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Enzyme-linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, and preparation method thereof and application thereof

An enzyme-linked immunosorbent reagent, troponin technology, applied in the fields of immunology and food safety analysis, can solve the problems of protein denaturation, loss of antigenicity and water solubility, and difficulties in immunological methods, and achieve high sensitivity and specificity

Active Publication Date: 2019-05-03
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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  • Enzyme-linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, and preparation method thereof and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, and preparation method thereof and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, and preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 Preparation of bovine or sheep skeletal muscle troponin I antigen according to the present invention

[0051] Take skeletal muscle of cattle or sheep to remove fat and connective tissue, grind and mix evenly, weigh 40g and add 0.15M NaCL solution (1:2w / v); after further mixing, ultrasonically extract for 5min (50W, 20KHz), and then use After heating in boiling water for 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. Treatment solution 1 and treatment solution 2 were identified by SDS-PAGE ...

Embodiment 2

[0052] Embodiment 2 Preparation of bovine or sheep skeletal muscle troponin I monoclonal antibody of the present invention

[0053] 1. Animal immunization: select the extracted immune antigen, immunize female Balb / c mice aged 6-8 weeks, and immunize once every 2 weeks. After 3 times of immunization, blood is taken from the tail to determine the titer and inhibition rate, and the immune result is selected. Optimal mouse preparation for fusion;

[0054] 2. Cell fusion: take the splenocytes of the mouse selected in step 1 and the mouse myeloma SP2 / 0 cells for fusion, measure the supernatant by indirect ELISA method, select the wells with high positive, and sub-select the positive wells by the limiting dilution method. Cloning until the establishment of a hybridoma cell line producing a single monoclonal antibody against bovine or sheep skeletal muscle troponin I;

[0055] 3. Large-scale preparation of monoclonal antibodies: select large female Balb / c mice, use the method of in v...

Embodiment 3

[0056] Example 3 Identification of Capture Antibody and Detection Antibody Characteristics of the Present Invention

[0057] 1. Potency determination

[0058] Dilute the detection antigen to 5 μg / ml with pH 9.6 carbonate buffer to coat the detection plate, and dilute the purified monoclonal antibody at 1:2000, 1:4000, 1:8000, ... 1:1024000, Add it into the well of the microtiter plate, add HRP-labeled goat anti-mouse secondary antibody after the reaction, and finally use TMB to develop the color. The results show that the purified bovine skeletal muscle troponin I monoclonal antibody has an effect The price reaches 1:10 6 , the titer of the purified goat skeletal muscle troponin I monoclonal antibody concentration was 1mg / mL reached 1:2.56×10 5 .

[0059] 2. Subtype determination

[0060] The mouse monoclonal antibody subtype identification kit purchased from Sigma was used to determine the subtype. The results showed that the subtype of bovine skeletal muscle troponin I m...

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Abstract

The invention, which belongs to the technical fields of immunology and food safety analysis, relates to an enzyme-linked immunosorbent assay kit for detecting skeletal muscle troponin I of cattle or sheep, and a preparation method thereof and application thereof. The kit comprises an enzyme label plate coated with a capture antibody, a horseradish-peroxidase-labeled detection antibody, a skeletalmuscle troponin I standard solution of cattle or sheep, a substrate coloured solution, a stop solution, and a concentrated washing solution. The capture antibody is obtained by secretion of a hybridoma cell line 3A8 with the preservation number of CCTCC NO:C2018217; and the detection antibody is obtained by secretion of a hybridoma cell line 3D3 with the preservation number of CCTCC NO:C2018218. The kit has advantages of high sensitivity, high precision, high accuracy, and low cross-reaction rate and is suitable for large-scale sample detection.

Description

technical field [0001] The invention relates to an ELISA kit for detecting bovine or sheep skeletal muscle troponin I and a preparation method and application thereof, which belong to the technical field of immunology and the technical field of food safety analysis. 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, many laboratories at home and abroad are using molecular biology techniqu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/535G01N33/577G01N33/58
Inventor 李春生李玉静刘静静杜顺丰张岩张静吴萌曹秀梅
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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