CN103278635A - Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit - Google Patents
Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit Download PDFInfo
- Publication number
- CN103278635A CN103278635A CN2013102138115A CN201310213811A CN103278635A CN 103278635 A CN103278635 A CN 103278635A CN 2013102138115 A CN2013102138115 A CN 2013102138115A CN 201310213811 A CN201310213811 A CN 201310213811A CN 103278635 A CN103278635 A CN 103278635A
- Authority
- CN
- China
- Prior art keywords
- brucella
- kit
- strain
- monoclonal antibody
- brucellosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 206010006500 Brucellosis Diseases 0.000 title claims abstract description 29
- 238000002965 ELISA Methods 0.000 title claims abstract description 24
- 241001465754 Metazoa Species 0.000 title claims abstract description 21
- 230000002860 competitive effect Effects 0.000 title claims abstract description 8
- 238000001514 detection method Methods 0.000 title abstract description 29
- 241000589562 Brucella Species 0.000 claims abstract description 50
- 239000000427 antigen Substances 0.000 claims abstract description 31
- 102000036639 antigens Human genes 0.000 claims abstract description 31
- 108091007433 antigens Proteins 0.000 claims abstract description 31
- 239000002158 endotoxin Substances 0.000 claims abstract description 25
- 229920006008 lipopolysaccharide Polymers 0.000 claims abstract description 25
- 241001333951 Escherichia coli O157 Species 0.000 claims abstract description 13
- 241001494479 Pecora Species 0.000 claims abstract description 11
- 230000035945 sensitivity Effects 0.000 claims abstract description 6
- 239000004744 fabric Substances 0.000 claims description 17
- 201000010099 disease Diseases 0.000 claims description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 13
- 244000005700 microbiome Species 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000004321 preservation Methods 0.000 claims description 7
- 238000009007 Diagnostic Kit Methods 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 4
- 241000607447 Yersinia enterocolitica Species 0.000 claims description 3
- 230000004907 flux Effects 0.000 claims description 3
- 229940098232 yersinia enterocolitica Drugs 0.000 claims description 3
- 241001148106 Brucella melitensis Species 0.000 claims description 2
- 229940038698 brucella melitensis Drugs 0.000 claims description 2
- 244000052769 pathogen Species 0.000 claims description 2
- 230000001717 pathogenic effect Effects 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 22
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 241000607734 Yersinia <bacteria> Species 0.000 abstract description 4
- 208000010227 enterocolitis Diseases 0.000 abstract description 3
- 241001148111 Brucella suis Species 0.000 abstract description 2
- 241000283690 Bos taurus Species 0.000 abstract 1
- 241000282887 Suidae Species 0.000 abstract 1
- 244000052616 bacterial pathogen Species 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 49
- 238000012360 testing method Methods 0.000 description 49
- 210000004027 cell Anatomy 0.000 description 20
- 206010003445 Ascites Diseases 0.000 description 19
- 238000010790 dilution Methods 0.000 description 17
- 239000012895 dilution Substances 0.000 description 17
- 230000004520 agglutination Effects 0.000 description 16
- 239000007788 liquid Substances 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 9
- 241001148129 Yersinia ruckeri Species 0.000 description 9
- 239000000523 sample Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000012856 packing Methods 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 238000001556 precipitation Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000004523 agglutinating effect Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 238000007689 inspection Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 5
- 230000009849 deactivation Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 229920000136 polysorbate Polymers 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000002405 diagnostic procedure Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 4
- 229940033663 thimerosal Drugs 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 241001416149 Ovis ammon Species 0.000 description 3
- 241000607142 Salmonella Species 0.000 description 3
- 238000003149 assay kit Methods 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 239000013068 control sample Substances 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000589567 Brucella abortus Species 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 241000282376 Panthera tigris Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 229940056450 brucella abortus Drugs 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- KGRVJHAUYBGFFP-UHFFFAOYSA-N 2,2'-Methylenebis(4-methyl-6-tert-butylphenol) Chemical compound CC(C)(C)C1=CC(C)=CC(CC=2C(=C(C=C(C)C=2)C(C)(C)C)O)=C1O KGRVJHAUYBGFFP-UHFFFAOYSA-N 0.000 description 1
- -1 8 μ L2.5mMdNTPs Substances 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 244000208060 Lawsonia inermis Species 0.000 description 1
- 244000131099 Macrotyloma uniflorum Species 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 101150028926 bp26 gene Proteins 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 101150004747 omp31 gene Proteins 0.000 description 1
- 230000010494 opalescence Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a fast, efficient and accurate animal brucellosis diagnosis method. On one hand, the method adopts a 3E4 strain monoclonal antibody specifically combined with brucella; in brucellosis antibody detection, the monoclonal antibody 3E4 strain can generate specific competition with an antibody generated by the brucella, but does not compete with other pathogenic bacteria, and particularly does not compete with enterocolitis yersinia O9 and escherichia coli O157 antiserum in a strong cross reaction with brucella; the technical defect that the existing commercialized brucellosis competitive ELISA (enzyme linked immunosorbent assay) kits can not distinguish the interferences of enterocolitis yersinia O9 from escherichia coli O157 is effectively overcome, and the detection specificity is improved; and on the other hand, the kit provided by the invention adopts brucella suis (S2 strain) lipopolysaccharide (LPS) with good reactionogenicity with pigs, cattle and sheep as an envelope antigen, and increases the output of LPS, reduces the production cost and improves the detection sensitivity.
Description
Technical field the present invention relates to a kind of animal brucellosis diagnostic method---and animal brucellosis competitive ELISA antibody assay kit belongs to biological products detection technique field.
