CN101581726B - New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit - Google Patents
New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit Download PDFInfo
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Abstract
The invention aims to provide a new-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit which meets animal epidemic prevention requirements in China, is sensitive and specific and can accurately diagnose brucellosis. In the invention, the lipopolysaccharide of smooth type brucella is used as an antigen-coated enzyme-labeled plate, inactivated bacilli liquid is used for immunizing or artificially infecting a healthy cattle to prepare positive control sera and standard weak positive sera, healthy non-immune cattle sera are used as negative control sera, a novel monoclonal antibody is used as a competition antibody, the protein AG enzyme-labeled substance of an anti-Fc receptor is used as an assistant antibody, serum diluent, a washing solution, a substrate solution A, a substrate solution B, H2O2 and a stop solution are prepared, and all raw materials are assembled to form the kit. The invention is a competition ELISA kit which is sensitive and specific and can accurately diagnose brucellosis. The invention meets animal epidemic prevention requirements in China, and carries out last diagnosis on the brucellosis by means of a laboratory diagnosis technique.
Description
(1) technical field
The present invention relates to the immunological test technology, is exactly New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test 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 Theoretical Design.Interpenetrate along with interdisciplinary, the scope that immunology relates to constantly enlarges, and new immunological detection method emerges in an endless stream.The range of application of immunological method not only becomes the important method of various clinical medical diagnosis on disease also at expanding day, also provides convenience for the multi-disciplinary research of crowd.
Antigen and corresponding antibodies meet specific binding can occur, and present certain reacting phenomenon under the impact of external condition, and as aggegation or precipitation, available known antigens (or antibody) detects unknown antibody (or antigen) by this.The antibody that test is adopted often is present in serum, therefore is referred to as again serological reaction (serological reaction).Antigen type is various, can divide graininess and solubility two classes by its physical behavior.The former phalangeal cell antigen (comprising bacterial antigens), its preparation is comparatively easy, generally is made into finite concentration with new fresh cell after with stroke-physiological saline solution or phosphate buffer washing.If be bacterial antigens, get the fresh cultured thing, be handled as follows through the collection bacterium, H antigen is because of thermo-labile use 0.3%～0.5% formaldehyde treated, and O antigen is used after 100 ℃ of 2h remove H antigen by heat-resisting the heating.Soluble antigen can be the cell ingredients such as cell membrane, cytoplasm, nucleus and nuclear membrane, may be also some soluble factors to the body fluid through emiocytosis.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.In body fluid, the soluble antigen of (as serum etc.) can directly obtain required composition with biochemical means.Some soluble antigen only has immunoreactivity, and non-immunogenicity, this type of antigen is still needed can become comlete antigen with the carrier coupling.
Brucellosis (Brucellosis) claim again the Mediterranean remittent fever, Malta fever, and undulant fever or undulant fever are the infecting both domestic animals and human whole body infectious diseases that is caused by brucella, its clinical characters is long-term fever, hidrosis, arthralgia and hepatosplenomegaly etc.Britain medical officer Bruce isolated " brucella " on the island, Malta from soldier's spleen of dying from " Malta fever " in 1886, clear and definite first pathogen that should disease.Agglutinating reaction can occur in Wright in 1897 and its colleague's Discover the patients serum and brucellar culture, is called the Wright agglutinating reaction, thereby has set up so far the still serological diagnostic method of use.In Ancient Times in China doctor nationality, though this disease is had description, until Boone in 1905 makes formal report in Chongqing to this disease.Should disease distribute in the world at present, only have several countries to eliminate this disease, and in the northeast of China, North China, northwest one are with popular and distribution, distribute in other area, and increasingly extensive serious with harm.Animal husbandry and the mankind are come serious economic loss.Brucella is a Gram negative bacillus pumilis of class, and endotoxin is important morbid substance.Brucella has strong invasiveness, and bacterium can enter the host by intact skin and mucous membrane.Brucella has 6 bions, and what China was popular is Brucella melitensis, and three kinds of Brucella abortus and Brucella suis are wherein the most common with Brucella melitensis.In the nature situation, have 60 many animals can infect brucella, it is mainly goat, sheep, and ox and pig, take miscarriage as main, the pregnancy period animal sense of being advisable most.The mankind are to the brucella susceptible, and after bacterium entered human body, protracted course of disease showed effect repeatedly, and heating is wave, and as not treating, consequence is serious.
According to clinical symptoms, epidemic and characteristic pathology, be not difficult to make the tentative diagnosis of brucellosis, but because there is similar symptom in Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157 infection clinically, must could make a definite diagnosis at last brucellosis by the laboratory diagnosis technology.
Be fit to that this disease diagnosed and quick, responsive, special, the method accurately of epidemiology survey in order to set up, domestic and international many scholars have carried out large quantity research, and have obtained significant achievement.Successively set up in cause of disease evaluation, fluorescence antibody and the detection method such as detection.Yet the brave red flat board that China uses at present and tube agglutination method are that method is eliminated in international trade, and backward in technique, false positive is also relatively high.
