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Enzyme linked immunosorbent assay kit detecting folic acid and application thereof

An enzyme-linked immunosorbent reagent and kit technology, applied in biological testing, material inspection products, etc., can solve the problems of inability to operate on site, high detection cost, complicated pretreatment, etc. Simple pre-processing

Inactive Publication Date: 2014-10-01
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Current detection methods for folic acid include: gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS-MS), liquid chromatography, colorimetry, thin-layer chromatography, microbiological methods, and isotope radiation. Immunoassay, etc. Instrumental methods are sensitive, accurate, specific, and good in separation, and can simultaneously determine multiple drugs, but require expensive instruments, sample pretreatment is complicated, tedious and time-consuming, and the cost of detection is high, so it cannot be operated on site. And it needs to be operated by professionals, so its application is limited

Method used

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  • Enzyme linked immunosorbent assay kit detecting folic acid and application thereof
  • Enzyme linked immunosorbent assay kit detecting folic acid and application thereof
  • Enzyme linked immunosorbent assay kit detecting folic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Preparation of kit components

[0027] 1. Antigen preparation

[0028] Immunogen preparation - Folic acid was coupled with bovine serum albumin (BSA) to obtain immunogen.

[0029] Take 8mg of folic acid and dissolve it in 1ml of dimethylformamide (DMF); take 15mg of carbodiimide (EDC) and dissolve it fully in 0.2ml of water, then add it to the folic acid solution, stir at room temperature for 24 hours, and you can get the reaction solution A; Weigh 30mg of BSA, fully dissolve it in 3ml 0.1mol / L CB (pH 9.6), slowly add the reaction solution A dropwise to the protein solution, and stir at room temperature for 24h, add 0.01mol / L PBS 4 Dialyze at ℃ for 3 days, and change the dialysate 3 times a day to remove unreacted small molecules; aliquot and store at -20℃ for later use.

[0030] Coating source preparation - folic acid and ovalbumin (OVA) were coupled to obtain the immunogen.

[0031] Take 10mg of folic acid and dissolve it in 1ml of DMF; take 0.1ml of g...

Embodiment 2

[0045] Example 2 Formation of an enzyme-linked immunosorbent assay kit for detecting folic acid

[0046] Set up the enzyme-linked immunosorbent assay kit for detection of folic acid, so that it contains the following components:

[0047] (1) Enzyme plate coated with folic acid-conjugated antigen;

[0048] (2) 6 bottles of folic acid standard solution, the concentrations are 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, 81μg / L;

[0049] (3) Folic acid monoclonal antibody working solution;

[0050] (4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0051] (5) The substrate chromogenic solution is composed of liquid A and liquid B, liquid A is hydrogen peroxide, and liquid B is tetramethylbenzidine;

[0052] (6) The stop solution is 2mol / L sulfuric acid;

[0053] (7) The washing solution has a pH value of 7.4, contains 0.5%~1.0% Tween-20, biological preservatives, and 0.1~0.3mol / L phosphate buffer, and the percentages are weight and volume percentages;

[0054] (...

Embodiment 3

[0055] Example 3 Detection of folic acid in samples of finished milk, milk powder and grains (rice, millet, corn, soybeans, flour, etc.)

[0056] 1. Sample pretreatment

[0057] Pretreatment method for finished milk samples: Measure 100 μl of milk samples into 2ml polystyrene centrifuge tubes; add 50 μl of 2% sodium chloride and 850 μl of reconstitution working solution respectively; vortex with a vortexer at 3000r / min for 30s to make the samples Mix well; take 50 μl for analysis.

[0058] Milk powder, grain (rice, millet, corn, soybean, flour, etc.) sample pretreatment method: Weigh 0.5g±0.05g homogenized sample into a 10ml polystyrene centrifuge tube, add 5ml 2% sodium chloride; Vortex at 3000r / min for 2min to make the sample fully mixed; let it stand for 5min; pipette 100μl supernatant into a 2ml polystyrene centrifuge tube, add 900μl reconstitution working solution, and use a vortexer at 3000r / min Vortex for 30s to fully mix the sample; take 50μl for analysis.

[0059] ...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit detecting folic acid. The enzyme linked immunosorbent assay kit comprises an elisa plate coated with a coating antigen, a folic acid antibody working solution, an enzyme marker, a folic acid standard substance solution, a substrate developing solution, a stop solution, a scrubbing solution and a compound solution. The coating antigen is a folic acid coupling antigen, and the enzyme marker is an enzyme marking antibody. The invention further discloses a method applying the enzyme linked immunosorbent assay kit detecting the folic acid. The method comprises the steps that sample pretreatment is carried out at first, then the kit is used for detection, and finally the detection result is analyzed. The enzyme linked immunosorbent assay kit can be used for detecting the content of the folic acid in finished milk, milk powder and cereals such as rice, millet, corns, soybeans and flour. The kit is easy and convenient to operate, low in cost, high in sensitivity, capable of monitoring the folic acid on site, and suitable for screening a large number of samples.

Description

technical field [0001] The present invention relates to enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting folic acid, which is especially suitable for determining the content of folic acid in finished milk, milk powder and grains (rice, millet, corn, soybeans, flour, etc.) detection. Background technique [0002] Folic acid is a compound composed of pterinidine, p-aminobenzoic acid and glutamic acid, and is a water-soluble B vitamin. Folic acid has an important nutritional effect on the human body. A lack of folic acid can cause megaloblastic anemia and leukopenia, as well as physical weakness, irritability, loss of appetite, and psychotic symptoms. In addition, folic acid is especially important for pregnant women. For example, lack of folic acid in the first 3 months of pregnancy can lead to fetal neural tube development defects, thereby increasing the incidence of split-brain and anencephaly. Now th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/82
CPCG01N33/82
Inventor 何方洋万宇平冯才伟罗晓琴杨秀贤贾芳芳冯静蒲小容
Owner BEIJING KWINBON BIOTECH
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