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Monoclonal antibody resisting duck skeletal troponin I and application of monoclonal antibody

A monoclonal antibody, troponin technology, applied in the field of food safety analysis and immunology, can solve the problems of loss of antigenicity and water solubility, protein denaturation, difficult immunological methods, etc., to achieve strong controllability and repeatability Effect

Active Publication Date: 2020-04-28
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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  • Monoclonal antibody resisting duck skeletal troponin I and application of monoclonal antibody
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  • Monoclonal antibody resisting duck skeletal troponin I and application of monoclonal antibody

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Effect test

Embodiment 1

[0035] The preparation of embodiment one described duck skeletal muscle troponin I antigen of 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 vortex mixing, ultrasonic extraction for 5min (100W), after boiling for 20min, 5000g Centrifuge for 20 min; remove the precipitate, take the supernatant and filter it as the detection antigen. Take another 20g of ground skeletal muscle and process it according to the above method. After centrifugation, take the supernatant and put it at 121°C for 30min under high pressure, then centrifuge at 5000g for 30min; filter the supernatant with Whatman No. 1 filter paper, and add 90% ethanol to the filtrate (1:3.74V / V) , let stand for 2h; the mixture was centrifuged at 7000g for 20min, and the precipitate was dried to obtain the immunogen extract. Select separation gel concentration to be 12%, stacking gel to be 5%, carry out ...

Embodiment 2

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

[0038](1) Animal immunization: immunize 6-8 week-old female Balb / c mice with the prepared immune antigen, and immunize once every 2 weeks. 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 bleed from the eyeballs, and the serum was used as a positive control. After the neck was killed, the spleen was taken out under aseptic conditions to prepare spleen cells, which were fused with SP2 / 0 cells by PEG at a ratio of 5:1. The fused cell suspension was added to a 96-well plate with feeder cells, and placed in a 37°C, 5% CO 2 cultivated in an incubator;

[0042] (3) Screening of positive hybridoma cell lines: the fused cells were checked for contamination the next day, and replaced with HT medium on the 10th day after fusion. 2-3 days after cha...

Embodiment 3

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

[0045] (1) Titer determination adopts indirect ELISA method, and the specific steps are as follows:

[0046] Coating: Dilute the coating material with carbonate buffer to a concentration of 5 μg / mL, 100 μL / well of a 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 hour; pour off the blocking solution, wash 3 times, and pat dry;

[0049] Add the primary antibody: use the washing solution to dilute the monoclonal antibody at 1:2000 times, add 100 μL to each well, and set a blank control well (PBS) and a negative control (negative serum),...

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Abstract

The invention relates to a monoclonal antibody resisting duck skeletal troponin I and application of the monoclonal antibody, and belongs to the technical field of immunology and the technical field of food safety analysis. The monoclonal antibody is secreted by a hybridoma cell strain with a preservation number of CCTCC NO: C202003, the titer of the antibody is 1:10<5>, the subtype is IgG1, and the affinity constant Ka is 5.9x10<5> L / mol. The monoclonal antibody can be used for preparing an enzyme linked immunosorbent assay kit and a colloidal gold chromatography test strip for detecting theduck skeletal troponin I in fresh meat and products thereof, and achieves the purpose of quickly and sensitively detecting duck meat-derived components in animal-derived foods.

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 Food safety analysis technology field. Background technique [0002] In meat products, due to reasons such as price, religion, and health, many countries have enacted regulations requiring food labels to truly and clearly indicate the source of meat and prohibit adulteration to protect the interests of consumers. However, the confusion of meat varieties in the market The phenomenon is still very common. Adulteration methods include blending, mixing, extraction, counterfeiting, etc., especially the adulteration and adulteration of beef and mutton products are the most common, and these adulteration behaviors have greatly damaged the interests of consumers. . Due to the short breed...

Claims

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Application Information

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Patent Type & Authority Applications(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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