(3) summary of the invention
The object of the present invention is to provide a kind of satisfy China's animal epidemic prevention demand, sensitivity, special, New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit that can the Accurate Diagnosis brucellosis.
The object of the present invention is achieved like this: it is to utilize the brucellar lipopolysaccharides of smooth type to make antigen coated ELISA Plate; prepare positive control serum, standard weak positive serum with inactivated bacterial liquid immune health ox or the healthy ox of artificial challenge; make negative control sera with the nonimmune cow's serum of health; make competition antibody with novel monoclonal antibody; the a-protein G enzyme mark thing of anti-Fc acceptor is anti-as two, preparation serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling forms.
New-generation brucellosis antibody competition ELISA detection kit of the present invention also has following technical characterictic:
1. described antigen coated ELISA Plate is preparation like this:
(1) coated: under aseptic condition, freeze-dried antigen is suspended to volume before freeze-drying with distilled water, by dilution in 1: 1000, with 100 μ l/ holes, coated polystyrene 96 orifice plates added and are placed on 4 ℃ and spend the night with the carbonic acid buffer of 0.05M pH9.6;
(2) washing: with 0.01mol/L, the pH value is that 7.2 PBST makes cleansing solution, adds the effect of capacity room temperature to get 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;
(3) sealing: with the 0.01mol/L that contains 1% bovine serum albumin(BSA), the pH value is that 6.3 PBST is by the 100ul/ hole, be added in 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 repeated 3 times, or repeated to wash 3 times with washing the plate machine, pat dry till the no-watermark, natural drying;
(4) packing: will seal good ELISA Plate and be put in aluminium foil bag, add the drying agent of 1g packing, with vacuum packing machine, ELISA Plate be packaged, labelled.
2. the preparation method of described positive control serum is as follows:
(1) animal is used in manufacturing: the ox of preparation positive serum need to detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, infect without Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157, and without the healthy ox of the negative antibodies such as propagating system infection; Observe a week;
(2) immunogene preparation: with the flat bottle of Brucella ox kind S1119.3 inoculation potato immersion liquid agar, putting 37 ℃ cultivated 48 hours, during one deck bacterium colony to be formed, choose pure flat bottle, agglutinating water is abandoned in suction, with the sterile saline that contains 0.5% phenol, lawn is washed, in the neutral glass bottle of impouring, adding formalin to final concentration is 0.2%, puts 37 ℃ of vibration deactivations 48 hours, qualified through steriling test, centrifugal 10 minutes with 20000 rev/mins in 4 ℃, get precipitation, do 10 times of dilutions with physiological saline, as immunogene.Standby in 2 ℃～8 ℃ Refrigerator stores;
(3) immune programme for children: press minute 4 intramuscular injection of 5ml/ point with immunogene, immune animal, after 14 days, with the method immunity once, then blood sampling afterwards on the 14th, 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;
(4) serum manufacturing: 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: 128 dilutions are done to get serum OD to be checked with reference to carrying out the ELISA test with Quality Control strong positive serum
650nm/ Quality Control strong positive serum OD
650nmBe worth the serum dilution to be checked of=1 o'clock as serum diluting multiple, with serum dilution, serum diluted;
(5) packing: serum is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion under aseptic condition, then quantitative separating, freeze-drying under aseptic condition, labelled;
(6) the standard weak positive serum is made: strong positive serum is done dilution in 1: 1.5 with serum dilution.
3. the preparation method of described negative control sera is as follows:
(1) manufacturing is used animal with animal with the manufacturing of positive control serum;
(2) the serum manufacturing is taken a blood sample with conventional method, separates its serum;
(3) serum is processed with packing serum is carried out the suction filtration degerming with the filter of 0.45 μ m, adds thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion under aseptic condition, and then quantitative separating under aseptic condition, labelled.
4. described novel animal brucellosis method for preparing monoclonal antibody is as follows:
(1) cultivation of hybridoma cell strain: hybridoma cell strain is taken out from liquid nitrogen, being placed in 37 ℃ of water-baths melts rapidly, 1000 rev/mins of centrifugation cells, remove cryopreserving liquid, add the 10mlMEM nutrient culture media, be divided into two culture flasks and cultivate in 37 ℃ of CO2gas incubator, after cell covered with at the bottom of culture flask in 2-3 days, carry out cell count;
(2) preparation of ascites novel monoclonal antibody: press 0.5ml/ intraperitoneal injection of mice norphytane, after three days, then by 5 * 10
5-6/ cell concentration lumbar injection hybridoma only approximately produces ascites after ten days, adopts every day once, can adopt 2-3 time continuously, after ascites was taken out, 3000 turned/the centrifugal 10min of min, collect supernatant and mix, in 56 ℃ of water bath processing 10 minutes ,-80 ℃ frozen was semi-manufacture;
(3) product check in the middle of: steriling test is undertaken by " veterinary drug allusion quotation ", answers asepsis growth;
(4) titration: the antigen coated ELISA Plate of animal brucellosis of taking out quality inspection, every hole adds the sample diluting liquid 45ul of animal brucellosis antibody competition ELISA diagnostic kit, animal brucellosis standard female serum 5ul, add 2 rounds, standard positive serum 5ul adds 2 rounds, and the animal brucellosis monoclonal antibody was with 1: 10 doubling dilution, each dilutability adds 2 row, hatches 30 minutes for 26 ℃. and antibody titer is OD
450It is the monoclonal antibody extension rate of 0.60 to 1.80 o'clock;
(5) finished product preparation: by the bioactivity result, monoclonal antibody is diluted, be distributed into the 0.2ml/ bottle, the sealing bottleneck posts label, dated batch.
