation method are as follows:
(1) preparation of positive control serum:
Manufacturing need detect through the laboratory with the ox that animal prepares positive control serum, 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;
Immunogen preparing is 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, and it is qualified to be undertaken by Chinese veterinary drug allusion quotation through steriling test, 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;
Immune programme for children is pressed minute 4 intramuscular injection of 5ml/ point with immunogene, immune animal, and after 14 days, with the method immunity once, and blood sampling after 14 days again, separation of serum detects with ELISA.Serum OD
450nmWith negative serum OD
450nmRatio (P/N)＞1 can take a blood sample in a large number.As disqualified upon inspection, booster immunization once again;
Serum is made
Strong positive serum is made: with the conventional method blood sampling, separate its serum, it as serum to be checked, is done 1: 2 with serum dilution, 1: 4......1: 126 dilutions are done to get serum OD to be checked with reference to carrying out the ELISA test with Quality Control strong positive serum
450nm/ Quality Control strong positive serum OD
450nmThe serum dilution to be checked that is worth at=1 o'clock is diluted serum with serum dilution as serum diluting multiple;
Weak positive serum is made: strong positive serum is done dilution in 1: 1.5 with serum dilution; Degerming and packing are carried out the suction filtration degerming with serum 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, quantitative packing, freeze-drying under the aseptic condition then.
The preparation method of described negative control sera is as follows:
Make and use animal: use animal with the positive control serum manufacturing, observe a week;
Serum is made: with the conventional method blood sampling, separate its serum;
Serum is handled and packing: serum is carried out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition adding thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion, and quantitative packing under the aseptic condition then is labelled.
Described enzyme labeling the acceptor that combines with mammal Fc--the preparation method of a-protein G is as follows: use pH7.2, PBS solution is as the enzyme conjugates dilution, presses 0.5mg/ml dilution enzyme labeling bond.Filtration sterilization.Enzyme is marked bond carry out the suction filtration degerming with the filter of 0.45 μ m, 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 mark bond down.
Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit of the present invention, quick, responsive, special, the method accurately that set up to be fit to brucellosis diagnosis and epidemiology survey, it is objective, easy and simple to handle that the result judges, both be fit to extensive shaker test, what can be used for brucellosis again makes a definite diagnosis diagnosis at last.
(4) embodiment
Below the invention will be further described.
Embodiment 1, Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit of the present invention, 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 serum, standard weak positive serum with S1119.3 immune health ox, make negative control sera with the non-immune cattle serum of health, with enzyme labeling combine with mammal Fc section acceptor--a-protein G makes the enzyme labeling bond, be equipped with serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling is formed.
Described antigen coated ELISA Plate preparation method is as follows:
(1) bacterial classification is made antigen and is respectively B.abortus S1119.3 and B.abortus RB51 with bacterial classification; B.abortus S1119.3 strain was hatched the beginning for 37 ℃ and can be grown transparent, colourless or light yellow, entire bacterium colony, colony diameter 1-2mm on potato immersion liquid agar through 48 hours; B.abortus RB51 strain was hatched the beginning for 37 ℃ and can be grown that transparency is low, the edge is rough, the bacterium colony of dry tack free on potato immersion liquid agar through 48 hours, be dark white or yellow under refract light, colony diameter 1-2mm;
(2) preparation of antigen first order seed and evaluation: freeze-drying B.abortus S1119.3 strain and B.abortus RB51 strain are dissolved with the equivalent sterile saline, inoculate potato immersion liquid agar slant respectively, cultivated 48 hours at 37 ℃; Observe colonial morphology, choose the bacterium colony of smooth type and rough type cultural character respectively, transplant in the flat bottle of the potato immersion liquid agar that contains 5% calf serum and 0.1% yeast extract, inoculating surfaces was cultivated 72 hours in 37 ℃ of incubators downwards, with 0.5% phenol physiological saline results bacterial cultures, carry out biological purity check, qualified in first order seed, put-20 ℃ of preservations;
The secondary seed breeding:
Get two kinds of bacterial strain first order seeds and be inoculated in 37 ℃ of cultivations of potato immersion liquid agar 72 hours, results bacterial cultures respectively; Carry out checks such as form and biochemical characteristic, cultural character, serological characteristic, variation inspection, pure check respectively; Satisfactory bacterial classification is put 2 ℃～8 ℃ preservations;
(3) antigen preparation
The bacteria suspension preparation: B.abortus S1119.3 strain and the B.abortus RB51 strain secondary seed with accreditation is inoculated in the flat bottle of the potato immersion liquid agar that contains 5% calf serum and 0.1% yeast extract respectively, cultivates 72 hours for 37 ℃; Check living contaminants will be arranged or grow atypical flat bottle to discard through variation; Aggegation water in the flat bottle is abandoned in suction, and every bottle of adding contains the physiological saline 20ml of 0.5% phenol, washes culture, is collected in the good glass bottle of autoclaving; Be heated to 80 ℃ with collecting good bacteria suspension, kept 90 minutes; Be stored in 4 ℃; Should carry out activity check to inactivated bacterial liquid, 37 ℃ of incubations bacterial growth that do not have after 10 days;
(4) antigen extracts respectively two kinds of inactivated bacterial liquid with 3000g centrifugal 75 minutes, collecting precipitation; 50g weight in wet base bacterium is added the 170ml distilled water, be heated to 66 ℃, add 66 ℃ 90% phenol solution then; 66 ℃ continue to be stirred 15 minutes, in 4 ℃ with 10, centrifugal 15 minutes of 000g; Abandon the henna phenol phase of lower floor with long tube suction, in case of necessity, remove big bacterial chip with No. 1 filter paper filtering of Whatman; Add 500ml then and contain the cold methanol of 1% saturated acetic acid sodium, hatched 2 hours for 4 ℃, centrifugal 10 minutes of 10000g abandons supernatant, will precipitate with the 80ml distilled water resuspended, after 18 hours, 4 ℃ 10, centrifugal 10 minutes of 000g; Supernatant is in 4 ℃ of preservations; Precipitation is suspended in the 80ml sterile purified water, stirred again 2 hours in 4 ℃; Comply with the centrifugal supernatant that obtains of last method, and mix with aforementioned supernatant; Subsequently, add the 8g trichloroacetic acid in supernatant, stirring at room is after 15 minutes, and by 10, centrifugal 15 minutes of 000g discards precipitation, and supernatant spends the night with distill water dialysis, changes liquid 2 times (at least 4000 milliliters each time), then with bag filter content-crude antigen freeze-drying;
(5) antigen coated microplate
The preparation of antigen coated microplate: bag is by 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;
Washing is that 7.2 PBST (seeing 5 cleansing solutions) makes cleansing solution with 0.01mol/l pH value, adds the effect of capacity room temperature and gets rid of after 3 minutes, as above repeats 3 times, or repeats to wash 3 times with washing the plate machine, pats dry till the no watermark;
Sealing is that 6.3 PBST (seeing 4 serum dilutions) presses the 100ul/ hole with the 0.01mol/l pH value that contains 0.1% bovine serum albumin(BSA), 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.
Described positive control serum, weak positive serum preparation method are as follows:
(1) preparation of positive control serum:
Manufacturing need detect through the laboratory with the ox that animal prepares positive control serum, 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;
Immunogen preparing is 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, and it is qualified to be undertaken by Chinese veterinary drug allusion quotation through steriling test, 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;
Immune programme for children is pressed minute 4 intramuscular injection of 5ml/ point with immunogene, immune animal, and after 14 days, with the method immunity once, and blood sampling after 14 days again, separation of serum detects with ELISA.Serum OD
450nmWith negative serum OD
450nmRatio (P/N)＞1 can take a blood sample in a large number.
As disqualified upon inspection, booster immunization once again;
Serum is made
Strong positive serum is made: with the conventional method blood sampling, separate its serum, it as serum to be checked, is done 1: 2 with serum dilution, 1: 4......1: 126 dilutions are done to get serum OD to be checked with reference to carrying out the ELISA test with Quality Control strong positive serum
450nm/ Quality Control strong positive serum OD
450nmThe serum dilution to be checked that is worth at=1 o'clock is diluted serum with serum dilution as serum diluting multiple;
Weak positive serum is made: strong positive serum is done dilution in 1: 1.5 with serum dilution; Degerming and packing are carried out the suction filtration degerming with serum 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, quantitative packing, freeze-drying under the aseptic condition then.