Technical background
Brucellosis (cloth disease) is by brucella or what claim that brucella (Brucella) causes is the Amphixenosis of feature with miscarriage and heating, is seriously threatening the life and health of people and multiple animal.This disease not only has serious harm to breeding and the production performance of animal, the more important thing is, after the people infects brucella, often is difficult to cure, thereby causes serious public health problem.Therefore in the popular country of brucella, eliminate the cloth disease is one of most important target in the public health program always.
There is existing history remote in the cloth disease in the world, the mankind progressively deepen this sick study and cognition, and diagnostic level improves constantly, and along with the progress of animal cloth disease, the sick serological detection method of animal cloth is updated and improved.Traditional agglutination test (as: brave red antigen plate agglutination test, the agglutination test of test tube antigen, the agglutination test of breast ring) is replaced by the ELISA method of susceptibility height, high specificity, fluorescence polarization test new detection techniques such as (FPA) gradually.
Agglutination test is the method a kind of commonly used of brucellosis diagnosis, comprise serum aggegation experiment, breast ring precipitation test and the test of anti-human immunoglobulin(HIg), wherein Jing Dian standard tube agglutination test (STAT), plate agglutination test (PAT), above method is stopped using substantially in developed country, the substitute is buffering brucellergen agglutination test such as the red plate agglutination test of tiger (RBPT).The red dull and stereotyped aggegation antigen of tiger is through cultivating with the good brucella bacterial strain of antigenicity, deactivation, make with being suspended in the lactic acid buffer after the brave red dyeing behind the centrifugal collection thalline, this method is highly sensitive, low price, easy to operate, detect fast, be suitable for the generaI investigation of animal population brucellosis.Be the appointment test of ox, sheep, traum's disease detection in international trade, also be used for the primary dcreening operation of people's brucellosis monitoring in China.The precipitation test of breast ring still is the main method of cow brucella monitoring, and this method directly detects cow's milk, can carry out screening to milk cow colony, and this method is easy fast.Agglutination test mainly detects the antibody of anti-brucella LPS-O chain, and the common antigen that brucellar LPS-O chain is multiple Gram-negative bacteria, therefore the cross-infection of brucella and other Gram-negative bacterias existence can't be differentiated with tube agglutination test, and this also is a great problem that the brucellosis diagnosis exists.
Consistently in the brucellosis serologic test think that complement fixation test (CFT) (CFT) is better than additive method in specificity, often be used to tube agglutination test and brave red plate agglutination test test positive or suspicious case are carried out qualitative detection.Complement fixation test (CFT) is considered to the sick diagnostic method of the most effective cloth always, does not still have a kind of diagnostic method can replace the status of complement fixation test (CFT) in serodiagnosis so far.But the preparation difficulty of the needed hemolysin of complement fixation test (CFT), test operation is loaded down with trivial details, is difficult to generally use clinically, and because the complement in the pig body can disturb the effect of GPC, causes susceptibility to descend.
ELISA is a kind of diagnostic method suitable with the CFT effect, and this method is easy to operate, and is highly sensitive, and specificity is better, once can finish the detection of a large amount of samples, both can be used as shaker test and has been applied to the animal population quarantine, can also be as confirmed diagnosis test.ELISA not only can be applied to the detection of serum antibody, also can be applicable to detection of antibodies in the newborn sample.The brucellosis ELISA detection method of ox has been one of detection method of appointment in the international trade, and the brucellar ELISA detection method of other kinds also is the focus of research.At present, be used for ELISA that brucellosis detects two kinds of indirect ELISA (iELISA) and competitive ELISAs (cELISA) are arranged.
Be to be at war with at the antibody at the cloth disease in the monoclonal antibody specific of brucella LPS and the animal blood serum to be checked because cELISA uses, use two anti-be enzyme labelled antibody at mouse IgG, so for iELISA, cELISA can detect the serum of pig, ox and sheep simultaneously, and order iELISA can only be at a certain diagnostic kit of setting up respectively wherein.Therefore, the cELISA kit has host's adaptability widely.But the major technique defective that present business-like cELISA kit exists is effectively to get rid of the serum interference of Yersinia ruckeri O9 and Escherichia coli O 157 infection animal, thereby its specificity is to a certain degree influenced.
Have high homology by lipopolysaccharides (LPS) in Yersinia ruckeri O9 and Escherichia coli O 157 (the particularly O157:H7 type) bacterium with brucella LPS, disturbed the diagnosis of brucellosis.Up to the present, no matter be traditional agglutination test, the ELISA kit that still is widely used now all can not effectively be distinguished the serum of above 2 kinds of bacterial infection animals, thereby it is potential non-specific to have brought for the diagnosis of cloth disease.There is research to intend using albumen such as brucellar outer membrane protein OMP28, OMP31 to set up brucellar diagnostic kit, but all there is strict dependence because of the kind of the generation of above protein specific antibody and bacterial infection and the kind of infected animal, cause its usable range seriously limited, further be developed to the possibility of kit thereby lost it.
The objective of the invention is in addition, external similar technology antigen adopts ox kind brucella A99 strain more, mainly for detection of the Brucella abortus disease.
Summary of the invention
The objective of the invention is to prepare a kind of detection kit that can effectively get rid of interference that yersinia enterocolitica O9 and Escherichia coli O 157 detect brucellosis and all can detect the brucellosis of pig, ox, sheep.