The New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit of the present invention is a kind of sensitivity, special, competitive ELISA kit that can the Accurate Diagnosis brucellosis, the present invention satisfies the demand of China's animal epidemic prevention, by the laboratory diagnosis technology, brucellosis is made a definite diagnosis at last.
(4) embodiment
Below the invention will be further described.
Embodiment 1; the New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit of the present invention; it is to utilize the brucellar lipopolysaccharides of smooth type to make antigen coated ELISA Plate; prepare positive control serum, standard weak positive serum with inactivated bacterial liquid immune health ox or the healthy ox of artificial challenge; make negative control sera with the nonimmune cow's serum of health; make competition antibody with novel monoclonal antibody; the a-protein G enzyme mark thing of anti-Fc acceptor is anti-as two, preparation serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling forms.
New-generation brucellosis antibody competition ELISA detection kit of the present invention also has following technical characterictic:
1. described antigen coated ELISA Plate is preparation like this:
(1) coated: under aseptic condition, freeze-dried antigen is suspended to volume before freeze-drying with distilled water, by dilution in 1: 1000, with 100 μ l/ holes, coated polystyrene 96 orifice plates added and are placed on 4 ℃ and spend the night with the carbonic acid buffer of 0.05M pH9.6;
(2) washing: with 0.01mol/L, the pH value is that 7.2 PBST makes cleansing solution, adds the effect of capacity room temperature to get 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;
(3) sealing: with the 0.01mol/L that contains 1% bovine serum albumin(BSA), the pH value is that 6.3 PBST is by the 100ul/ hole, be added in 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 repeated 3 times, or repeated to wash 3 times with washing the plate machine, pat dry till the no-watermark, natural drying;
(4) packing: will seal good ELISA Plate and be put in aluminium foil bag, add the drying agent of 1g packing, with vacuum packing machine, ELISA Plate be packaged, labelled.
2。The preparation method of described positive control serum is as follows:
(1) animal is used in manufacturing: the ox of preparation positive serum need to detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, infect without Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli O:157, and without the healthy ox of the negative antibodies such as propagating system infection; Observe a week;
(2) immunogene preparation: with the flat bottle of Brucella S1119.3 inoculation potato immersion liquid agar, putting 37 ℃ cultivated 48 hours, during one deck bacterium colony to be formed, choose pure flat bottle, agglutinating water is abandoned in suction, with the sterile saline that contains 0.5% phenol, lawn is washed, in the neutral glass bottle of impouring, adding formalin to final concentration is 0.2%, puts 37 ℃ of vibration deactivations 48 hours, qualified through steriling test, centrifugal 10 minutes with 20000 rev/mins in 4 ℃, get precipitation, do 10 times of dilutions with physiological saline, as immunogene.Standby in 2 ℃～8 ℃ Refrigerator stores;
(3) immune programme for children: press minute 4 intramuscular injection of 5ml/ point with immunogene, immune animal, after 14 days, with the method immunity once, then blood sampling afterwards on the 14th, 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;
(4) serum manufacturing: 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: 128 dilutions are done to get serum OD to be checked with reference to carrying out the ELISA test with Quality Control strong positive serum
650nm/ Quality Control strong positive serum OD
650nmBe worth the serum dilution to be checked of=1 o'clock as serum diluting multiple, with serum dilution, serum diluted;
(5) packing: serum is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion under aseptic condition, then quantitative separating, freeze-drying under aseptic condition, labelled;
(6) the standard weak positive serum is made: strong positive serum is done dilution in 1: 1.5 with serum dilution.
3. the preparation method of described negative control sera is as follows:
(1) manufacturing is used animal with animal with the manufacturing of positive control serum;
(2) the serum manufacturing is taken a blood sample with conventional method, separates its serum;
(3) serum is processed with packing serum is carried out the suction filtration degerming with the filter of 0.45 μ m, adds thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion under aseptic condition, and then quantitative separating under aseptic condition, labelled.
