Animal brucellosis competition ELISA antibody test kit

A technology for brucellosis and brucellosis, applied in the field of biological product detection, can solve problems such as non-specificity, inability to effectively distinguish serum, loss, etc., to achieve the effect of improving specificity

Active Publication Date: 2014-10-22
CHINA INST OF VETERINARY DRUG CONTROL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, neither the traditional agglutination test nor the widely used ELISA kit can effectively distinguish the sera of animals infected with the above two bacteria, thus bringing potential non-specificity to the diagnosis of brucellosis
Some studies intend to use the outer membrane protein OMP28, OMP31 and other proteins of Brucella to establish a diagnostic kit for Brucella, but the production of specific antibodies to the above proteins is related to the species of the infected bacteria and the species of the infected animal. Strict dependence leads to a severe limitation of its scope of use, thus losing the possibility of its further development into a kit

Method used

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  • Animal brucellosis competition ELISA antibody test kit
  • Animal brucellosis competition ELISA antibody test kit
  • Animal brucellosis competition ELISA antibody test kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] -Assembly of kit

[0059] Preparation of antigen-coated plates Use 0.05mol / L carbonate buffer (pH9.6, containing 0.01% thimerosal by volume) to dilute the LPS antigen to 1 μg / ml, and coat 100 / μl wells on the enzyme plate (Corning ), 2~8°C for more than 18h. The antigen coating solution in the well was discarded, and then blocked with 5% gelatin (containing 0.01% thimerosal) (dissolved in PBS, pH 7.4), 100 μL / well, 37°C for 2 hours.

[0060] After drying and discarding the blocking solution, dry at 37°C for 2 hours, add a desiccant and put it into a Xibo bag, and vacuum seal it.

[0061] Store at 2-8°C, and the validity period is tentatively set at 12 months.

[0062] The inspection packaging bag of the antigen-coated plate is well sealed, the bottom of the well is clean and transparent, and there is no foreign matter.

[0063] Dispensing of kit components

[0064] Strong positive control serum, weak positive control serum, negative control serum (provided by China V...

Embodiment 2

[0070] ———Kit sensitivity test

[0071]After the positive reference serum was serially diluted 1:2 to 1:32, 50 μL of each dilution was added to the antigen-coated wells, and then 50 μL of the monoclonal antibody diluted according to the requirements of the kit was added. The inhibition rate (PI) of the positive reference serum was ≥30% after 1:32 dilution. At the same time, strong positive serum control, weak positive serum control, negative serum control and serum blank control were set up. Two parallel wells were made for each sample. The serum PI of the strong positive control is 80%-110%; the PI of the weak positive control serum is 30%-70%; the PI of the negative control serum is 10%-15%. 450nm between 0.75 and 2.0.

[0072] After 10 parts of Brucella positive reference serum doubling dilutions, the Brucella antibody cELISA detection kit that laboratory trial-manufacture is carried out sensitivity test respectively, the cELISA kit prepared by the present invention dete...

Embodiment 3

[0076] ———— Kit specificity test

[0077] Yersinia enterocolitica O:9 (Y.E.O:9), Salmonella (S.E.) 045-2 (Group D), C500 (Group C1), AE616 (Group B), Paratyphi B (S.paratyphiB ) and Escherichia coli O:157 (933 strains) positive sera, as well as 30 negative reference sera, were used as test samples for cELISA to verify its specificity. Among them, Yersinia enterocolitica O:9 (Y.E.O:9), Salmonella (S.E.) 045-2 (Group D), C500 (Group C1), AE616 (Group B), and Bacillus paratyphi (S.paratyphi B) and 30 copies of negative reference serum were provided by the Bacterial Products Testing Office of China Veterinary Drug Control Institute, and Escherichia coli O:157 (933 strains) positive serum was provided by Professor Gao Song of the Veterinary College of Yangzhou University.

[0078] Results The kit prepared by the present invention detects the positive serum of Yersinia enterocolitica O: 9, and the positive serum of Escherichia coli O: 157, and its PI is less than 6%; detects Salmon...

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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

[0001] Technical field The present invention relates to a method for diagnosing animal brucellosis—animal brucellosis competitive ELISA antibody detection kit, which belongs to the technical field of biological product detection. technical background [0002] Brucellosis (brucellosis) is a zoonotic disease characterized by abortion and fever caused by Brucella or Brucella, which seriously threatens the life and health of humans and various animals. . This disease not only has serious harm to the reproduction and production performance of animals, but more importantly, people are often difficult to cure after being infected with Brucella, thus causing serious public health problems. Elimination of brucellosis has therefore been one of the most important goals of public health programs in countries where Brucella is endemic. [0003] The existence of brucellosis in the world has a long history. Human beings have gradually deepened their understanding of the disease, and their d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
Inventor 丁家波秦爱建王芳王楠赵妮冯忠泽顾进华冯忠武张广川郭辉程君生毛开荣
Owner CHINA INST OF VETERINARY DRUG CONTROL
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