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Mycoplasma hyopneumoniae antibody detection kit and manufacture method thereof

A detection technology for mycoplasma hyopneumoniae and antibody, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem that is not high, and needs to be further verified by using a large number of clinical samples in my country, and it is difficult to accurately detect and determine specific mycoplasma hyopneumoniae, Complicated serological diagnosis of Mycoplasma hyopneumoniae, etc., to achieve the effect of promoting the use, reducing non-specific background readings, and fast diagnosis

Active Publication Date: 2013-02-13
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the detection results obtained by using imported ELISA detection kits are relatively uniform, the foreign kits use the whole bacterial protein of Mycoplasma hyopneumoniae or the protein expressed by genetic engineering as the coating antigen, so the detection of this type of kit The specificity is not high, and it needs to be further verified by using a large number of clinical samples in my country. In addition, the price of imported kits is generally expensive; the judgment of the results of the IHA test kits of the China Veterinary Drug Administration requires certain experience, and the sensitivity is low. Market Occupation is not large
In addition, experiments have proved that in many serological assays, the serological diagnosis of Mycoplasma hyopneumoniae becomes very complicated due to the cross-reaction of Mycoplasma hyopneumoniae antibody with Mycoplasma hyopneumoniae, and it is difficult to accurately detect and determine the specific Mycoplasma hyopneumoniae

Method used

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  • Mycoplasma hyopneumoniae antibody detection kit and manufacture method thereof
  • Mycoplasma hyopneumoniae antibody detection kit and manufacture method thereof
  • Mycoplasma hyopneumoniae antibody detection kit and manufacture method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0046] 2) Preparation of conjugated Mycoplasma hyopneumoniae specific polypeptide

[0047] Weigh 10mg of any one of the above-mentioned Mycoplasma hyopneumoniae specific reactogenic polypeptides, add 50μL DMSO to dissolve, then add 10ml double distilled water, weigh 1mg polysaccharide glucosamine, add 1ml PBS to dissolve, add 10mg coupling EDC was mixed evenly, then mixed with the peptide solution, and stirred at room temperature for 3 hours. Use a dialysis bag (pore size MWCO 10000-12000) to remove the coupling agent (EDC) not involved in the binding, purify and freeze-dry to obtain the conjugated Mycoplasma hyopneumoniae specific polypeptide.

Embodiment 1

[0049] A detection kit for mycoplasma hyopneumoniae antibody, prepared by the following method:

[0050] 1) Choose any of the above-mentioned coating solutions for coupling Mycoplasma hyopneumoniae specific polypeptide antigens according to the following (coating solution is 1.59g Na 2 CO 3 , 2.93 g NaHCO 3 Dissolve in 800mL double-distilled water, adjust the pH value to 9.6, and adjust the volume to 1000mL) for proportional dilution of 5μg / ml, and then coat the diluted conjugated polypeptide antigen on an 8-well plate, 100μL per well, and coat overnight at 4°C. The next day, wash 3 times with PBST washing solution 250 μl / well, pat dry, use blocking solution 150 μl / well, block at 37°C for 1 hour, wash 3 times with washing solution PBST 250 μl / well, pat dry, put into vacuum bag, add desiccant , evacuated, and stored at 4°C;

[0051] 2) Preparation of Antibody Diluent (AD): Weigh 1.0g BSA (0.1%), add PBS to 1000ml, store at 4°C;

[0052] 3) Preparation of substrate liquid ch...

Embodiment 2

[0058] A detection kit for mycoplasma hyopneumoniae antibody, prepared by the following method:

[0059] 1) Choose any of the above-mentioned coating solutions for coupling Mycoplasma hyopneumoniae-specific polypeptide antigens (the coating solution is 1.59g Na 2 CO 3 , 2.93 g NaHCO 3 Dissolve in 800mL double-distilled water, adjust the pH value to 9.6, and set the volume to 1000mL) according to the ratio of 10μg / ml to dilute, and then coat the diluted conjugated Mycoplasma hyopneumoniae specific polypeptide antigen on a 48-well plate, 100μL per well, 4 Coat overnight at ℃, wash 3 times with PBST washing solution 250 μl / well the next day, pat dry, use blocking solution 150 μl / well, block at 37 °C for 1 hour, wash 4 times with washing solution PBST 250 μl / well, pat dry, put into vacuum bag , add desiccant, vacuumize, and store at 4°C;

[0060] 2) Preparation of Antibody Diluent (AD): Weigh 1.0g BSA (0.1%), add PBS to 1000ml, store at 4°C;

[0061] 3) Preparation of chromoge...

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Abstract

The invention belongs to the detection field of biotechnology and particularly relates to a mycoplasma hyopneumoniae antibody detection kit and a manufacture method thereof. A solid carrier is wrapped by a link-coupled mycoplasma hyopneumoniae peculiar polypeptide antigen, and the manufactured mycoplasma hyopneumoniae peculiar polypeptide antigen enzyme-linked immuno sorbent assay (ELISA) detection kit is used for clinical detection of mycoplasma hyopneumoniae infection, epidemiological investigation and immunologic surveillance. The non-protein polymer directional link-coupled mycoplasma hyopneumoniae peculiar polypeptide antigen serves as the wrapping solution, the combination efficiency of polypeptide and a target antibody is effectively improved, accordingly, detection sensitivity is remarkably improved, non-singular background value reading is remarkably reduced simultaneously, the specificity is high, and the detection kit has the advantages of being easy to operate, fast in diagnosis, economic and convenient in large-scale detection and the like. The manufacture method is convenient to popularize and has wide application prospect. When the kit is used for detecting samples, the using amount of the wrapping antigen can be reduced to 10ng / ml, so that cost is reduced, and popularization and using are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and specifically relates to a Mycoplasma hyopneumoniae antibody detection kit and a preparation method thereof. The Mycoplasma hyopneumoniae specific polypeptide antigen is coated with a solid-phase carrier by using Mycoplasma hyopneumoniae-coupled Mycoplasma hyopneumoniae-specific polypeptide antigen Antigen ELISA kit is used for clinical detection of Mycoplasma hyopneumoniae infection, epidemiological investigation and immune monitoring. Background technique [0002] Mycoplasmal Pneumonia of Swine (MPS, also known as "asthma") is a chronic, contact, high morbidity and low mortality infectious disease caused by Mycoplasma hyopneumoniae (Mhp) . Mycoplasma hyopneumoniae mainly infects the respiratory tract and damages cilium and epithelial cells. An important factor of its pathogenicity is the interaction between mycoplasma and lymphocytes. Mycoplasma infection changes the phagocytic functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
Inventor 李其昌白方方邵国青陈善真李中圣刘小琴罗均赵焱陈克宏王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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