4. described novel animal brucellosis method for preparing monoclonal antibody is as follows:
(1) cultivation of hybridoma cell strain: hybridoma cell strain is taken out from liquid nitrogen, being placed in 37 ℃ of water-baths melts rapidly, 1000 rev/mins of centrifugation cells, remove cryopreserving liquid, add the 10mlMEM nutrient culture media, be divided into two culture flasks and cultivate in 37 ℃ of CO2gas incubator, after cell covered with at the bottom of culture flask in 2-3 days, carry out cell count;
(2) preparation of ascites novel monoclonal antibody: press 0.5ml/ intraperitoneal injection of mice norphytane, after three days, then by 5 * 10
5-6/ cell concentration lumbar injection hybridoma only approximately produces ascites after ten days, adopts every day once, can adopt 2-3 time continuously, after ascites was taken out, 3000 turned/the centrifugal 10min of min, collect supernatant and mix, in 56 ℃ of water bath processing 10 minutes ,-80 ℃ frozen was semi-manufacture;
(3) product check in the middle of: steriling test is undertaken by " veterinary drug allusion quotation ", answers asepsis growth;
(4) titration: the antigen coated ELISA Plate of animal brucellosis of taking out quality inspection, every hole adds the sample diluting liquid 45ul of animal brucellosis antibody competition ELISA diagnostic kit, animal brucellosis standard female serum 5ul, add 2 rounds, standard positive serum 5ul adds 2 rounds, and the animal brucellosis monoclonal antibody was with 1: 10 doubling dilution, each dilutability adds 2 row, hatches 30 minutes for 26 ℃. and antibody titer is OD
450It is the monoclonal antibody extension rate of 0.60 to 1.80 o'clock;
(5) finished product preparation: by the bioactivity result, monoclonal antibody is diluted, be distributed into the 0.2ml/ bottle, the sealing bottleneck posts label, dated batch.
5. described serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer is like this preparation:
The preparation of (1) 10 times of washing lotion (PBST):
Sodium chloride, 8 grams; Potassium dihydrogen phosphate, 0.2 gram; Sodium hydrogen phosphate (12H
2O), 6.2.9 gram; 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 (2) 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 (3) 10 times of substrate A solution:
Get citric acid 4.2g, sodium acetate 13.5g, peroxidating urine 0.5g adds deionized water 100ml, adjust pH 4.0, filtration sterilization; Packing: the packing of 1.2ml/ bottle, sealing bottleneck, adhesive label;
(4) preparation of substrate B solution:
Get TMB 0.2192g, dimethyl sulfoxide (DMSO) 20ml dissolves under room temperature; Packing: the 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
(5) H
2O
2The preparation of liquid:
Packing: produced by U.S. VWR company, the 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
The preparation of (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;
Packing: 1.2 milliliters/bottle are sub-packed in bottle, the sealing bottleneck, adhesive label, batch.
Embodiment 2, and just the relevant issues of the New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit of the present invention are explained as follows:
1 selects and standard about bacterial classification
Bacterial classification is selected and is planted subalgebra and separated a lot of brucella both at home and abroad, and different serotype is arranged.The brucella that I preserved and used at present is ox type S1119.3 strain, and this bacterial strain is the reference culture of international preparation brucellosis detectable antigens, and this bacterial strain is easy to cultivate, and is difficult for occuring thick the change.Freeze-drying is stored in-70 ℃, storage life at least 10 years.
According to the inheritance stability Journal of Sex Research of this bacterium, bacterium is after passing for 10 generations continuously, and SLPS antigen site structure is more stable.
2 prepare about antigen
2.1 it is the reference culture that international preparation detects brucellosis antigen that the bacterial reproduction condition is controlled S1119.3, is easy to judge germ propagation situation in growth course, and is difficult for thick the change.But no matter which kind of serological type strain, have certain appeal to humans and animals, and therefore, bacterial reproduction must carry out in the three-grade biological safety laboratory.
2.2 bacteria inactivation can reduce the bio-safety risk of production with bacteria inactivation.Studies show that, brucella S1119.3 gathers in the crops bacterial cultures with 0.5% carbolic acid physiological saline, is loaded in sterilization container.Be heated to 80 ℃ and kept 90 minutes, after the killing bacteria deactivation, on extracting not impact of SLPS.
2.3 the purity of antigen purification, concentrated antigen determines envelope antigen actual content and detection limit, therefore, must purify to antigen; Purifying, concentrated method adopt simple and easy to do conventional method, utilize brucellergen sLPS can be present in characteristic in the organic phase carbolic acid, then use sodium acetate formalin Precipitation Antigen sLPS, water dialysis, freeze drying antigen sLPS.Again through ultrasonic treatment, thereby obtain the antigen of purifying.
2.4 the titration purified antigen is for being used for coated elisa plate, need to measure antigen valence with the monoclonal antibody measuring of anti-sLPS, according to experimental study, because the selectivity of monoclonal antibody identification form one antigen site, antigen purity by the producting rule preparation meets the kit requirement substantially, but in the manufacture process of every batch of antigen, the amount of sLPS is not definite constant, so every batch of antigen all need carry out titration.Use the monoclonal antibody through demarcating, when the antigen amount is enough, its OD should be 1.5～2.0, and this interval of developing the color is that ELISA is the most responsive, and the antigen amount slightly changes, its OD namely changes rapidly, the antigen amount is during less than standard, and the OD value may be on the low side, tests insensitive, the sensing range of microplate reader may appear exceeding in OD value higher (greater than 3.0).