Technology path of the present invention is to choose the exclusive brucella S2 strain of China, pig, ox, sheep all had good antigenicity, it is the brucellosis vaccine strain that a strain can be used for pig, ox and sheep, LPS with this bacterial strain extraction, brucellosis to pig, ox, sheep detects, better susceptibility is all arranged, also be more suitable for China's brucellosis prevention and control demand simultaneously.This research finds that simultaneously extracting its output of LPS with brucella S2 strain is higher than output with A99 and S1119 strain, can effectively reduce production costs;
The present invention is the Monoclonal Antibody technology routinely, utilize brucella S2 strain as immunogene, immunity Balb/C mouse, after getting its spleen cell and mouse myeloma cell line SP20 fusion, 127 strain monoclonal antibody specific cell lines have been obtained with brucella aggegation antigen selection, further carry out the agglutination test screening by Yersinia ruckeri O9 and Escherichia coli 933 strains (O157:H7 type), final screening has obtained a strain only has agglutinating reaction with brucella, and the sick cELISA diagnostic kit of the cloth of researching and developing on this basis with the cell strain of monoclonal antibody of Yersinia ruckeri O9 and the no agglutinating reaction of Escherichia coli O 157 (H7 type), remedy the defective of existing commercialization competition kit, improved the specificity that detects.Therefore:
1. this kit mainly contains: pig kind brucella S2 strain LPS antigen coated microplate and specificity brucella monoclonal antibody 3E4, wherein:
(1) envelope antigen that uses in this kit is the LPS of pig kind brucella S2 strain extraction, and the pathogen of the pig of this LPS, ox, Brucella melitensis disease has good sensitivity, has improved the susceptibility that kit detects;
(2) used specificity brucella monoclonal antibody 3E4 in this kit, this monoclonal antibody can specific recognition the brucella lipopolysaccharides, can effectively get rid of the interference that yersinia enterocolitica O9 and Escherichia coli O 157 detect brucellosis; The brucella cell strain of monoclonal antibody of preparation brucella monoclonal antibody 3E4 was delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 31st, 2013, and deposit number is CGMCCNo.7706.
2. using this kit to carry out high flux to pig, ox and sheep cloth brucellosis detects.Embodiment of the present invention:
1. the application relates to and has set up the quality standard of antigen preparation with bacterial classification pig kind brucella S2 strain (B.suisS2).
(1) the colonial morphology colony edge is neat, mellow and full, reveal to drip shape, and the skew ray irradiation, backlight is observed the blue opalescence of little band.
(2) the dyeing form is coccobacillus, and single being dispersed in do not form gemma and pod membrane.Size is between 0.3～0.6 μ m.Gram is negative, and Ke's Albert'stain Albert is red.
(3) undertaken by existing " Chinese veterinary drug allusion quotation " appendix purely, should be pure.
(4) specificity
1) serological specificity is made antigen with culture, aggegation should occur with smooth type brucella positive serum, aggegation do not occur with rough type serum.
2) synthetic following 4 primers of PCR specificity are made into primer with it and mix storage liquid, and each primer concentration is 25 μ M.
Feri:5 '-GCGCCGCGAAGAACTTATCAA-3 ' (sequence 1)
Reri:5 '-CGCCATGTTAGCGGCGGTGA-3 ' (sequence 2)
F
Suis:5 '-GCGCGGTTTTCTGAAGGTTCAGG-3 ' (sequence 3)
R
IS711:5 '-TGCCGATCACTTAAGGGCCTTCAT-3 ' (sequence 4)
Template: S2 cultivates the S2 genomic DNA of bacterium colony or kit extraction.
The PCR reaction system: in the reaction system of 50 μ L, contain 5 μ L10 * Buffer, 8 μ L2.5mMdNTPs, mix primer storage liquid 2 μ L, Taq enzyme 2U, template DNA 1 μ L(or picking colony a little).
PCR response procedures: behind 95 ℃ of 5min, carry out 28 circulations, last 72 ℃ of 10min by 94 ℃ of 1min, 60 ℃ of 1.5min, 72 ℃ of 1.5min.
The result: amplified production carries out electrophoresis with 1.5% Ago-Gel and identifies that 2 specific PCR bands should occur, size is respectively 178bp and 285bp(sees accompanying drawing 1).
2. the application has set up the extracting method of envelope antigen LPS.
(1) the bacteria suspension preparation is inoculated in the B.suisS2 strain secondary seed of accreditation in the flat bottle of Yi Shi agar, cultivate after 48 hours for 37 ℃, every bottle of adding contains more than the physiological saline 20ml immersion thalline surface 10min of 0.5% phenol, washes culture, is collected in the aseptic vial.
(2) deactivation of bacterium liquid and deactivation check will be collected the bacteria suspension of getting well and will be heated to 80 ℃, keep 120min, treat that it cools off naturally after, be stored in 4 ℃.Should carry out the deactivation check to inactivated bacterial liquid.Get inactivated bacterial liquid 0.1ml coating Yi Shi agar or TSA flat board, cultivated 7 days, and do not answer asepsis growth for 37 ℃.
(3) antigen extracts bacteria suspension that deactivation is up to the standards with the centrifugal 20min of 10000g, collecting precipitation.Weighing is also calculated the thalline weight in wet base, with the thalline weight in wet base in 1:3(W/W) ratio adds in the sterile purified water, fully behind the mixing, is heated to 66 ℃, adds the 90%(V/V of 66 ℃ of preheatings then) phenol solution.(66 ℃) continue to stir 15min under this temperature, put after room temperature cools off naturally, in 4 ℃ of centrifugal 15min of 10000g.Abandon the henna phenol phase of lower floor with long tube suction, water is filtered with Whatman1 filter paper remove big bacterial chip.Measure the phenol phase volume with graduated cylinder, add the methyl alcohol (containing the saturated sodium acetate of 1% methyl alcohol) of precooling below 3 times of volumes-15 ℃ then, hatched 2 hours for 4 ℃, 4 ℃ of centrifugal 10min of 10000g, abandon supernatant, will precipitate with former water 1/2 volume distilled water resuspended, 4 ℃ of centrifugal 10min of 10000g.Collect supernatant in 4 ℃ of preservations.To precipitate and use the equal-volume sterile purified water resuspended again, 4 ℃ were stirred 2 hours again, complied with the centrifugal supernatant that obtains of last method, and mixed with aforementioned supernatant.Subsequently, the adding final concentration is 5% trichloroacetic acid in supernatant, behind the stirring at room 15min, the centrifugal 15min of 10000g discards precipitation, and supernatant spends the night with distill water dialysis, change liquid 2 times (each time 4000ml) at least, collect the bag filter content, this is the LPS of purifying.
