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A kind of anti-duck skeletal muscle troponin i monoclonal antibody and its application

A technology of monoclonal antibody and troponin, which is applied in the field of food safety analysis and immunology, and can solve the problems of difficult immunological methods, loss of antigenicity and water solubility, protein denaturation, etc.

Active Publication Date: 2022-07-08
BIOLOGY INST OF HEBEI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, cooked meat products are generally subjected to high temperature and high pressure treatment. During this process, many proteins are denatured and lose their antigenicity and water solubility, which brings difficulties to the establishment of immunological methods.

Method used

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  • A kind of anti-duck skeletal muscle troponin i monoclonal antibody and its application
  • A kind of anti-duck skeletal muscle troponin i monoclonal antibody and its application
  • A kind of anti-duck skeletal muscle troponin i monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of duck skeletal muscle troponin I antigen according to the present invention

[0036] Take duck skeletal muscle to remove fat and connective tissue, grind and mix well, weigh 20g and add 0.15M NaCl solution (1:2 m / V); after vortexing and mixing, ultrasonic extraction for 5min (100W), after boiling for 20min, 5000g Centrifuge for 20 min; remove the precipitate and filter the supernatant as the detection antigen. Another 20 g of ground skeletal muscle was taken and treated according to the above method. After centrifugation, the supernatant was centrifuged at 121 °C for 30 min under high pressure and centrifuged at 5000 g for 30 min; , let stand for 2h; the mixture was centrifuged at 7000g for 20min, and the precipitate was dried to obtain the immunogen extract. Select the concentration of separating gel to be 12% and the concentration of stacking gel to be 5%, and identify the antigen by SDS-PAGE. figure 1 It can be seen that there are 2 with cle...

Embodiment 2

[0037] Example 2 Preparation of anti-duck skeletal muscle troponin I monoclonal antibody of the present invention

[0038](1) Animal immunization: 6-8 week-old female Balb / c mice were immunized with the prepared immunization antigen, and immunized once every 2 weeks. The immunization process is shown in Table 1. After three immunizations, blood was taken from the tail to determine the titer and inhibition. rate, select the mouse with the best immune result to prepare for fusion;

[0039] Table 1 Immunization process

[0040]

[0041] (2) Cell fusion: The fused mice were exsanguinated by removing the eyeballs, and the serum was used as a positive control. The spleen was taken out under aseptic conditions after decapitation to prepare spleen cells, which were fused with SP2 / 0 cells at a ratio of 5:1 by PEG. The fused cell suspension was added to a 96-well plate that had been plated with feeder cells, and was placed in 37°C, 5% CO. 2 cultured in an incubator;

[0042] (3) S...

Embodiment 3

[0044] Embodiment 3 Characteristic identification of the anti-duck skeletal muscle troponin I monoclonal antibody of the present invention

[0045] (1) The indirect ELISA method is adopted for titer determination, and the concrete steps are as follows:

[0046] Coating: Dilute the original coating with carbonate buffer to a concentration of 5 μg / mL, 100 μL / well of 96-well microtiter plate, overnight at 4°C;

[0047] Washing: Return the coated plate to room temperature, pour off the coating solution, add 300 μL of washing solution to each well, let stand for 1 min each time, wash 3 times, and pat dry for the last time;

[0048] Blocking: add 200 μL of washing solution containing 10% calf serum to each well, 37°C for 1 h; pour off the blocking solution, wash 3 times, and pat dry;

[0049] Add primary antibody: start doubling dilution of monoclonal antibody at 1:2000 times with washing solution, add 100 μL to each well, and set blank control wells (PBS) and negative control (neg...

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PUM

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Abstract

The invention relates to an anti-duck skeletal muscle troponin I monoclonal antibody and an application thereof, belonging to the technical field of immunology and the technical field of food safety analysis. Described anti-monoclonal antibody is secreted and produced by the hybridoma cell line whose deposit number is CCTCC NO: C202003, and the antibody titer is 1:10 5 , the subtype is IgG1, the affinity constant Ka=5.9×10 5 L / mol. The monoclonal antibody can be used to prepare an enzyme-linked immunosorbent assay kit for detecting duck skeletal muscle troponin I in fresh meat and its products, and a colloidal gold test chromatographic test strip to achieve rapid and sensitive detection of animal origin. The purpose of detection of duck meat-derived components in food.

Description

technical field [0001] The invention relates to an anti-duck skeletal muscle troponin I monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and the application of the antibody in detecting duck skeletal muscle troponin I, belonging to the technical field of immunology and The field of food safety analysis technology. Background technique [0002] In meat food, due to price, religion, health and other reasons, many countries have formulated regulations to require food labels to truly and clearly indicate the source of meat, and prohibit adulteration to protect the interests of consumers. The phenomenon is still very common. The methods of adulteration include mixing, mixing, extraction, counterfeiting and other means, especially the adulteration and adulteration of beef and mutton products are the most common behaviors, which greatly damage the interests of consumers. . Duck, chicken and other poultry have a short feeding cycle and low price. At th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/20G01N33/68G01N33/577
CPCC07K16/18G01N33/6887G01N33/577C07K2317/92C07K2317/33G01N2333/4712
Inventor 李春生贾春旭李玉静刘静静张静吴萌杜顺丰
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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