3 about novel monoclonal antibody
3.1 the monoclonal antibody that this competitive ELISA of hybridoma cell strain uses is the monoclonal antibody specific of anti-SLPS antigen site.Hybridoma cell strain is adopted as the sLPS immunity BalB/C mouse after the purifying deactivation, after mouse boosting cell and the fusion of SP2/0 oncocyte, screening obtains, through being accredited as the specific monoclonal antibody cell line of anti-sLPS, through experimental identification, the specificity of the novel monoclonal antibody that this cell and OIE generally acknowledge is consistent, is characterized in not the anti-FC receptors bind with enzyme labeling.Be stored at present my company, preserve in liquid nitrogen, and have the special messenger to be responsible for keeping an eye on.
3.2 the processing of ascites novel monoclonal antibody is dense with the monoclonal antibody of cell strain of monoclonal antibody injection mouse preparation, dilute concentration is higher in use, after antibody goes out through 56 ℃, active enzyme wherein is inactivated, can remove suspension in ascites with differential centrifugation, after degerming, packing can be measured the use of tiring after filtration.
4 to prepare the method for negative serum and positive serum about standard female serum and positive serum a lot, prepare effect with this animal comparatively speaking best, and specificity is stronger.Because at present domestic animal is most by the different brucella vaccine of immunity, negative serum picks up from the clinical health animal, except requiring the Brucella antibody feminine gender, requirement is without Caused by Yersinia enterocolitica antibody, Bacillus paratyphosus B antibody, Escherichia coli O:157 antibody so more can react the actual conditions of China's Healthy animal.With the dead brucella of heavy dose of heat kill repeatedly the method for immunity prepare positive serum, bacterium kind amount and program, the immunity time can carry out according to condition about practice examining.After the zoogenetic infection brucella, begin to produce anti-sLPS antibody, its antibody horizontal peaked about 30 days, longer duration; Use in addition the serum of making a definite diagnosis diagnosis natural infection brucella animal through aseptic filtration, also be conducive to react the actual infection conditions of Chinese animal.The quality standard of the positive serum of working out according to OIE and the concrete condition of China are carried out, thereby guarantee with the prepared antibody of brucella and with the accuracy of positive serum as standard control serum.
Standard positive serum is that in kit, whether checking ELISA test operation is correct, and whether reaction a special important component, is also that this reagent of reaction closes the index of whether working.According to the sensitivity experimental study of animal brucellosis antibody competition ELISA test, standard positive serum to the competition inhibiting rate of novel monoclonal antibody near 100%, colour developing fully, in experimental phase OD value never over 1.80.Standard female serum is not competed inhibition to monoclonal antibody, and due to the impact of serum, its OD value just is slightly less than blank in the competitive ELISA experiment.Blank, its OD value is 0.05-0.50 in novel competitive ELISA experiment.
5 determining about critical value (cut off), determine that the yin and yang attribute limit value is to determine the very important index of testing result accuracy, in this experiment, the competitive ELISA kit of using preparation detects understands 30 parts of brucellosis Zhunyin property serum, 37 parts of immune animal brucella serum and 16 parts of OIE positive serums, use the CELISA kit that OIE has in the world confirmed accuracy, testing result is compared, and has determined that positive rate is more than 20%.Detectability should be and detects a large amount of negative serum samples in theory, carry out positive rate distribution research, affect specificity to get rid of nonspecific reaction, but in real work, be difficult to obtain a large amount of definite negative samples, can only be by comparing to determine with other detection reagent, in this experiment, 30 parts of its positive rates of definite negative sample of detection are below 20%.
6 is to determine one of important indicator that whether accurate diagnostic result is about specificity check specificity.For verifying its specificity, make the negative recall rate of sample detection animal brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit with OIE standard positive serum, standard female serum, negative reference serum, immune serum, result is 100%.The negative recall rate of carrying out kit with Bacillus paratyphosus B, Caused by Yersinia enterocolitica, Escherichia coli O:157 positive and negative serum detects, and result is 96.5%.Test findings shows, animal brucellosis antibody competition ELISA detection kit has higher specificity.
