A kind of mycoplasma bovis double-antibody sandwich ELISA detection kit and its application

A detection kit, Mycoplasma bovis technology, applied in the field of double-antibody sandwich ELISA, double-antibody sandwich ELISA kit, can solve the problem of inability to quickly diagnose Mycoplasma bovis infection, inability to achieve high-throughput detection, low detection specificity and sensitivity, etc. problems, to achieve the effect of simple operation, rapid detection and analysis, and realization of detection and analysis

Active Publication Date: 2020-09-15
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because Mycoplasma bovis belongs to facultative anaerobic organisms, has strict nutritional requirements on the medium for isolation, is often affected by clinical antibiotics, and has a long culture period (subculture usually takes more than 72 hours), the separation is particularly time-consuming; After that, further biochemical identification or biological identification is required, and it cannot be used as a method for early and rapid diagnosis of Mycoplasma bovis infection
The PCR detection method is highly sensitive, but requires complex processing of disease samples, and may cause false positives due to contamination
In addition, certain laboratory conditions and equipment are required, and high-throughput detection cannot be achieved.
[0004] Most of the Mycoplasma bovis ELISA kits currently on the market can only detect Mycoplasma bovis antibodies, and the detection specificity and sensitivity are very low

Method used

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  • A kind of mycoplasma bovis double-antibody sandwich ELISA detection kit and its application
  • A kind of mycoplasma bovis double-antibody sandwich ELISA detection kit and its application
  • A kind of mycoplasma bovis double-antibody sandwich ELISA detection kit and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 kit

[0036] 1. Preparation of rabbit anti-Mycoplasma bovis polyclonal antibody

[0037] 1.1 Mycoplasma bovis immunization animals

[0038] Japanese big-eared white rabbits were selected, and ear vein blood was collected before immunization as a negative control. The immunization dose of each rabbit was 0.8 mg, emulsified with an equal volume of adjuvant for 30 minutes, and injected subcutaneously, and immunized 4 times in total. Freund's complete adjuvant was used for the first time, and Freund's incomplete adjuvant was used for the last three times, and the interval between each immunization was 2 weeks. After the final immunization, blood was collected to measure its titer by indirect ELISA method, and when the titer reached the requirement, blood was collected from the heart to collect serum. The titer of polyclonal antibody against Mycoplasma bovis was determined to be 100×2 by indirect ELISA 11 .

[0039] 1.2 Western blot analy...

Embodiment 2

[0057] Example 2 Sample Detection

[0058] 1. Pre-enrichment of the sample to be tested: filter the sample to be tested through a 0.45 μm filter, inoculate it in the PPLO liquid medium of Mycoplasma bovis at a volume ratio of 1:10, and place it in a medium containing 5% volume of CO 2 incubator at 37°C for 36 hours;

[0059] 2. Centrifuge the enriched sample and wash it 3 times with PBS, add to the microtiter plate, 100 μL / well, and incubate at 37°C for 90 minutes;

[0060] 3. Shake off the sample solution, wash the plate 3 times with the washing solution, add 1:800 dilution of horseradish peroxidase-labeled monoclonal antibody to Mycoplasma bovis MbovP579 protein, 100 μL / well, and incubate at 37°C for 90 minutes;

[0061] 4. Shake the solution in the well, wash the plate 3 times, add color developing solution, 200 μL / well, and develop color at room temperature in the dark;

[0062] 5. Add stop solution to stop the reaction, 50 μL / well;

[0063] 6. Measure the OD450 value w...

Embodiment 3

[0065] Embodiment 3 detection condition optimization

[0066] 1. Optimization of capture antibody coating concentration

[0067] The polyclonal antibody against Mycoplasma bovis was coated on the microtiter plate according to 2000ng / well, 1000ng / well, 500ng / well, 250ng / well, 100ng / well, 50ng / well and 25ng / well, and the monoclonal antibody of Mycoplasma bovis MbovP579 protein was used as gradient dilution. To detect antibodies, a double-antibody sandwich ELISA detection method was established according to the aforementioned procedure. Judgment criteria: measure the OD450nm value, stipulate that the measurement hole / negative hole ≥ 2.1 is positive, and the rest of the holes are negative. When the M. bovis polyantibody coating concentration is 250ng / well, the P / N value is 9.936, and the P value is 1.354, as shown in the following table 1, so 250ng / well is selected as the coating concentration of the capture antibody.

[0068] Table 1 Capture antibody coating concentration selec...

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Abstract

The invention discloses a mycoplasma bovis dual-antibody sandwich ELISA detection kit. The kit comprises an elisa plate coated with a mycoplasma bovis polyclonal antibody, and a monoclonal antibody ofanti-mycoplasma bovis MbovP759 protein labelled by horseradish peroxidase, the monoclonal antibody is secreted by a hybridoma cell strain 1A2, and the hybridoma cell strain 1A2 is preserved in the China Center for Type Culture Collection with a preservation number of CCTCCC NO: C 2021919. The mycoplasma bovis dual-antibody sandwich ELISA detection kit disclsoed by the invention realizes the specific detection of mycoplasma bovis antigen, and the sensitivity for detecting the mycoplasma bovis is 108 CFU / mL. By performing pre-enrichment treatment on a sample, a mycoplasma bovis sample containing as low as 1 CFU / mL can reach the detection sensitivity standard. Compared with the prior art, the mycoplasma bovis dual-antibody sandwich ELISA detection kit disclosed by the invention has the advantages of simple and convenient operation, low requirements for the technical level of personnel, small subjective factor error, simple equipment and the like, and can quickly detect and analyze samples in large batches.

Description

technical field [0001] The invention belongs to the field of pathogenic microorganism detection, and in particular relates to a double-antibody sandwich ELISA kit for detecting bovis mycoplasma, and the invention also relates to a double-antibody sandwich ELISA method for non-diagnostic detection of bovis mycoplasma. Background technique [0002] Mycoplasma bovis (M.bovis) is a pathogenic microorganism that can infect cattle of any age, and can cause severe respiratory disease, reproductive system disease, keratitis, mastitis and arthritis in cattle. In 1961, Mycoplasma bovis was isolated from mastitis-affected cattle in the United States for the first time. Cases of economic loss caused by Mycoplasma bovis infection leading to dairy cattle mastitis have been reported all over the world. In my country, Mycoplasma bovis was first discovered in cattle suffering from "suspected bovine pneumonia" in Hubei in 2008, with an infection rate of 50%-100% and a case fatality rate of 10...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/577G01N33/569G01N33/543
CPCG01N33/543G01N33/56933G01N33/577G01N33/581G01N2469/10
Inventor 胡长敏郭爱珍陈颖钰张文劲杨莉陈曦李雨芮
Owner HUAZHONG AGRI UNIV
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