Kit for enzyme-linked immunosorbent assay of bovine abortion clamydia

An enzyme-linked immunoassay technology for detection of Chlamydia abortus, which is applied in the field of immunology, can solve the problems of general sensitivity and specificity, great influence of subjective factors, and long detection time, so as to improve specificity and sensitivity and reduce subjective factors Effects, effects with low instrument requirements

Active Publication Date: 2018-11-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of directly staining pathogens is simple to operate and fast to detect, but the sensitivity and specificity are average
The operation of immunohistochemical staining method is relatively complicated, and the judgment of the above two methods requires rich professional and technical experience, and the judgment result is greatly affected by subjectivity, especially when it is quite difficult to identify EB and RB of chlamydia
2) Pathogen isolation and identification method. Cell culture and isolation of pathogens. Immunohistochemical or immunological staining of chlamydia inclusion bodies requires poultry embryos or cells. The isolation process is more complicated and has higher requirements for experimental operations, experimental environments, and experimental facilities. General grassroots laboratories Difficult to carry out
The disadvantage is that the sensitivity is low, the price of fluorescence microscopy is expensive, and it is not suitable as a routine detection method
4) Although the polymerase chain reaction has high specificity and sensitivity, the template preparation is relatively complicated, and the sample size required is large, and expensive instruments such as PCR machines are required, and the detection reagents are expensive
5) The double-antibody sandwich enzyme-linked immunosorbent assay method has the characteristics of simplicity, sensitivity, and high efficiency, but currently there is no ELISA kit that can detect Chlamydia abortus antigen in China
Disadvantages are low sensitivity and expensive equipment
2) Enzyme-linked immunosorbent assay (ELISA) has the advantages of simple operation, high specificity and sensitivity, low technical requirements for operators, and little influence of subjective factors on experimental results, so it is widely used in clinical detection of serum antibody technology. No ELISA kit for Chlamydia abortus antibodies on the market
3) The operation procedure of the indirect hemagglutination test method is simple and does not require special equipment, but the judgment of the results is greatly affected by subjective factors, and most of the coupling antigens are whole bacteria, which may have cross-reactions with some Gram-negative bacteria, and the results are easy to produce false positive
However, the defects of complement fixation test, such as cumbersome operation, low sensitivity, long detection time, high professional knowledge and easy cross-reaction with other Gram-negative bacterial sera, are limited in clinical application.
5) The colloidal gold test strip method is simple and easy to operate, but the detection sensitivity is low
However, since the main components of these antigens are Chlamydia Major Outer Membrane Protein (MOMP) and lipopolysaccharide, they are the most common Chlamydia cross-reactive antigens, which are likely to cause cross-reaction, and can only be limited to the identification of Chlamydia antigens and cannot distinguish between different species. Chlamydia, i.e. no specific detection of Chlamydia bovis abortus

Method used

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  • Kit for enzyme-linked immunosorbent assay of bovine abortion clamydia
  • Kit for enzyme-linked immunosorbent assay of bovine abortion clamydia
  • Kit for enzyme-linked immunosorbent assay of bovine abortion clamydia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Synthesis of POMP90 protein polypeptide of Chlamydia abortus S26 / 3 strain

[0058] According to the POMP90 protein sequence of Chlamydia abortus S26 / 3 strain recorded in Genbank (Genbank Accession: U65943), DNAstar software was used to compare and analyze the gene sequences of different Chlamydia, and then a specific peptide sequence composed of 25 amino acids was selected for artificial synthesis. Quantitative peptides were aliquoted and stored at -20°C after freeze-drying.

Embodiment 2

[0059] Embodiment 2 The selection of antigen protection agent

[0060] Selection - sucrose, trehalose, dextran, polyethylene glycol 6000, glycerin, formulated as an antigen protection agent according to the formula in Table 1, coated with the Chlamydia abortus POMP90 protease plate with the best optimized conditions, sealed and washed respectively Add 200 μL / well of antigen stabilizer (prescription 1, prescription 2, prescription 3, prescription 4), overnight at 4°C, take out and discard the liquid in the well the next day, add PBST solution 300 μL / well, wash 3 times, 3 min each time, Pat dry with absorbent paper, irradiate with ultraviolet light for 1 hour, put into vacuum bag after drying and store at 37°C for continuous storage, take out the microplate plate on the 14th day and 21st day, detect the positive control substance according to the established ELISA procedure, repeat 4 wells , calculate its mean value, and compare its OD 450 of the decline. Select the now-coated...

Embodiment 3

[0066] Example 3 Preparation of an ELISA plate coated with Chlamydia abortus POMP90 protein polypeptide

[0067] 1. Dilute the synthesized Chlamydia abortus POMP90 protein polypeptide with sterile deionized water to a final concentration of 1 mg / mL.

[0068] 2. Dilute the diluted 1mg / mL polypeptide with pH9.6, 50mM carbonate buffer to a final concentration of 1.0μg / mL, add to each well of the microplate, incubate at 4°C for 16 hours, wash the enzyme with washing buffer PBST Mark the plate, then block with blocking solution (PBST solution containing 10% skimmed milk powder), block with 100 μL / well of blocking solution, incubate at 37°C for 2 hours, pour off the blocking solution, wash 3 times with PBST, and dry with 200 μL / well of blocking solution. The wells were sealed, incubated at 37°C for 2 hours, poured off the blocking solution, washed 3 times with PBST and dried;

[0069] 3. Add 200 microliters of antigen protection agent to each well of the sealed microwell plate, inc...

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Abstract

The invention relates to the field of immunology, and particularly discloses a kit for enzyme-linked immunosorbent assay of bovine abortion clamydia. The kit realizes enzyme-linked immunosorbent assayof the bovine abortion clamydia by artificially synthesizing polypeptide as shown in SEQ ID NO.1, taking the polypeptide as antigen and coating a solid phase carrier to the polypeptide. The kit takesthe artificially synthesized polypeptide antigen as coating antigen, and ensures that enzyme-linked immunosorbent assay has the characteristics of high specificity and good sensitivity. The kit further provides an excellent antigen protective agent after the antigen is coated with the solid phase carrier, protects the coated antigen, and prolongs the storage time of the antigen.

Description

technical field [0001] The invention relates to the field of immunology, in particular to an ELISA kit for detecting bovine chlamydia abortus. Background technique [0002] Bovine chlamydia abortus (Bovine chlamydia abortus) is caused by Chlamydophila abortus (Chlamydophila abortus). Infected cows infected with the disease mainly manifested as abortion, decreased reproductive ability, stillbirth, and retained placenta after abortion; at the same time, it was accompanied by decreased milk production, loss of appetite, and some sick cows had symptoms such as elevated body temperature and listlessness. In addition, experiments have confirmed that intrauterine inoculation of cows with Chlamydia abortifaciens can cause mastitis in cows. The breeding bull is infected with Chlamydia abortus, and the pathogen enters the semen, which can cause the decline of the breeding ability of the breeding bull. When humans contact or handle aborted fetuses or calves without proper protective ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/543G01N33/569
CPCG01N33/535G01N33/54306G01N33/56927
Inventor 何诚王艺晖郭永霞李顺琴
Owner CHINA AGRI UNIV
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