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Mycoplasma hyopneumoniae solid-phase competition ELISA (Enzyme Linked Immunosorbent Assay) kit, and preparation method and application thereof

A technology for mycoplasma hyopneumoniae and mycoplasma pneumoniae, which is applied in biological testing, material inspection products, measuring devices, etc., can solve the problems of long time-consuming virus challenge tests and difficulty in finding negative pigs, and achieve improved animal welfare, shortened detection cycle, and simple operation easy effect

Inactive Publication Date: 2019-07-23
JIANGSU NANNONG HI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The first object of the present invention is to provide a solid-phase competitive ELISA kit for detecting the relative content / efficacy of Mycoplasma hyopneumoniae antigen, and the second object is to provide a method for vaccine efficacy evaluation, which solves the problem that it is difficult to find pigs that are negative in the challenge protection test , the time-consuming problem of virus attack test

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  • Mycoplasma hyopneumoniae solid-phase competition ELISA (Enzyme Linked Immunosorbent Assay) kit, and preparation method and application thereof

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Embodiment 1

[0054] The preparation of embodiment 1 coating antigenic protein, negative and positive serum, relevant vaccine

[0055] For a better understanding of this description, only one method of antigen protein preparation is described here: the high-pressure homogeneous crushing method, the preparation of rabbit anti-Mycoplasma hyopneumoniae positive serum is only described by the preparation method of Freund's adjuvant; the test sample is described by vaccine.

[0056] 1.1 Preparation of coated antigen

[0057] Centrifuge the 3L Mycoplasma hyopneumoniae culture at 4°C and 10,000rpm for 60min after 3 times of expansion, and wash the precipitate with PBS buffer at pH 7.2 for 3 times to make a bacterial suspension of 1 / 10 of the original culture volume. Translucent, centrifuged at 10000rpm for 10min, a small amount of supernatant antigen protein was taken for protein concentration determination and specificity test. Aliquot the rest, 1.0ml / tube, and store at -40°C for later use.

[...

Embodiment 2

[0071] The establishment of embodiment 2 indirect ELISA method

[0072] 2.1 Determination of the best antigen coating concentration and the best negative and positive serum concentrations

[0073] The titration test was carried out according to the orthogonal matrix method, and the coating antigen was serially diluted to 2, 4, 8, 16, 32, 64, 128, 256 μg / ml, coated overnight at 4°C, 100 μl / well. Wash 3 times with 1× washing solution, add PBST containing 1% BSA, 200 μl / well, block at 37°C for 2 hours, add positive serum diluted 1:1000, 1:5000, 1:25000 and 1:50000 after washing, Negative serum diluted 1:80, 1:160 and 1:320, 100 μl / well, reacted at 37°C for 1 hour, after washing, added horseradish peroxidase-labeled goat anti-rabbit IgG diluted 1:20000, 100 μl / well, Act at 37°C for 1 hour, add TMB chromogenic solution after washing, 100 μl / well, act for 15 minutes at room temperature, add 1.5mol / LH 2 SO 4 100 μl / well to terminate the reaction, measure the OD value at a waveleng...

Embodiment 3

[0088] The establishment of embodiment 3 solid-phase competition ELISA method

[0089] 3.1 Samples and testing methods

[0090] 3.1.1 Samples and kits

[0091] Reference sample: Mycoplasma hyopneumoniae reference vaccine, batch number 20170401;

[0092] Samples to be tested: 6 batches of Mycoplasma hyopneumoniae vaccines with different antigen content; 0601, 0602, 0603, 0604, 0605 and 0606; 5 batches of Mycoplasma hyopneumoniae test vaccines, the batch numbers are 20170801, 20170802, 20170803, 20170804 and 20170801;

[0093] Solid-phase competitive ELISA kits: batch numbers are 20171101, 20171102, 20171103, 20171104, 20171105.

[0094] 3.1.2 Detection method:

[0095] (1) Sample processing

[0096] Freeze the Mycoplasma hyopneumoniae reference vaccine, 6 batches of Mycoplasma hyopneumoniae vaccines with different antigen contents, and 5 batches of Mycoplasma hyopneumoniae test vaccines below -70°C for at least 24 hours; take them out and place them in a warm bath at 37°C f...

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Abstract

The invention discloses a mycoplasma hyopneumoniae solid-phase competition ELISA (Enzyme Linked Immunosorbent Assay) kit, and a preparation method and application thereof. The kit comprises an ELISA plate coated with mycoplasma hyopneumoniae antibody protein, a reference sample, rabbit-anti mycoplasma hyopneumoniae positive serum and rabbit-anti mycoplasma hyopneumoniae negative serum. The kit disclosed by the invention can measure the relative content / efficacy of the mycoplasma hyopneumoniae antigen and has good specificity and repeatability. In addition, the provided solid-phase competitionELISA kit is simple in operation, consumes short time, does not need high-end instrument equipment, is easy in promotion and is suitable for applying to vaccine production inspection and laboratory research.

Description

technical field [0001] The invention relates to the technical field of veterinary medicine, in particular to a Mycoplasma hyopneumoniae solid-phase competition ELISA kit and a preparation method and application thereof. Background technique [0002] Mycoplasma pneumonia (Mycoplasma pneumonia of swine), also known as swine panting disease and porcine endemic pneumonia, is a chronic respiratory infectious disease caused by Mycoplasma hyopneumoniae (Mhp), which has a high incidence rate and low mortality rate. exist all over the world. Clinically, it is characterized by coughing, wheezing and typical "meat-like" or "shrimp-like" lesions in the lungs. Affected pigs have slow growth, low feed conversion rate, and normal body temperature. Mycoplasma hyopneumoniae mainly destroys the respiratory cilia of pigs through infection, leading to the shedding of cilia and causing pneumonia, which reduces the humoral and cellular immune functions of pigs, produces immunosuppression, and ma...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/68G01N33/543G01N33/535
CPCG01N33/56933G01N33/68G01N33/543G01N33/535
Inventor 车巧林肖澄缪芬芳董彦鹏徐凤耿晓眉胡静雅靳蒙蒙余姣
Owner JIANGSU NANNONG HI TECH
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