ELISA (enzyme linked immunosorbent assay) kit of folic acid

A kit, folic acid technology, applied in the field of detection and analysis of food and feed ingredients, can solve the problems of expensive and bulky instruments, time-consuming, difficult on-site operation, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2011-09-07
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the HPLC method has the characteristics of fast, accurate, and good reproducibility, the instruments used are expensive and bulky, and require ...

Method used

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  • ELISA (enzyme linked immunosorbent assay) kit of folic acid
  • ELISA (enzyme linked immunosorbent assay) kit of folic acid
  • ELISA (enzyme linked immunosorbent assay) kit of folic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, immunogen, synthesis of coated antigen and preparation of antibody

[0030] 1. Synthesis of immunogen

[0031] The immunogen was obtained by coupling folic acid and bovine serum albumin (BSA) by EDC method. Specifically include the following steps:

[0032] A. Weigh 22.7mg (51.5μmol) of folic acid, 98.7mg (514.7μmol) of water-soluble carbodiimide (EDC), 29.6mg (257.4μmol) of N-hydroxysuccinimide (NHS) and dissolve them in 5mL DMF in sequence , protected from light, and reacted with magnetic stirring at room temperature for 24 hours;

[0033] B. Dissolve 100mg (1.5μmol) BSA (molecular weight range: 6.7KDa~6.8 KDa) in 10mL phosphate buffer saline (PBS) (0.01M, pH7.4);

[0034] C. Slowly add the "product of step A" to the "product of step B", and react with magnetic stirring at room temperature for 3 hours; (0.01M, pH7.4) dialysis for 3 days, during which the dialysate was changed every 4-6 hours; followed by distilled water dialysis for 3 days, during wh...

Embodiment 2

[0042] Embodiment 2, the establishment of ELISA detection method

[0043] 1. Optimal concentration of antibody and coated antigen (square array method)

[0044] Coat the microtiter plate longitudinally with the coating antigen at serial concentrations of 8.0 μg / mL, 4.0 μg / mL, 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL and 0.25 μg / mL, 100 μL / well, 37 Place at ℃ for 2 hours, wash the plate three times with 300 μL / well of washing solution; then block with 250 μL / well of blocking solution, place overnight at 0-4°C, wash the plate three times; add 100 μL / well of a series of diluted antibodies horizontally (1: 2500 to 1:80000), placed at 37°C for 30 minutes, washed three times; added 100 μL / well of 1:2000 enzyme-labeled secondary antibody (horseradish peroxidase-labeled goat anti-rabbit antibody), placed at 37°C for 30 minutes, washed Plate three times; add 100 μL / well of substrate chromogenic solution, and measure the absorbance value. Specificity determination was carried out with the coat...

Embodiment 3

[0054] Embodiment 3, the assembly of the enzyme-linked immunoassay kit that detects folic acid

[0055] 1. The composition of the enzyme-linked immunoassay kit for detecting folic acid

[0056] A. A solid phase carrier (enzyme plate) coated with a coated antigen (a conjugate of folic acid and ovalbumin);

[0057] B. Folic acid antibody working solution (volume ratio concentration is 1:20000);

[0058] C. 6 bottles of folic acid standard solution, the concentrations are: 3ng / ml, 6 ng / ml, 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml;

[0059] D, horseradish peroxidase-labeled goat anti-rabbit IgG antibody working solution (working concentration is 1:2000);

[0060] E. Concentrated phosphate buffer solution contains 80g of NaCl and KH per liter 2 PO 4 2.0g, Na 2 HPO 4 12H 2 o 2 29.0g, KCl 2.0g aqueous solution.

[0061] F, concentrated washing solution: the above-mentioned concentrated phosphate buffer solution is added with Tween 20 (Tween20) with a volume ratio concentr...

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Abstract

An ELISA (enzyme linked immunosorbent assay) kit of folic acid belongs to the technical field of enzyme linked immunosorbent assay. The reagent components in the kit comprise: an enzyme label plate coated with a folic acid coating antigen (the antigen is prepared by coupling folic acid and ovalbumin); a folic acid polyclonal antibody; a goat anti-rabbit antibody marked by enzyme-labelled bi-antibody which is horseradish peroxidase; a folic acid standard solution; a concentrated phosphate buffer; a concentrated cleaning solution; a substrate colour developing solution A; a substrate colour developing solution B and a stop solution. The ELISA kit provided by the invention performs ELISA by using a polyclonal antibody, wherein IC50=15.5ng/ml. The lowest detecting limit in milk products is 11.9ng/ml; the coefficients of variation between batches and in batches are 11.8%; and the recovery is 89.1-110.3%. The ELISA kit provided by the invention has the characteristics of simple structure, fast operations, accurate detection result and high sensitivity; and the kit can be used for fast detecting folic acids contained in foods, feeds and vitamin products.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay kit for detecting folic acid content in food, feed and vitamin products, and belongs to the technical field of detection and analysis of food and feed components. Background technique [0002] Folic acid is a water-soluble B vitamin, also known as vitamin B 9 Or vitamin M, which is necessary for the growth and reproduction of the body's cells. Folic acid acts in the form of tetrahydrofolate in the body, and tetrahydrofolate participates in the synthesis and transformation of purine nucleic acid and pyrimidine nucleotide in vivo. Plays an important role in the manufacture of nucleic acids. Necessary substance when the human body utilizes sugar and amino acids. When folic acid is deficient, the form of deoxythymidylic acid and purine nucleotides and the interconversion of amino acids are blocked, the synthesis of DNA in cells is reduced, and the division and maturation of cells are hindered, causin...

Claims

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Application Information

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IPC IPC(8): G01N33/82
Inventor 郗日沫张太昌孟萌薛虎寅徐静张元阳
Owner NANKAI UNIV
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