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
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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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