3. the application has set up the check program of envelope antigen LPS.
(1) after purity testing is done the 1:10 dilution with the sLPS of purifying, gets 10 μ L and carry out the SDS-PAGE electrophoresis, with Coomassie brilliant blue dye liquor dyeing detection, should not have protein band electrophoresis is finished after.
(2) antigen freeze-drying and packing are got the qualified antigen of purity detecting by 4ml/ bottle packing 10ml penicillin bottle, carry out freeze-drying.Take out freeze-dried antigen, weigh, by the carbonic acid buffer of absolute mass with the 0.05MpH9.6 that contains 0.01% thimerosal it is dissolved, making its concentration is 1mg/ml.With the antigen packing, to preserve with below-70 ℃, the term of validity is tentative to be 24 months.
(3) after titration is made antigen respectively 1:1000,1:2000 and 1:4000 and is doubly diluted with the carbonic acid buffer of 0.05MpH9.6, bag is by 96 hole elisa plates, (China Veterinery Drug Inspection Office provides with the ELISA strong positive, the sick positive serum of cloth country is with reference to product, lot number: 201001), the weak positive (1:100 dilution strong positive serum) and negative serum (China Veterinery Drug Inspection Office provides) respectively 1 part as reference, carry out indirect ELISA method and detect (seeing note 1), after the cessation reaction, measure the OD450 value.ELISA strong positive reference serum OD450 〉=1.0; The weak positive reference serum OD450 of ELISA is that the negative reference serum OD450 of 0.4～0.7, ELISA＜0.1 is qualified.Antigen valence is answered 〉=1:2000.
4.. the application has set up the odd contradictive hydroperitoneum preparation procedure.
(1) cell strain of monoclonal antibody the present invention Monoclonal Antibody technology routinely, utilize brucella S2 strain as immunogene, immunity Balb/C mouse, after getting its spleen cell and mouse myeloma cell line SP20 fusion, 127 strain monoclonal antibody specific cell lines have been obtained with brucella aggegation antigen selection, further carry out the agglutination test screening by Yersinia ruckeri O9 and Escherichia coli 933 strains (O157:H7 type), final screening has obtained a strain only has agglutinating reaction with brucella, and with the cell strain of monoclonal antibody of Yersinia ruckeri O9 and the no agglutinating reaction of Escherichia coli O 157 (H7 type), called after 3E4 strain, this cell strain of monoclonal antibody was delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 31st, 2013, and deposit number is CGMCCNo.7706.
(2) the inoculation mouse is got monoclonal anti body cell 3E4 strain recovery from liquid nitrogen container, goes down to posterity by 1:4 cultivate 48～72h in complete 1640 nutrient culture media after, hybridoma is enlarged cultivate the back harvesting.Collect the hybridoma, the counting (seeing note 2) that enlarge cultivation, abdominal cavity inoculation female BALB/c mouse in 8～10 ages in week, dosage of inoculation is with 1 * 10
6Hybridoma/only.
(3) draw the interior ascites of mouse peritoneal with the 2.5mL syringe behind the about 10d of the collection of ascites, every mouse can be collected several times, and each 3～10mL does not wait, and collects ascites to dead mouse.The thimerosal of adding 0.01% is antibacterial in the ascites, and 12000g is centrifugal 2 times again, to remove cell fragment and lipid, packing ,-70 ° of C preservations.
(4) check of ascites
) purity check gets 5 μ L ascites samples and carry out the SDS-PAGE electrophoresis, coomassie brilliant blue staining is checked its purity, and qualified ascites should be 27KD and 52KD2 bar protein band.The content of protein density two chains of scanning (heavy chain and light chain) should account for more than 80% of total protein concentration.
2) check of tiring is seen note 3 with cELISA() detect tiring of ascites.Microplate reader is measured OD
450nm, by formula calculate inhibiting rate, determine tiring of ascites.Inhibiting rate (%) (PI)=( suppress blank according to OD
450nm-test sample OD
450nmIt is blank according to OD that)/not suppresses
450nm* 100%.Maximum dilution multiple when the PI of ascites 〉=30% is ascites and tires.Tiring should be greater than 1:400.
(5) Purification of Monoclonal Antibodies and check
1) purifying of odd contradictive hydroperitoneum is learnt from else's experience to tire and is detected qualified ascites, adopts precipitation method purified monoclonal antibody ascites.Successively add the CaCl that concentration is 0.2mol/LNaCl and 25mmol/L
2Filter paper filters, and adds 100 times of volumes pure water of sterilizing and places 4 ℃ to the filtrate 8～15h that dialyses, and changes water 1～2 time.Then with filtrate with the centrifugal 30min of 22000g, abandon supernatant; Precipitation is dissolved in the 0.1moL/LTris-HCl solution that contains 1mol/LNaCl (pH8.0), repeats above-mentioned dialysis and centrifugal 1 time; The protein concentration of precipitation is transferred to 5～10mg/mL.Get the monoclonal antibody of 5 μ L purifying and carry out SDS-PAGE, coomassie brilliant blue staining detects the purity of antibody, answers 〉=95%.
2) tire by the detection of cELISA method.Tire be the highly diluted multiple of blocking-up rate PI 〉=30% o'clock monoclonal antibody, ascites tire 〉=1:800 is qualified.