7 is to detect at present one of the most responsive method of anti-brucellosis antibody about susceptibility check ELISA, due to cross reaction being arranged, and the existence of vaccine type antibody, this makes Serology test be difficult to distinguish diagnosis.The extensive compulsory immunization animal of China particularly, the Shandong Salmonella can be distinguished and be made a definite diagnosis at last to the differentiation of vaccine type antibody and natural infection antibody must by the antibody test competitive ELISA sick, reduces mistaken diagnosis, avoids economic loss.On the basis of selecting the indirect ELISA Screening Diagnosis, adopt competitive ELISA to make a definite diagnosis diagnosis to test sample.In research process, 16 parts of OIE standard positive serums and 37 parts of artificial challenge's serum detect, and positive rate reaches 96.23%.Get 5 parts of international standard positive serums, make respectively serial doubling dilution with physiological saline, do respectively competitive ELISA, indirect ELISA, rose bengal precipitation test and complement fixation test (CFT), wherein the competitive ELISA test adopts the kit of 3 different batches to test, result shows that the competitive ELISA test is identical with the indirect ELISA test, the serum greatest dilution reaches 1: 5120, and detection sensitivity is far above the red dull and stereotyped aggegation experiment of tiger and complement fixation test.These experimental results can show that the animal brucellosis antibody competition ELISA detection kit detection sensitivity of preparation reaches international OIE level, has filled up domestic blank.
8 is closely related with judgement test method and test findings about usage, correct operation and appropriate judgement just can draw correct result, ELISA is highstrung detection method, often because experiment for improper causes testing result widely different, therefore to usage and as a result decision method also make concrete regulation: kit should return to room temperature before use, because joining, ice-cold diagnostic reagent need the time just can make reaction conditions reach 18～28 ℃ on ELISA Plate, so just cause the real reaction deficiency of time, affect the combination of antigen antigen.Washing under plate machine operation condition, washing plate for 4 times just enough.Result has designed two yin and yang attributes and blank with calculating mean value in calculating, and detects the accuracy of test.The computing method of positive percentage are general in the world computing method.
9 are bioactive ingredients about points for attention because of the composition in the ELISA detection kit, antibody wherein etc. is all again protein ingredient, can cause albuminous degeneration to affect the detection effect of kit thereby at room temperature reach frequent heating and cooling, so must deposit on request use; In addition, the developer in kit and stop buffer all contain the chemical substance that is pernicious to people, and should avoid skin contact in experiment.
10 about stability, storage and the term of validity when carrying out storage life research, respectively brucellosis antibody competition ELISA detection kit is positioned over 2～8 ℃, room temperature and 37 ℃, getting at regular intervals several coated good ELIAS strip tests, result shows that detection kit was 2～8 ℃ of preservations 12 months, room temperature preservation 6 months, preserved 9 for 37 ℃, its physical behavior, detection specificity and sensitivity are all unaffected, therefore, we are defined as 2～8 ℃ of preservations with storage life, the term of validity 1 year.
Embodiment 3, and the elementary combination test of Test in Diagnosis of Human Brucellosis comprises that fluorescence polarization detects and enzyme linked immunosorbent assay.Fluorescence polarization method is a kind of homogeneous phase method, does not need to remove not binding reagents, and operating speed is fast, just obtains result, and can eliminate some vaccine reactions in two minutes.It both can be used for the laboratory detects, and also can apply to pasture and field, is fit to the detection of wild animal brucellosis.The susceptibility of FPA is high, is outstanding shaker test.Relative low price, accuracy is elementary the same in conjunction with test with other.Such mensuration also is fit to robotization.Its principle is to measure the molecular reaction rate to change, and this change is to react with soluble antigen due to antibody in test specimen to cause.If the antibody conjugated antigen, the specific rotation of antigen will descend, and can measure this reaction.The ultimate principle of FPA is that molecule rotates Stochastic Interest Rates and its size be inversely proportional to (rotation of little molecule fast, larger molecule is slower) in solution.If a little molecule makes it become large by being attached on a larger molecule, just can measure micromolecular rotational speed and change.What FPA used is by being hydrolyzed the 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 in glass tube or 96 orifice plates after dilute serum, blood or milk, use the FPA analyser to remove the fluorescence reading of background.This reading be stored and the value of reading afterwards in deduct it.The FPA analyser is by the velocity of rotation of polarized light by specific measurement of angle molecule.Then add fluorescently-labeled specific polysaccharide antigen, after 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.
The enzyme linked immunosorbent assay of two types is used for the detection of antagonist.They are indirect enzyme-linked immunosorbent assay and competition enzyme-linked immunosorbent adsorption test.Indirect enzyme-linked immunosorbent assay is combined on solid phase with antibody, with a kind of reaction of the enzyme labeling reagent that can be combined with the antibody of selected or all isotypes.Because vaccine antibody also may detect with this method, it mainly is intended for shaker test.It has several advantages, comprises robotization, the business supply of material, accuracy, relatively short proving time, the adaptability such as relatively cheap.Indirect enzyme-linked immunosorbent assay (IELISA) can be used for most of mammal for detection of antibody level and smooth or that Rough Anti-Brucella produces.This method of inspection can be completed in 90 minutes and also can be used as outstanding shaker test, was used for the laboratory and detected.Its susceptibility is the highest, mainly contains in the elimination plan of brucella bacterium disease.
Indirect enzyme-linked immunosorbent assay (RIELISA), for detection of antibody level and smooth or that Rough Anti-Brucella produces, can be used for most of mammal fast.This method of inspection can be completed in 15 minutes and can be used as outstanding shaker test, and great advantage is can be in the pasture, field operation, quick.
