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Anti-goat skeletal muscle troponin I monoclonal antibody and application thereof

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

Active Publication Date: 2019-05-28
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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  • Anti-goat skeletal muscle troponin I monoclonal antibody and application thereof
  • Anti-goat skeletal muscle troponin I monoclonal antibody and application thereof
  • Anti-goat skeletal muscle troponin I monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 The preparation of sheep skeletal muscle troponin I antigen described in the present invention

[0038]Take sheep skeletal muscle to remove fat and connective tissue, grind and mix well, weigh 40g and add 0.15M NaCL solution (1:2w / v); after further mixing, ultrasonically extract for 5min (50W, 20KHz), and then heat with boiling water After 20 minutes, centrifuge at 2000g for 30 minutes; remove the precipitate, take half of the supernatant and filter it as treatment solution 1. The other half of the supernatant was subjected to high pressure at 121°C for 30min, centrifuged at 5000g for 30min, filtered with Whatman No. 1 filter paper, added 90% ethanol (1:3.74v / v) to the filtrate, and the mixture was centrifuged at 7000g for 20min, and the precipitate was dried at 37°C , after reconstitution with physiological saline, it becomes the treatment solution 2. After treatment solution 1 and treatment solution 2 were identified by SDS-PAGE electrophoresis, the iden...

Embodiment 2

[0039] Embodiment 2 Preparation of sheep skeletal muscle troponin I monoclonal antibody of the present invention

[0040] (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;

[0041] Table 1 Immunization flow chart

[0042]

[0043] (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;

[0044] (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

[0046] Example 3 Characteristic Identification of Goat Skeletal Muscle Troponin I Monoclonal Antibody of the Present Invention

[0047] (1) Titer determination adopts indirect ELISA method, and the specific steps are as follows:

[0048] 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;

[0049] 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;

[0050] 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;

[0051] 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), and plac...

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Abstract

The invention relates to an anti-goat skeletal muscle troponin I monoclonal antibody and application thereof, and belongs to the technical field of immunology and the technical field of food safety analysis. The monoclonal antibody is generated through secretion of a hybridoma cell line with the preservation number of CCTCC (China Center for Type Culture Collection) NO: C2018218; the titer of theantibody is 1 to 2.56*10<5>, a subtype is IgM, and an affinity constant Ka is equal to 7.6*10<8>L / mol. The monoclonal antibody provided by the invention can be used for preparing an enzyme linked immunosorbent assay kit and a colloidal gold chromatography test strip, which are used for detecting goat skeletal muscle troponin I in fresh mutton and products thereof, so that the aim of rapidly and sensitively detecting mutton derived components in animal derived foods is realized.

Description

technical field [0001] The invention relates to an anti-sheep skeletal muscle troponin I monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and the application of the antibody in detecting sheep 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. . [0003] At present,...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20G01N33/543G01N33/68
Inventor 李春生刘静静李玉静张静吴萌张岩
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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