The preparation method of described negative control sera is as follows:
Make and use animal: use animal with the positive control serum manufacturing, observe a week;
Serum is made: with the conventional method blood sampling, separate its serum;
Serum is handled and packing: serum is carried out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition adding thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion, and quantitative packing under the aseptic condition then is labelled.
Described enzyme labeling combine with mammal Fc section acceptor--the preparation method of a-protein G is as follows: use pH7.2, PBS solution is as the enzyme conjugates dilution, presses 0.5mg/ml dilution enzyme labeling bond.Filtration sterilization.Enzyme is marked bond carry out the suction filtration degerming with the filter of 0.45 μ m, 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 mark bond down.
Described serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer the preparation method as follows:
(1) serum dilution is the 0.05M carbon acid solution:
Sodium carbonate 1.93 grams; Sodium bicarbonate 3.80 grams; Adding distil water to 1000 milliliter, pH9.6,10 pounds of high pressure 15 minutes;
1% BSA solution
Get 1000 milliliters of PBST solution, add BSA 10 grams.Adjust pH 7.0, filtration sterilization;
(2) cleansing solution is 10 times of washing lotions
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;
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;
(3) substrate solution is 10 times of substrate solution A
Get citric acid 4.2g, sodium acetate 13.5g, peroxidating urine 0.5g adds deionized water 100ml, adjust pH 4.0, filtration sterilization;
(4) substrate solution B gets TMB 0.2192g, and dimethyl sulfoxide (DMSO) 20ml dissolves under the room temperature;
(5) H
2O
2Liquid
Produced by U.S. VWR company, the 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
(6) 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.
Embodiment 2, and just the relevant issues of Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit of the present invention are described 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
For overcoming traditional indirect ELISA method ELIAS secondary antibody host's unicity problem, adopt enzyme labeling thing a-protein G to combine as the ELIAS secondary antibody of homogeneous and with the Fc section of all mammal antibody.
4 negative control seras and positive control serum
Positive control serum is that whether checking ELISA test operation is correct in the kit, and the important component whether reaction is special also is the index whether this kit of reaction works.For reducing the non-specific of control serum, the animal of preparation negative control sera and positive control serum all need carry out Brucella antibody, enterocolitis yersinia enterocolitica antibody, Bacillus paratyphosus B antibody, Escherichia coli O:157 antibody test, and detect negative.
The method repeatedly immune with heavy dose of deactivation brucella prepares positive serum.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 serum 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 brucellosis antibody indirect ELISA test, positive control serum to the positive rate of monoclonal antibody near 100%P, colour developing fully basically.Negative control sera is not competed inhibition to monoclonal antibody, because influence of serum, its OD value just is slightly larger than blank in the indirect ELISA experiment.
5 determine that about critical value (cut off) the yin and yang attribute limit value is a decision testing result accuracy very important index.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 this research experiment, the indirect ELISA reagent kit of using development detects 30 parts of brucellosis negative serums, 53 parts of immune animal brucella infect serum and immune serum, use in the world OIE and confirmed that the indirect ELISA reagent kit testing result of accuracy compares, 30 parts of its positive percentages of definite negative sample are all below 20%, therefore, determine that detectability is that critical value is 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, detect with OIE standard positive serum, standard female serum, negative reference serum, artificial challenge's serum, immune serum, negative recall rate is 100%.Detect with Bacillus paratyphosus B, enterocolitis yersinia enterocolitica, Escherichia coli O:157 positive and negative serum, the specificity that overcomes cross reaction reaches 96.5%.Test findings shows that animal brucellosis indirect ELISA antibody assay kit has higher specificity.
Can not also there is no need when when practical application and to every batch of product, detecting ill the detection, so when working out specific criteria only to enterocolitis yersinia enterocolitica positive serum, Bacillus paratyphosus B positive serum, Escherichia coli O:157 positive serum, the checking of testing of vaccine type antibody, OIE standard positive and standard female serum.