Milk indirect enzyme-linked immunosorbent assay (MIELISA), for detection of the Brucella antibody in ruminant milk, this conventional sense method can be measured the antibody of smooth type brucella in milk from cows and goats.90 minutes consuming time, be the shaker test as the mixed milk sample.Because the milk that detects animal can be used for the monitoring of brucellosis, also reduced the stimulation to dairy animal simultaneously.
Competition enzyme-linked immunosorbent adsorption test uses the antibody competition in monoclonal antibody and check sample.It not only has the indirect enzyme-linked immunosorbent assay attribute, and the serological reaction of selecting suitable affinity competition antibody to cause in order to the antibody of eliminating due to vaccine and cross reaction is arranged.Competition enzyme-linked immunosorbent adsorption test (CELISA) for detection of the antibody that the smooth type brucella produces, is applicable to people and all animals almost.This detection method take high sensitive monoclonal antibody as the basis, is the test of making a definite diagnosis of well last brucellosis, is also for the unique method of distinguishing vaccine immunity and wild virus infection.
Claims (1)
1. brucellosis antibody; it is characterized in that: it is to utilize the brucellar lipopolysaccharides of smooth type to make antigen coated ELISA Plate; prepare positive control serum, standard weak positive serum with inactivated bacterial liquid immune health ox or the healthy ox of artificial challenge; make negative control sera with the nonimmune cow's serum of health; make competition antibody with monoclonal antibody; the a-protein G enzyme labeling thing of anti-Fc acceptor is two anti-, preparation serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer, assembling forms; Described antigen coated ELISA Plate is preparation like this:
(1) coated: under aseptic condition, freeze-dried antigen is suspended to volume before freeze-drying with distilled water, by dilution in 1: 1000, with 100 μ l/ holes, coated polystyrene 96 orifice plates added and are placed on 4 ℃ and spend the night with the carbonic acid buffer of 0.05M pH9.6;
(2) washing: with 0.01mol/L, the pH value is that 7.2 PBST makes cleansing solution, adds capacity, and the room temperature effect 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 the 0.01mol/L that contains 1% bovine serum albumin(BSA), the pH value is that 6.3 PBST is by the 100ul/ hole, be added in 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 repeated 3 times, or repeated to wash 3 times with washing the plate machine, pat dry till the no-watermark, natural drying;
(4) packing: will seal good ELISA Plate and be put in aluminium foil bag, add the drying agent of 1g packing, with vacuum packing machine, ELISA Plate be packaged, labelled;
The preparation method of described positive control serum is as follows:
(1) animal is used in manufacturing: the ox of preparation positive serum need to detect through the laboratory, must be the sexal maturity of 18 monthly ages, healthy ox, infect without Caused by Yersinia enterocolitica, Bacillus paratyphosus B, Escherichia coli 0:157, and without the healthy ox of the negative antibodies such as propagating system infection; Observe a week;
(2) immunogene preparation: with the flat bottle of Brucella S1119.3 inoculation potato immersion liquid agar, putting 37 ℃ cultivated 48 hours, during one deck bacterium colony to be formed, choose pure flat bottle, agglutinating water is abandoned in suction, with the sterile saline that contains 0.5% phenol, lawn is washed, in the neutral glass bottle of impouring, adding formalin to final concentration is 0.2%, puts 37 ℃ of vibration deactivations 48 hours, qualified through steriling test, in 4 ℃ with 20000 rev/mins centrifugal 10 minutes, get precipitation, do 10 times of dilutions with physiological saline, as immunogene, standby in 2 ℃～8 ℃ Refrigerator stores;
(3) immune programme for children: press minute 4 intramuscular injection of 5ml/ point with immunogene, immune animal, after 14 days, with the method immunity once, then blood sampling afterwards on the 14th, 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;
(4) serum manufacturing: with the conventional method blood sampling, separate its serum, it as serum to be checked, is done 1: 2 with serum dilution, 1: 4... ... dilution in 1: 128 is done to get serum OD to be checked with reference to carrying out the ELISA test with Quality Control strong positive serum
650nm/ Quality Control strong positive serum OD
650nmBe worth the serum dilution to be checked of=1 o'clock as serum diluting multiple, with serum dilution, serum diluted;
(5) packing: serum is carried out the suction filtration degerming with the filter of 0.45 μ m, add thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion under aseptic condition, then quantitative separating, freeze-drying under aseptic condition, labelled;
(6) the standard weak positive serum is made: strong positive serum is done dilution in 1: 1.5 with serum dilution;
The preparation method of described negative control sera is as follows:
(1) manufacturing is used animal with animal with the manufacturing of positive control serum;
