Method for detecting in-vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method

A technology of Mycoplasma hyopneumoniae and inactivated vaccine is applied in the field of testing the relative efficacy of Mycoplasma hyopneumoniae inactivated vaccine (DJ166 strain) in vitro, which can solve the problems of long time for animal testing, high testing cost, affecting the repeatability of testing, etc. Cost and labor intensity of animal testing, good sensitivity and specificity, good specificity and reproducibility

Inactive Publication Date: 2021-01-12
BEIJING KEMUFENG BIOLOGICAL PHARMA +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can intuitively evaluate the in vitro efficacy of the vaccine, it has the disadvantages that the animal experiment takes a long time, and the virus needs to be challenged during the experiment. Many factors, such as health status, feeding environment and feed nutrition, directly affect the accuracy and reliability of the test results, and inevitably affect the repeatability of the test; and the test cost is relatively high, and the efficacy test uses animals, operators, attack The preparation of the poisonous bacteria solution, the breeding environment and other conditions lead to the defects of large differences between the batches of the protection test against the virus
More importantly, the detection and result cycle of this method is as long as 2 months, which not only prolongs the research and development cycle of new vaccines, but also indirectly shortens the validity period of vaccines, increases the pressure on vaccine stocks, and seriously affects the production and production of vaccines. Sales

Method used

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  • Method for detecting in-vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method
  • Method for detecting in-vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method
  • Method for detecting in-vitro efficacy of mycoplasma hyopneumoniae inactivated vaccine based on ELISA method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This example is used to prepare Mycoplasma hyopneumoniae inactivated vaccine (DJ166 strain) and its reference vaccine (batch number ZYTCK-1801).

[0059] Mycoplasma hyopneumoniae inactivated vaccine (DJ166 strain) reference vaccine (batch number: ZYTCK-1801) is produced by the applicant in strict accordance with the current "Chinese Veterinary Pharmacopoeia" standard, and the inactivated mycoplasma hyopneumoniae vaccine (DJ166 strain) to be inspected (batch number: ZYT -191, ZYT-192) are also produced by the applicant in strict accordance with the current "Chinese Veterinary Pharmacopoeia" standards.

[0060] The reference vaccine ZYTCK-1801 and the inactivated vaccine of Mycoplasma hyopneumoniae (DJ166 strain) to be tested (batch number: ZYT-191, ZYT-192) were inoculated with the appropriate culture medium with Mycoplasma hyopneumoniae DJ-166 strain, harvested, concentrated and purified Afterwards, the antigen is prepared by thimerosal inactivation, multiple antigen co...

Embodiment 2

[0072] The preparation method of mycoplasma competitive coating antigen in this example includes the following steps: the concentrated and purified bacterial liquid of Mycoplasma hyopneumoniae DJ-166 strain is inactivated, centrifuged, and combined with TritonX-100 and KI solution. It is used to test the relative potency of Mycoplasma hyopneumoniae inactivated vaccine (DJ-166 strain) in vitro by indirect competitive inhibition ELISA test.

[0073] Coating antigen quality control

[0074] Properties: After dissolving and shaking well, it becomes a milky white liquid;

[0075] Sterility test: should grow sterile;

[0076] Potency determination: carry out agar diffusion test with positive serum of Mycoplasma hyopneumoniae, the agar expansion titer should not be lower than 1:2;

[0077] Determination of protein concentration: use a commercial BCA kit for detection, and the protein concentration should not be lower than 1.0mg / ml;

[0078] Specificity: There should be a clear whi...

Embodiment 3

[0081] The preparation method of the positive serum in this example includes the following steps: immunize healthy rabbits with the inactivated Mycoplasma hyopneumoniae vaccine, and inoculate once with the same dose and route 2 weeks after the immunization, when the antibody titer is not lower than 1:16 It is prepared by aseptic blood collection, serum separation, filter sterilization, inactivation and purification, and freeze-drying in vacuum. Positive serum for indirect competitive inhibition ELISA test for inactivated Mycoplasma hyopneumoniae inactivated vaccine (DJ-166 strain) in vitro, which can be freeze-dried in batches.

[0082] Positive serum freeze-dried quality control standard

[0083] Properties: It should be a white spongy loose mass;

[0084] Sterility test: should grow sterile;

[0085] Titer determination: carry out agar diffusion test with Mycoplasma hyopneumoniae antigen, the agar expansion titer should not be lower than 1:2;

[0086] Specificity: There s...

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Abstract

The invention belongs to the technical field of detection of biological products for animals, and particularly relates to a method for detecting in-vitro relative efficacy of a mycoplasma hyopneumoniae inactivated vaccine (DJ166 strain) based on an indirect competitive inhibition ELISA detection method. The method for detecting the in-vitro relative efficacy of the mycoplasma hyopneumoniae inactivated vaccine (DJ166 strain) based on the indirect competitive inhibition ELISA method comprises the following steps: inoculating the mycoplasma hyopneumoniae DJ-166 strain into a suitable culture medium for culture, concentrating and purifying the harvested culture, and inactivating with thiomersalate to prepare an antigen and an inactivated reference vaccine, and enabling a vaccine to be detectedand a reference vaccine to be subjected to indirect competitive inhibition ELISA antigen detection, namely in vitro relative potency ELISA detection. The detection method disclosed by the invention is simple to operate and short in consumed time, has good sensitivity and specificity and good detection result stability, specificity and repeatability, and is a convenient, rapid, specific and sensitive potency determination method meeting the development trend.

Description

technical field [0001] The invention belongs to the technical field of biological product testing for animals, and in particular relates to a method for testing the relative effectiveness of Mycoplasma hyopneumoniae inactivated vaccine (DJ166 strain) in vitro based on an indirect competitive inhibition ELISA detection method. Background technique [0002] Mycoplasma swine pneumonia (MPS), commonly known as swine panting disease, also known as porcine endemic pneumonia, is a chronic contact respiratory infectious disease caused by Mycoplasma hyopneumoniae. The clinical symptoms of mycoplasma swine pneumonia are mainly coughing and wheezing. Generally, the death rate of pigs is not high, but the feed conversion rate is seriously reduced, and the long-term growth and development are poor, causing huge economic losses. At present, immunization against Mycoplasma hyopneumoniae vaccine is one of the important measures to prevent and control MPS. Vaccination can effectively reduce ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/58G01N33/543
CPCG01N33/54306G01N33/56933G01N33/581G01N2469/10
Inventor 徐慧敏卢晓楠于萍萍王美君汤波韩昱鹏刘飞刘奇张渊魁王敏
Owner BEIJING KEMUFENG BIOLOGICAL PHARMA
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