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 at present one of the most responsive method of anti-brucellosis antibody, but we are badly in need of a kind ofly can being applied to field, sensitivity, the little screening detection method of as far as possible avoiding immune antiboidy to disturb again of cross reaction in work such as actual detected generaI investigation.
Select for use artificial brucella to attack 37 parts of malicious serum in the research process, 16 parts of OIE standard positive serums, 8 parts of OIE standard female serum, the immune vaccine positive serum detects with the animal brucella indirect ELISA reagent kit respectively for 5 parts, 51 parts of brucellosis positive serums 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 antigen-antibody combination.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 stability, storage and the term of validity when carrying out storage life research, be placed on 2～8 ℃ and room temperature after brucellosis antibody I-ELISA detection kit respectively, 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 (4)
1. Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit, it is characterized in that: 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 serum, standard weak positive serum with S1119.3 immune health ox, make negative control sera with the non-immune cattle serum of health, with enzyme labeling combine with mammal Fc section acceptor--a-protein G makes the enzyme labeling bond, be equipped with serum 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 antibody assay kit according to claim 1 is characterized in that described antigen coated ELISA Plate preparation method is as follows:
(1) bacterial classification
Make antigen and be respectively B.abortus S1119.3 and B.abortus RB51 with bacterial classification; B.abortus S1119.3 strain was hatched the beginning for 37 ℃ and can be grown transparent, colourless or light yellow, entire bacterium colony, colony diameter 1-2mm on potato immersion liquid agar through 48 hours; B.abortus RB51 strain was hatched the beginning for 37 ℃ and can be grown that transparency is low, the edge is rough, the bacterium colony of dry tack free on potato immersion liquid agar through 48 hours, be dark white or yellow under refract light, colony diameter 1-2mm;
(2) antigen
First order seed preparation and evaluation: freeze-drying B.abortus S1119.3 strain and B.abortusRB51 strain are dissolved with the equivalent sterile saline, inoculate potato immersion liquid agar slant respectively, cultivated 48 hours at 37 ℃; Observe colonial morphology, choose the bacterium colony of smooth type and rough type cultural character respectively, transplant in the flat bottle of the potato immersion liquid agar that contains 5% calf serum and 0.1% yeast extract, inoculating surfaces was cultivated 72 hours in 37 ℃ of incubators downwards, with 0.5% phenol physiological saline results bacterial cultures, carry out biological purity check, qualified in first order seed, put-20 ℃ of preservations;
The secondary seed breeding:
Get two kinds of bacterial strain first order seeds and be inoculated in 37 ℃ of cultivations of potato immersion liquid agar 72 hours, results bacterial cultures respectively; Carry out checks such as form and biochemical characteristic, cultural character, serological characteristic, variation inspection, pure check respectively; Satisfactory bacterial classification is put 2 ℃～8 ℃ preservations;
(3) antigen preparation
The bacteria suspension preparation:
B.abortus S1119.3 strain and B.abortus RB51 strain secondary seed with accreditation is inoculated in the flat bottle of the potato immersion liquid agar that contains 5% calf serum and 0.1% yeast extract respectively, cultivates 72 hours for 37 ℃; Check living contaminants will be arranged or grow atypical flat bottle to discard through variation; Aggegation water in the flat bottle is abandoned in suction, and every bottle of adding contains the physiological saline 20ml of 0.5% phenol, washes culture, is collected in the good glass bottle of autoclaving; Be heated to 80 ℃ with collecting good bacteria suspension, kept 90 minutes; Be stored in 4 ℃; Should carry out activity check to inactivated bacterial liquid, 37 ℃ of incubations bacterial growth that do not have after 10 days;
(4) antigen extracts
