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Tyramine artificial antigen and antibody, and preparation methods and application thereof
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An artificial antigen, tyramine hapten technology, applied in chemical instruments and methods, animal/human proteins, serum albumin and other directions, to achieve good application prospects, low cost, and high detection efficiency.
Inactive Publication Date: 2016-03-16
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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Tyramide artificial antigens and specific antibodies and the establishment of immunoassay methods based on them have not been reported yet
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Embodiment 1
[0052] (1) Preparation of tyramide artificial antigen
[0053] a. Preparation of cationized BSA (cBSA):
[0054] Accurately measure 20 mL of PBS, slowly add 18.0 mg of ethylenediamine (EDA) while stirring in an ice bath, and adjust the pH to 7.4 with 1 mol / L HCl; weigh 1.00 g of BSA and 56.0 mg of EDC and add them to the above solution, and react at room temperature for 2 hours; use a glass sand funnel Suction filtration, take the supernatant and put it into a dialysis bag; use 0.01mol / L PBS for continuous dialysis at 4°C for 3 days, then dry it with a vacuum freezer at -20°C, and finally store it in a -20°C refrigerator .
[0056] Accurately weigh 20.0 mg cBSA into a 25 mL round bottom flask, add 4 mL of 0.01 mol / L PBS to dissolve it, accurately weigh 4.2 mg tyramine into a 5 mL small brown bottle, add 200 μL DMSO to dissolve it. Slowly add tyramine to the dissolved protein. Add 5 μL of 37% aqueous formaldehyde solu...
Embodiment 2
[0070] Use the tyramide antibody that embodiment 1 obtains to carry out immunodetection
[0071] (1) Coating: Dilute the coating antigen appropriately with the coating solution, then coat it on a 96-well microplate at 100 μL / well, and incubate overnight at 4°C or 3h at 37°C.
[0072] (2) Plate washing: add washing solution PBST, 250 μL / well, shake on the shaker for 2 minutes, discard PBST, repeat plate washing 3 times;
[0073] (3) Blocking: Add 200 μL of blocking solution to each well, block at 37°C for 1 hour, then discard the blocking solution, and wash the plate three times with PBST;
[0074] (4) Adding samples: first add 50 μL of tyramide standard solution or sample solution to each well, and then add 50 μL of antibody each. After adding the samples, perform a competitive reaction at 37°C for 1 hour, and then wash the plate 4 times with PBST;
[0075] (5) Add goat anti-rabbit HRP-labeled secondary antibody: Dilute the goat anti-rabbit HRP-labeled secondary antibody to a...
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Abstract
The invention provides a tyramineartificial antigen and antibody, and preparation methods and an application thereof, and relates to preparation of the tyramineartificial antigen and antibody and establishment of an immunoassay method. The problems that a traditional physical and chemical analysis method is tedious and complex, relatively high in cost and slow in analysis speed are overcome, and a simple, rapid, sensitive and accurate immunoassay technique is provided. With adopting of a formaldehyde method and a glutaraldehyde method, tyramine is coupled respectively with cationized bovine serum albumin and ovalbumin, and the tyramine artificial antigen and a tyramine coated antigen are successfully prepared. The artificial antigen is subjected to animal immunization, blood acquirement and separation purification to obtain the specific antibody. A detection method established by using the antibody and used for detection of tyramine in food has good specificity and sensitivity, and the detection limit can be stabilized to 0.02 mg / L; the detection method is low in cost, simple to operate, and suitable for rapid detection of tyramine in the food.
Description
technical field [0001] The invention belongs to the technical field of immunochemistry and trace residue analysis of small molecular compounds; it relates to organic synthesis, immunochemistry, biochemistry and physical and chemical testing technology, etc.; it particularly relates to the design and synthesis of tyramine haptens and artificial antigens of biological amine small molecule compounds, And the preparation of specific antibody of immunized animals and the establishment of immunoassay method. Background technique [0002] Tyramine is an effective physiologically active substance existing in biological organisms. It is a vasoactive amine and plays an irreplaceable physiological function in the normal metabolism of biological organisms. A small amount of tyramine has an important regulatory effect on the cerebral cortex and its mental activities, and has obvious effects on the relaxation and contraction of blood vessels and muscles. In addition, tyramine, phenethyla...
Claims
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