3) packing and freeze-drying with freezing drying protective agent (seeing note 4) as dilution, will be through the qualified monoclonal antibody of check purity by tiring of measuring, being diluted to tires is 1: 5.Monoclonal antibody after the dilution is carried out packing, freeze-drying by every bottle of 1mL dosage.
Description of drawings
The PCR of Fig. 1 Brucella suis S1330 identifies among the figure, 1:DNAMarker; 2: the Escherichia coli negative control; The pcr amplification of 3:S2 bacterial strain.
The microbial resources information that the present invention relates to
Pig kind brucella S2 strain (B.suisS2), this bacterial strain is the production bacterial strain of animal brucella live vaccine, China Veterinery Drug Inspection Office's preservation, be numbered: CVCC70502(China Veterinery Drug Inspection Office, China veterinary microorganism culture presevation administrative center, Chinese animal doctor's bacterial classification catalogue second edition, Scientia Agricultura Sinica technology publishing house, 2002, p35); One strain only has agglutinating reaction with brucella, and with the cell strain of monoclonal antibody of Yersinia ruckeri O9 and the no agglutinating reaction of Escherichia coli O 157 (H7 type), called after 3E4 strain, this cell strain of monoclonal antibody was delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 05 31st, 2013, and deposit number is CGMCCNo.7706.
Advantage of the present invention
The present invention utilize China exclusive and pig, ox, sheep all had good antigenicity pig kind brucella S2 strain extract lipopolysaccharides (LPS) as envelope antigen, brucellosis competitive ELISA detection method has been set up in the brucella monoclonal antibody specific 3E4 strain (this monoclonal antibody and Yersinia ruckeri O9 and Escherichia coli O 157 are reactionless) that utilizes the present invention to screen.This kit can carry out high flux to pig, ox, sheep cloth disease simultaneously and detect, and overcome the technical vulnerability that the sick cELISA diagnostic method of existing cloth can not effectively be distinguished Yersinia ruckeri O9 and Escherichia coli O 157 infection, the equal very desirable detection kit of specificity and susceptibility during to be that present animal cloth is sick detect is for the sick prevention and control of China's brucellosis provide new technological means.
Embodiment
---the assembling of kit
The preparation of antigen coated microplate becomes 1 μ g/ml with 0.05mo1/L carbonate buffer solution (pH9.6 contains the thimerosal of volume ratio 0.01%) with the LPS antigen diluent, and by 100/ μ l hole coated elisa plate (Corning), 2～8 ° of C bags are by more than the 18h.Discard the antigen coated liquid in the hole, use again 5% gelatin (containing 0.01% thimerosal) sealing (be dissolved in PBS, pH7.4), 100 μ L/ holes, 37 ° of C effect 2h.
Drying discards 37 ° of dry 2h of C behind the confining liquid, adds drying agent and puts into the Xibe bag together, vacuumizes sealing.
Preserve 2～8 ° of C and preserve, the term of validity is tentative to be 12 months.
The test package bag sealing of antigen coated microplate is good, clean transparent at the bottom of the hole, no foreign matter.
The packing of kit component
With strong positive control serum, weak positive control serum, negative control sera (China Veterinery Drug Inspection Office provides), brucella monoclonal antibody 3E4(China Veterinery Drug Inspection Office provides), HRP mark mountain sheep anti mouse IgG(Sigma, Lot:A4416-1), PBS-Tween cleansing solution (20 *), substrate colour developing liquid and stop buffer are distributed into the used dosage of kit.
The kit assembling is loaded the bottle number with above-mentioned component by each kit quantity put into the kit plastic stent.Shrouding film, cryodesiccated monoclonal antibody and instructions are put into kit, post outer label and side label.
Each the kit component according to the form below 1 that is up to the standards is assembled into kit.
The component of table 1 kit involved in the present invention
| (1) antigen coated microplate | 2 (96 holes/piece) |
| (2) strong |
1 pipe (100 μ L/ pipe) |
| (3) weak |
1 pipe (100 μ L/ pipe) |
| (4) |
1 pipe (100 μ L/ pipe) |
| (5) brucella |
2 bottles (1mL/ bottle) |
| (6) HRP mark mountain sheep |
1 bottle (30mL/ bottle) |
| (7) substrate |
1 bottle (20mL/ bottle) |
| (8) stop |
1 bottle (20mL/ bottle) |
| (9) PBS-Tween cleansing solution (20 *) | 1 bottle (100mL/ bottle) |
| (10) shrouding film | 4 |
| (11) |
1 part |
[0068] Embodiment 2
---the kit sensitivity tests
Behind 1:32, every dilutability is got 50 μ L respectively and is added in the antigen coated hole with the continuous doubling dilution of positive reference serum 1:2, adds 50 μ L again by the monoclonal antibody of kit requirement dilution.Positive reference serum 1:32 doubly dilutes its inhibiting rate of back (PI) all 〉=30%.Establish the contrast of strong positive serum, weak positive serum contrast, negative serum contrast and serum blank simultaneously.Each sample is all made 2 parallel holes.Strong positive control serum PI is all 80%～110%; Weak positive control serum PI is all 30%～70%; Negative control sera PI is all 10%～15%, blank OD
450nmBetween 0.75～2.0.
Behind the positive reference serum doubling dilution of 10 parts of brucella, respectively the Brucella antibody cELISA detection kit of laboratory trial-production is carried out sensitivity tests, the prepared cELISA kit of the present invention as a result detects positive maximum dilution multiple to positive reference serum and is 640～1280 times (table 2), the kit that shows trial-production has good sensitivity, meets the detection kit requirement.