Respectively with two kinds of inactivated bacterial liquid centrifugal 75 minutes with 3000g, collecting precipitation; 50g weight in wet base bacterium is added the 170ml distilled water, be heated to 66 ℃, add 66 ℃ 90% phenol solution then; 66 ℃ continue to be stirred 15 minutes, in 4 ℃ with 10, centrifugal 15 minutes of 000g; Abandon the henna phenol phase of lower floor with long tube suction, in case of necessity, remove big bacterial chip with No. 1 filter paper filtering of Whatman; Add 500ml then and contain the cold methanol of 1% saturated acetic acid sodium, hatched 2 hours for 4 ℃, centrifugal 10 minutes of 10000g abandons supernatant, will precipitate with the 80ml distilled water resuspended, after 18 hours, 4 ℃ 10, centrifugal 10 minutes of 000g; Supernatant is in 4 ℃ of preservations; Precipitation is suspended in the 80ml sterile purified water, stirred again 2 hours in 4 ℃; Comply with the centrifugal supernatant that obtains of last method, and mix with aforementioned supernatant; Subsequently, add the 8g trichloroacetic acid in supernatant, stirring at room is after 15 minutes, and by 10, centrifugal 15 minutes of 000g discards precipitation, and supernatant spends the night with distill water dialysis, changes liquid 2 times (at least 4000 milliliters each time), then with bag filter content-crude antigen freeze-drying;
(5) antigen coated microplate
The preparation of antigen coated microplate:
Bag is by 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;
Washing is that 7.2 PBST (seeing 5 cleansing solutions) makes cleansing solution with 0.01mol/l pH value, adds the effect of capacity room temperature and gets rid of after 3 minutes, as above repeats 3 times, or repeats to wash 3 times with washing the plate machine, pats dry till the no watermark;
Sealing is that 6.3 PBST (seeing 4 serum dilutions) presses the 100ul/ hole with the 0.01mol/l pH value that contains 0.1% bovine serum albumin(BSA), 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.
3. a kind of Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit according to claim 1 is characterized in that described positive control serum, weak positive serum preparation method are as follows:
(1) preparation of positive control serum:
Manufacturing need detect through the laboratory with the ox that animal prepares positive control serum, 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;
Immunogen preparing is 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, it is qualified to be undertaken by Chinese veterinary drug allusion quotation through steriling test, 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;
Immune programme for children is pressed minute 4 intramuscular injection of 5ml/ point with immunogene, immune animal, and after 14 days, with the method immunity once, and blood sampling after 14 days again, separation of serum detects with ELISA; Serum OD
450nmWith negative serum OD
450nmRatio (P/N)＞1 can take a blood sample in a large number; As disqualified upon inspection, booster immunization once again;
Serum is made
Strong positive serum is made: with the conventional method blood sampling, separate its serum, it as serum to be checked, is done 1: 2 with serum dilution, 1: 4......1: 126 dilutions are done to get serum OD to be checked with reference to carrying out the ELISA test with Quality Control strong positive serum
450nm/ Quality Control strong positive serum OD
450nmThe serum dilution to be checked that is worth at=1 o'clock is diluted serum with serum dilution as serum diluting multiple;
Weak positive serum is made: strong positive serum is done dilution in 1: 1.5 with serum dilution; Degerming and packing are carried out the suction filtration degerming with serum 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, quantitative packing, freeze-drying under the aseptic condition then;
The preparation method of described negative control sera is as follows:
Make and use animal: use animal with the positive control serum manufacturing, observe a week;
Serum is made: with the conventional method blood sampling, separate its serum;
Serum is handled and packing: serum is carried out the suction filtration degerming with the filter of 0.45 μ m, and aseptic condition adding thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion, and quantitative packing under the aseptic condition then is labelled.
4. a kind of Brucellosis indirect enzyme-linked immunosorbent assay antibody assay kit according to claim 1, it is characterized in that described enzyme labeling combine with mammal Fc section acceptor--the preparation method of a-protein G is as follows:
Use pH7.2, PBS solution is pressed 0.5mg/ml dilution enzyme labeling bond, filtration sterilization as the enzyme conjugates dilution; Enzyme is marked bond carry out the suction filtration degerming with the filter of 0.45 μ m, aseptic condition adds thimerosal and gentamicin down makes its final concentration be 0.02% with anticorrosion; 0.2ml/ bottle packing enzyme mark bond is pressed in packing freeze-drying, aseptic condition down.
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