ELISA (enzyme-linked immunosorbent assay) reagent kit for detecting IgM antibodies of brucellosis and application of ELISA reagent kit

A brucella and kit technology, applied in the field of medicine, can solve the problems of short duration and difficult detection

Inactive Publication Date: 2018-02-02
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because it is a pentamer, it has a strong immune effect, which is 100 times that of IgG, b...

Method used

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  • ELISA (enzyme-linked immunosorbent assay) reagent kit for detecting IgM antibodies of brucellosis and application of ELISA reagent kit
  • ELISA (enzyme-linked immunosorbent assay) reagent kit for detecting IgM antibodies of brucellosis and application of ELISA reagent kit
  • ELISA (enzyme-linked immunosorbent assay) reagent kit for detecting IgM antibodies of brucellosis and application of ELISA reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 is used to detect the preparation and the assembly of the ELISA kit of brucellosis IgM antibody

[0049] 1. Preparation of coated antigen

[0050] 1.1 The CCVV12 strain of Brucella abortus was introduced by China Veterinary Inspection Institute and preserved by Lanzhou Veterinary Research Institute.

[0051] 1.2 Aseptically operate under biosafety environment conditions, inoculate 1 mL of the preserved F2 culture of CVCC12 strain into 100 mL of TSB liquid medium, seal the container mouth, and shake it at 37 °C for about 48 hours until it grows to the logarithmic phase. Inactivate the bacteria liquid by acting at 80°C for 30min, and collect the precipitate by centrifuging at 8000rpm for 20min at 4°C after inactivation. Weigh 1 g of the precipitate and dilute it with 30 mL of PBS solution, sonicate, centrifuge at 12,000 rpm at 4°C for 30 min, and collect the supernatant, which is the coated antigen.

[0052] 2. Preparation of negative and positive serum

...

Embodiment 2

[0111] Embodiment 2 is used to detect the determination of the negative and positive critical value of the ELISA kit of brucellosis IgM antibody

[0112] Application test kit of the present invention detects 120 parts of brucella negative serums that are all negative through tiger red, test tube agglutination, and competition ELISA method to measure antibody kit detection before immunization, and every part of serum is repeatedly measured two holes, according to embodiment 1 The operating instructions are for detection, read out the OD450nm value, and calculate the OD average and standard deviation of 120 serums. The negative and positive critical value = the negative serum OD average + 3 times the standard deviation, thereby calculating the negative and positive critical value. According to the principle of statistics, when the OD450nm value ≥ X+3SD, it can be judged as positive at the level of 99.9%, so the judgment standard of the experimental results can be obtained, and the ...

Embodiment 3

[0116] Embodiment 3 is used for detecting the specificity test of the ELISA kit of brucellosis IgM antibody

[0117] Use the kit of the present invention to detect Salmonella (S.E.), Yersinia enterica 0:9, Listeria, B. 10 negative sera were tested for kit specificity. Among them, Salmonella, Yersinia small intestine, and Paratyphi B were provided by the Bacterial Products Testing Office of China Veterinary Drug Administration; Listeria and Escherichia coli were provided by the herbivorous animal bacterial disease team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; Brucella Bacillus negative serum is kept by our laboratory.

[0118] The test results are shown in Table 3-Table 5. The results show that: Salmonella (S.E.), Yersinia enterica 0:9, Listeria, Paratyphi b and Escherichia coli 0:157 were positively detected by this kit The OD450nm values ​​of serum and cattle and sheep negative serum were all <0.37, which was judged as negative. T...

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Abstract

The invention discloses an ELISA (enzyme-linked immunosorbent assay) reagent kit for detecting IgM antibodies of brucellosis and application of the ELISA reagent kit. The ELISA reagent kit comprises brucella antigen coated microporous plates, HRP (horseradish peroxide) labeled brucella antigens, negative reference substances, positive reference substances, color developing agents A, color developing agents B, stop solution and concentrated cleaning solution. The application of the ELISA reagent kit on the basis of double-antigen sandwich immunoassay principles includes adding to-be-detected samples into the microporous plates, binding unknown antibodies in the samples with solid-phase antigens, carrying out washing to form stable unknown antibody-antigen complexes, adding labeled antigensinto the unknown antibody-antigen complexes to bind the labeled antigens with blank sites of IgM antibodies so as to form labeled antigen-IgM antibody-antigen complexes, washing the labeled antigen-IgM antibody-antigen complexes, and then adding the color developing agents into the labeled antigen-IgM antibody-antigen complexes; measuring absorbance OD (optical density) values under the conditionof detection wavelengths of 450 nm and then judging the to-be-detected samples according to critical values. The shade of colors and the degrees of concentration are in positive correlation function relationships. The ELISA reagent kit and the application have the advantage that the ELISA reagent kit can be used for monitoring early acute infection to detect the brucellosis of pigs, cattle and sheep.

Description

technical field [0001] The invention relates to an ELISA kit and application thereof, in particular to an ELISA kit for detecting brucellosis IgM antibody and application thereof. The invention belongs to the technical field of medicine. Background technique [0002] Brucella Blues are Gram-negative intracellular parasites without flagella, plasmids, and capsules. According to different hosts and pathogenicity, it can be divided into pigs (Bsuis), cattle (B.abortus), sheep (B.melitensis), gerbils (B.neotomae), sheep (B.ovis), dogs (B. .canis) 6 species and 19 types of Brucella, of which 3 species and 16 types of pig, cattle and sheep can infect humans through aerosol, parasitize in mononuclear macrophages, and destroy the body's immune system. kind. The diseases caused by Brucella are collectively called brucellosis, which can infect a variety of animals, leading to premature birth, miscarriage or even infertility in female animals, orchitis and dysplasia in male animals....

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911G01N2333/23
Inventor 陈妍尚佑军吴锦艳王光祥尹双辉曹小安刘湘涛张志东
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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