Enzyme linked immunosorbent assay kit for detecting chicken or duck skeletal troponin I, manufacturing method and application of enzyme linked immunosorbent assay kit

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

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] Cooked meat products are generally processed under high temperature and high pressure. 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 chicken or duck skeletal troponin I, manufacturing method and application of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit for detecting chicken or duck skeletal troponin I, manufacturing method and application of enzyme linked immunosorbent assay kit
  • Enzyme linked immunosorbent assay kit for detecting chicken or duck skeletal troponin I, manufacturing method and application of enzyme linked immunosorbent assay kit

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 The preparation of chicken or duck skeletal muscle troponin I antigen described in the present invention

[0045] Take chicken (or duck) skeletal muscle to remove fat and connective tissue, grind and mix evenly, weigh 20g and add 0.15M NaCL solution (1:2w / v); after further mixing, ultrasonic extraction for 5min (50W, 20KHz), After heating with 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, the supernatant was filtered with Whatman No. 1 filter paper, 90% ethanol (1:3.74v / v) was added to the filtrate, 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 b...

Embodiment 2

[0046] Embodiment 2 Preparation of chicken or duck skeletal muscle troponin I monoclonal antibody according to the present invention

[0047] 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 specificity, and the immune result is selected. Optimal mouse preparation for fusion;

[0048] 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 skTnI-3E7 that produces a single monoclonal antibody against chicken or duck skeletal muscle troponin I;

[0049] 3. Large-scale preparation of monoclonal antibodies: select large female Balb / c mice, us...

Embodiment 3

[0050] Embodiment 3 Preparation of chicken or duck skeletal muscle troponin I polyclonal antibody according to the present invention

[0051] 1. Animal immunization: Select the extracted immune antigen, immunize New Zealand white rabbits, and immunize once every 2 weeks. After 3 times of immunization, take blood from the ear vein to determine the titer and specificity;

[0052] 2. Mass preparation of polyclonal antibodies: Collect blood from the jugular vein of immunized rabbits, separate the serum and measure the volume, add ammonium sulfate (0.313g per milliliter of serum), stir for 30min, let stand for 2h, centrifuge at 8000g for 20min, take the precipitate and use 0.01M PBS Dissolve, dialyze for 1 day, and store at -20°C to obtain chicken or duck skeletal muscle troponin I polyclonal antibody.

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Abstract

The invention relates to a double-antibody sandwich enzyme linked immunosorbent assay kit for detecting chicken or duck skeletal troponin I, a manufacturing method and an application of the double-antibody sandwich enzyme linked immunosorbent assay kit, and belongs to the technical field of immunology and the technical field of food safety analysis. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a capture antibody, a horse radish peroxidase labeled detection antibody, a chicken or duck skeletal troponin I standard product solution, a substrate developing solution, a stop solution and a concentrated washing solution. The capture antibody is secreted by a hybridoma cell strain skTnI-3E7 with a preservation number of CCTCC NO:C202003, and the detection antibody is an anti-chicken skeletal troponin I polyclonal antibody. The enzyme linked immunosorbent assay kit has advantages that sensitivity, precision and accuracy are high; a cross reaction rate is low;and the kit is suitable for large-batch sample detection and so on.

Description

technical field [0001] The invention relates to an ELISA kit for detecting troponin I in chicken or duck skeletal muscle 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. . In meat products, due to reasons such as price, religion, and health, many countries have e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/58G01N33/577G01N33/543
CPCG01N33/6887G01N33/581G01N33/577G01N33/54306G01N2333/4712Y02A50/30
Inventor 李春生刘鹏茹刘静静张静徐冬梅吴萌杜顺丰
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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