Table 2cELISA kit is to the testing result of the positive reference serum of different dilutabilitys
---the test of kit specificity
With YE O:9(Y.E.O:9), salmonella (S.E.) 045-2(D group), C500(C1 group), AE616(B group), Bacillus paratyphosus B (S.paratyphiB) and Escherichia coli O:157(933 strain) positive serum, and 30 parts of negative reference serums, carry out cELISA as test sample, verify its specificity.YE O:9(Y.E.O:9 wherein), salmonella (S.E.) 045-2(D group), C500(C1 group), AE616(B group), 30 parts of Bacillus paratyphosus B (S.paratyphiB) and negative reference serums provide Escherichia coli O:157(933 strain by bacterial preparation sensing chamber of China medicine supervision for animals institute) positive serum provides by the high ancient name for Chinese cabbage professor of Yangzhou University's veterinary college.
The prepared kit of the present invention as a result detects the positive serum of YE O:9, and Escherichia coli O:157 positive serum, and its PI is all less than 6%; Detect salmonella (S.E.) 045-2(D group), C500(C1 group), AE616(B group) and Bacillus paratyphosus B (S.paratyphiB) PI all less than 10%; PI is all greater than 80% when detecting the Brucella abortus positive serum, all positive (table 3); Detect the PI of ox negative serum all less than 3%, all negative (table 4).The sick cELISA kit of the cloth of Bi Dui 2 different manufacturers productions but can not effectively be distinguished the positive serum of Yersinia O:9 simultaneously, and Escherichia coli O:157 positive serum (the results are shown in Table 5, table 6).This result of study shows, can effectively get rid of by enterocolitis yersinia genus O:9 based on the cELISA antibody assay kit that cloth disease-specific monoclonal antibody 3E4 sets up, the false positive reaction that Escherichia coli O 157 and other gramnegative bacterium cause, the antibody cELISA detection kit that trial-production is described has good specificity, can be used for the clinical detection of Brucella antibody.
The cELISA detection kit specificity test testing result of table 3 the application research and development
Annotate: PI=100-(the average OD of test sample or control sample
450nm* 100)/blank according to OD
450nm
Table 4cELISA detection kit is to 30 parts of negative reference serum testing results
| The serum numbering | Measurement result | The serum numbering | Measurement result | The serum numbering | Measurement result |
| 1 | － | 11 | － | 21 | － |
| 2 | － | 12 | － | 22 | － |
| 3 | － | 13 | － | 23 | － |
| 4 | － | 14 | － | 24 | － |
| 5 | － | 15 | － | 25 | － |
| 6 | － | 16 | － | 26 | － |
| 7 | － | 17 | － | 27 | － |
| 8 | － | 18 | － | 28 | － |
| 9 | － | 19 | － | 29 | － |
[0083]?
| 10 | － | 20 | － | 30 | － |
| Positive control | 0.112 | Negative control | 1.363 | —— | —— |
Table 5 commercialization cELISA detection kit (1) specificity test testing result
Annotate: PI=100-(the average OD of test sample or control sample
450nm* 100)/blank according to OD
450nm
Table 6 commercialization cELISA detection kit (2) specificity test testing result
Annotate: PI=100-(the average OD of test sample or control sample
450nm* 100)/blank according to OD
450nm
Note
1.iELISA method
The LPS antigen of purifying is diluted to 1 μ g/mL with the carbonate buffer solution of 0.05M, by 100 μ L/ holes bag by 96 hole ELISA Plate (Corning, 2592), more than 4 ℃ of effect 18h.Add 37 ° of C sealings of 5% gelatin 2h by 100 μ L/ holes.Discard coating buffer, add 200 μ L1 * PBS-Tween cleansing solution and wash plate 4 times.Get the ELISA strong positive reference serum of 1:3200 dilution, the weak positive reference serum of 1:3200 dilution and each 100 μ L of negative serum of 1:100 dilution and add in the hand-hole, do a parallel hole, room temperature effect 45min.Discard liquid, add 200 μ L1 * PBS-Tween cleansing solution and wash plate 4 times, each 3min.Add anti-ox two anti-(A9425, Sigma) 1:10000 dilution, the every hole 100 μ L of HRP mark.Incubated at room 45min.Discard liquid, add 200 μ L1 * PBS-Tween cleansing solution and wash plate 4 times, each 3min.Add substrate colour developing liquid (860336, Sigma), every hole 50 μ L, room temperature lucifuge reaction 5～10min.
2. cell count
Blood counting chamber and cover plate are tried totally with wiping, and cover plate is covered on tally.With the cell suspension sucking-off a little, drip at the cover plate edge, suspension is full of between cover plate and the tally.Leave standstill 3min.Observe count plate four big lattice total cellular score, on the left of the line ball cell is only counted and top under the mirror.Be calculated as follows then: the big lattice total cellular score of cell number/ml=4/4 * 10000.Attention: the accidental cell mass of being formed by two above cells under the mirror, should calculate by individual cells, if cell mass accounts for more than 10%, illustrate that dispersion is bad, need prepare cell suspension again.
3.cELISA method and step
Get bag quilt and the LPS antigen diluent through being up to the standards and become 1 μ g/ml, bag is by 96 hole enzymes mark elisa plates (Corning), 100 μ l/ holes.Behind the 18h, with 5% gelatin (Sigma) sealing (be dissolved in PBS, pH7.4), 100 μ L/ holes, 37 ° of C effect 2h.Wash 4 times.Behind the continuous doubling dilution of ascites/supernatant/monoclonal antibody, each dilutability is got 100 μ L(and is done the repetition of 2 holes) add in the hand-hole.Every hole adds the brucella OIE strong positive standard serum 50 μ L of 1:10 dilution, incubated at room 45min immediately then.After the cleaning (method is the same) 4 times, with HRP mark mountain sheep anti mouse IgG(Sigma) the 1:10000 dilution, every hole adds every hole 100 μ L, and 37 ° of C are hatched 45min, wash 4 times again.(TMB860336, Sigma) 50 μ L behind colour developing 5～10min, add stop buffer (0.5MH to add substrate colour developing liquid
2SO
4) 50 μ L.Establish the weak positive criteria serum of brucella OIE, the negative standard serum contrast of OIE simultaneously.Other establishes the blank that does not add ascites/supernatant/monoclonal antibody.Microplate reader is measured OD
450nm, by formula calculate inhibiting rate, determine tiring of ascites.Inhibiting rate (%) (PI)=( suppress blank according to OD
450nm-test sample OD
450nmIt is blank according to OD that)/not suppresses
450nm* 100%.Maximum dilution multiple when the PI of ascites/supernatant/monoclonal antibody 〉=30% is ascites/supernatant/monoclonal antibody and tires.