(2) the serum manufacturing is taken a blood sample with conventional method, separates its serum;
(3) serum is processed with packing serum is carried out the suction filtration degerming with the filter of 0.45 μ m, adds thimerosal and gentamicin to make its final concentration be 0.02% with anticorrosion under aseptic condition, and then quantitative separating under aseptic condition, labelled;
Described novel animal brucellosis method for preparing monoclonal antibody is as follows:
(1) cultivation of hybridoma cell strain: hybridoma cell strain is taken out from liquid nitrogen, being placed in 37 ℃ of water-baths melts rapidly, 1000 rev/mins of centrifugation cells, remove cryopreserving liquid, add the 10mlMEM nutrient culture media, be divided into two culture flasks and cultivate in 37 ℃ of CO2gas incubator, after cell covered with at the bottom of culture flask in 2-3 days, carry out cell count;
(2) preparation of ascites novel monoclonal antibody: press 0.5ml/ intraperitoneal injection of mice norphytane, after three days, then press 5X10
5-6/ only cell concentration lumbar injection hybridoma approximately produces ascites after ten days, adopts every day once, adopts 2-3 time continuously, and after ascites was taken out, 3000 turned/the centrifugal 10min of min, collected supernatant and mixing, and in 56 ℃ of water bath processing 10 minutes ,-80 ℃ frozen was semi-manufacture;
(3) product check in the middle of: steriling test is undertaken by " veterinary drug allusion quotation ", answers asepsis growth;
(4) titration: the antigen coated ELISA Plate of animal brucellosis of taking out quality inspection, every hole adds the sample diluting liquid 45ul of animal brucellosis antibody competition ELISA diagnostic kit, animal brucellosis standard female serum 5ul, add 2 rounds, standard positive serum 5ul adds 2 rounds, and the animal brucellosis monoclonal antibody was with 1: 10 doubling dilution, each dilutability adds 2 row, hatches 30 minutes for 26 ℃. and antibody titer is OD
450It is the monoclonal antibody extension rate of 0.60 to 1.80 o'clock;
(5) finished product preparation: by the bioactivity result, monoclonal antibody is diluted, be distributed into the 0.2ml/ bottle, the sealing bottleneck posts label, dated batch;
Described serum dilution, cleansing solution, substrate solution A, substrate solution B, H
2O
2, stop buffer is like this preparation:
The preparation of (1) 10 times of washing lotion PBST:
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 (2) 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 (3) 10 times of substrate A solution:
Get citric acid 4.2g, sodium acetate 13.5g, urea peroxide 0.5g adds deionized water 100ml, adjust pH 4.0, filtration sterilization; Packing: the packing of 1.2ml/ bottle, sealing bottleneck, adhesive label;
(4) preparation of substrate B solution:
Get TMB 0.2192g, dimethyl sulfoxide (DMSO) 20ml dissolves under room temperature; Packing: the 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
(5) H
2O
2The preparation of liquid:
Packing: produced by U.S. VWR company, the 0.2ml/ bottle is sub-packed in black bottle, sealing bottleneck, adhesive label;
The preparation of (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;
Packing: 1.2 milliliters/bottle are sub-packed in bottle, the sealing bottleneck, adhesive label, batch.
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| CN102391993B (en) * | 2011-11-16 | 2013-12-18 | 中国人民解放军疾病预防控制所 | Hybridoma cell strain 17C8 and monoclonal antibody generated therefrom used in Brucella detection |
| CN102680681A (en) * | 2012-05-25 | 2012-09-19 | 北京金豪制药股份有限公司 | Novel I-type human immunodeficiency virus (HIV-1) infection enzyme test-free reagent kit |
| CN103278635B (en) * | 2013-05-31 | 2014-10-22 | 中国兽医药品监察所 | Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody 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 |
| CN107475347A (en) * | 2017-10-12 | 2017-12-15 | 天康生物股份有限公司 | A kind of method of quick analysis Brucella sp strain variation rate |
| CN108812548A (en) * | 2018-09-19 | 2018-11-16 | 天康生物股份有限公司 | The method and purposes of brucella vaccine immune cattle |
| CN112611864B (en) * | 2020-12-03 | 2024-03-12 | 重庆市畜牧科学院 | System and method for screening germ model |
| CN113281502B (en) * | 2021-05-14 | 2024-02-02 | 遵义医科大学附属医院 | ELISA (enzyme-linked immunosorbent assay) simulation method |
| CN117538549A (en) * | 2023-09-27 | 2024-02-09 | 上海交通大学医学院附属瑞金医院 | Prediction system for evaluating clinical efficacy of plasma exchange and DSA removal |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632585A (en) * | 2004-04-02 | 2005-06-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof |
-
2009
- 2009-03-26 CN CN 200910071634 patent/CN101581726B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632585A (en) * | 2004-04-02 | 2005-06-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper for detecting brucellar infection and preparing process thereof |
Non-Patent Citations (2)
| Title |
|---|
| Anastasios Minas等.《Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats》.《The veterinary Journal》.2008,第177卷(第3期),材料和方法部分，尤其是竞争ELISA部分. * |
| 张东林等.《蛋白AG检测多种弓形虫抗体AG-ELISA的效果评价》.《湖北农业科学》.2008,第47卷(第6期),1.6节. * |
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