4. freezing drying protective agent prescription: monoclonal antibody is dissolved in PBS(pH=7.4), every ml wherein contains 10% sweet mellow wine, 1.5% glycocoll, 1% glucose, 1%BSA.
Claims (2)
1. the competitive ELISA diagnostic kit of an animal brucellosis is characterized in that this kit mainly contains: pig kind brucella S2 strain LPS antigen coated microplate, and specificity brucella monoclonal antibody 3E4, wherein:
(1) envelope antigen that uses in this kit is the LPS of pig kind brucella S2 strain extraction, and the pathogen of the pig of this LPS, ox, Brucella melitensis disease has good sensitivity, has improved the susceptibility that kit detects;
(2) used specificity brucella strain monoclonal antibody 3E4 in this kit, this monoclonal antibody can specific recognition the brucella lipopolysaccharides, can effectively get rid of the interference that yersinia enterocolitica O9 and Escherichia coli O 157 detect brucellosis; The brucella monoclonal antibody 3E4 cell line of preparation brucella strain monoclonal antibody 3E4 is delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City day in 2013 05 month, and deposit number is CGMCCNo.7706.
2. the application of the competitive ELISA diagnostic kit of the described animal brucellosis of claim 1 is characterized in that using this kit to carry out high flux to pig, ox and sheep cloth brucellosis and detects.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310213811.5A CN103278635B (en) | 2013-05-31 | 2013-05-31 | Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310213811.5A CN103278635B (en) | 2013-05-31 | 2013-05-31 | Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103278635A true CN103278635A (en) | 2013-09-04 |
| CN103278635B CN103278635B (en) | 2014-10-22 |
Family
ID=49061214
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310213811.5A Expired - Fee Related CN103278635B (en) | 2013-05-31 | 2013-05-31 | Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103278635B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103995123A (en) * | 2014-06-16 | 2014-08-20 | 扬州大学 | Method for detecting bovine brucellosis by competence ELISA (Enzyme-Linked Immunosorbent Assay) kit |
| CN105067812A (en) * | 2015-08-06 | 2015-11-18 | 中国兽医药品监察所 | Bovine brucella indirect ELISA antibody detection kit |
| CN105137073A (en) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | Bovine Brucella colloidal gold antibody detection test paper strip |
| CN105445473A (en) * | 2015-11-13 | 2016-03-30 | 中国检验检疫科学研究院 | ELISA detection kit for bovine Brucella |
| CN106771208A (en) * | 2016-11-10 | 2017-05-31 | 中国兽医药品监察所 | Brucella antibody test strip |
| CN112300254A (en) * | 2020-11-05 | 2021-02-02 | 中国动物卫生与流行病学中心 | Polypeptide for brucellosis diagnosis and application thereof |
| CN113092755A (en) * | 2021-05-18 | 2021-07-09 | 中国兽医药品监察所 | Colloidal gold test strip for detecting brucella antigen by sandwich method |
| CN115267203A (en) * | 2022-08-02 | 2022-11-01 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting content of specific IgG and IgM of brucellosis in sheep and application |
| CN115541887A (en) * | 2022-08-02 | 2022-12-30 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting content of specific IgG and IgM of brucellosis of cattle and application |
| CN116083502A (en) * | 2022-12-19 | 2023-05-09 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Antigen for detecting brucella as well as preparation method and application thereof |
| CN116121199A (en) * | 2022-12-27 | 2023-05-16 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Brucella monoclonal antibody 6A5, application thereof and kit |
| CN117405884A (en) * | 2023-08-14 | 2024-01-16 | 珠海朗泰生物科技有限公司 | Sheep whole blood brucella antibody detection kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037663A (en) * | 2006-09-08 | 2007-09-19 | 中国兽医药品监察所 | Production method for rude type Brucella and its bacterin by using gene recombination technique |
| US20090166200A1 (en) * | 2007-11-30 | 2009-07-02 | William Edward Lee | Serum components that bind to threat agents |
| CN101581726A (en) * | 2009-03-26 | 2009-11-18 | 张晓艳 | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit |
| US20120012197A1 (en) * | 2009-03-31 | 2012-01-19 | Watos Corea Co., Ltd. | Valve for controlling amount of water in rim-side supply pipe of fill valve in water toilet |
-
2013
- 2013-05-31 CN CN201310213811.5A patent/CN103278635B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037663A (en) * | 2006-09-08 | 2007-09-19 | 中国兽医药品监察所 | Production method for rude type Brucella and its bacterin by using gene recombination technique |
| US20090166200A1 (en) * | 2007-11-30 | 2009-07-02 | William Edward Lee | Serum components that bind to threat agents |
| CN101581726A (en) * | 2009-03-26 | 2009-11-18 | 张晓艳 | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit |
| US20120012197A1 (en) * | 2009-03-31 | 2012-01-19 | Watos Corea Co., Ltd. | Valve for controlling amount of water in rim-side supply pipe of fill valve in water toilet |
Non-Patent Citations (3)
| Title |
|---|
| 梅建军: "布鲁氏菌特异性单克隆抗体的研制和抗原捕捉ELISA方法的建立", 《农业科技辑》, 30 September 2006 (2006-09-30), pages 050 - 44 * |
| 王加兰: "光滑型布鲁氏菌LPS单克隆抗体的制备及竞争ELISA方法的建立", 《中国硕士论文数据库》, 31 December 2009 (2009-12-31) * |
| 王加兰等: "光滑型布鲁氏菌LPS单克隆抗体的制备及鉴定", 《中国预防兽医学报》, vol. 31, no. 8, 31 August 2009 (2009-08-31), pages 642 - 649 * |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103995123B (en) * | 2014-06-16 | 2016-08-17 | 扬州大学 | The preparation method of a kind of ELISA kit of detection B rucella melitensis antibody and application |
| CN103995123A (en) * | 2014-06-16 | 2014-08-20 | 扬州大学 | Method for detecting bovine brucellosis by competence ELISA (Enzyme-Linked Immunosorbent Assay) kit |
| CN105067812A (en) * | 2015-08-06 | 2015-11-18 | 中国兽医药品监察所 | Bovine brucella indirect ELISA antibody detection kit |
| CN105137073A (en) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | Bovine Brucella colloidal gold antibody detection test paper strip |
| CN105445473A (en) * | 2015-11-13 | 2016-03-30 | 中国检验检疫科学研究院 | ELISA detection kit for bovine Brucella |
| CN105445473B (en) * | 2015-11-13 | 2017-11-03 | 中国检验检疫科学研究院 | A kind of Brucella abortus ELISA detection kit |
| CN106771208A (en) * | 2016-11-10 | 2017-05-31 | 中国兽医药品监察所 | Brucella antibody test strip |
| CN112300254B (en) * | 2020-11-05 | 2022-08-19 | 中国动物卫生与流行病学中心 | Polypeptide for brucellosis diagnosis and application thereof |
| CN112300254A (en) * | 2020-11-05 | 2021-02-02 | 中国动物卫生与流行病学中心 | Polypeptide for brucellosis diagnosis and application thereof |
| CN113092755A (en) * | 2021-05-18 | 2021-07-09 | 中国兽医药品监察所 | Colloidal gold test strip for detecting brucella antigen by sandwich method |
| CN115267203A (en) * | 2022-08-02 | 2022-11-01 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting content of specific IgG and IgM of brucellosis in sheep and application |
| CN115541887A (en) * | 2022-08-02 | 2022-12-30 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting content of specific IgG and IgM of brucellosis of cattle and application |
| CN116083502A (en) * | 2022-12-19 | 2023-05-09 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Antigen for detecting brucella as well as preparation method and application thereof |
| CN116083502B (en) * | 2022-12-19 | 2024-09-20 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Antigen for detecting brucella as well as preparation method and application thereof |
| CN116121199A (en) * | 2022-12-27 | 2023-05-16 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Brucella monoclonal antibody 6A5, application thereof and kit |
| CN116121199B (en) * | 2022-12-27 | 2024-06-21 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | Brucella monoclonal antibody 6A5, application thereof and kit |
| CN117405884A (en) * | 2023-08-14 | 2024-01-16 | 珠海朗泰生物科技有限公司 | Sheep whole blood brucella antibody detection kit |
| CN117405884B (en) * | 2023-08-14 | 2024-05-17 | 珠海朗泰生物科技有限公司 | Sheep whole blood brucella detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103278635B (en) | 2014-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103278635B (en) | Animal brucellosis competitive ELISA (enzyme linked immunosorbent assay) antibody detection kit | |
| FORGIE et al. | Etiology of acute lower respiratory tract infections in Gambian children: II. Acute lower respiratory tract infection in children ages one to nine years presenting at the hospital | |
| CN105067812A (en) | Bovine brucella indirect ELISA antibody detection kit | |
| CN106556701B (en) | Brucella melitensis indirect ELISA antibody assay kit | |
| Escobar et al. | Etiology of respiratory tract infections in children in Cali, Colombia | |
| CN105137073A (en) | Bovine Brucella colloidal gold antibody detection test paper strip | |
| CN105203754B (en) | Method and kit for fast detection of moraxella catarrhalis based on magnetic resolution and quantum dot labelling | |
| CN106771182A (en) | Brucella abortus IgM subclass antibodies indirect ELISA testing kits | |
| Roca et al. | Surveillance of acute bacterial meningitis among children admitted to a district hospital in rural Mozambique | |
| CN101581726B (en) | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit | |
| CN104007269A (en) | Riemerella anatipestifer indirect coagulation antibody detection kit as well as application thereof | |
| CN110218668A (en) | A kind of inert carrier salmonella and its potential application | |
| CN104789500A (en) | Pullorum staining agglutination antigen as well as preparation method and application thereof | |
| CN106596958B (en) | Brucellosis CF-ELISA antibody assay kits | |
| CN106771208A (en) | Brucella antibody test strip | |
| CN107446851A (en) | A kind of white diarrhea aggegation antigen and preparation method thereof | |
| CN101592661A (en) | The brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit | |
| CN105784999A (en) | Method and kit for immunological detection of mycobacterium tuberculosis complex | |
| CN101799470A (en) | Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit | |
| CN105158463A (en) | Colloidal gold test paper bar for antibody detection of sheep Brucella | |
| CN106841609A (en) | A kind of porcine circovirus 2 type blocking ELISA antibody assay kits and preparation method thereof | |
| CN109975541B (en) | Detection card for rapidly detecting canine distemper virus antigen and preparation method thereof | |
| Kadanali et al. | Bacteremic and nonbacteremic brucellosis: clinical and laboratory observations | |
| CN106353496A (en) | Brucella fluorescence polarization (FPA) detection kit | |
| CN101846681A (en) | FPA antibody detection kit for animal brucellosis fluorescence polarization detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141022 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |