Enzyme-linked immunoassay method for food preservative nisin
A nisin and enzyme-linked immunosorbent assay technology, which is applied in the field of nisin content determination, can solve the problems of low sensitivity, low detection limit, difficult to popularize and use by detection institutions, and achieves the effect of highlighting technological progress.
Active Publication Date: 2012-04-04
COMPREHENSIVE TECH SERVICE CENT YILI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology
[0006] At present, there are two methods for the determination of nisin in my country, one is the agar diffusion method, which is based on the enterprise standard QB 2394-2007 "Food Additives Nisin", using the principle of agar diffusion method to display the antibacterial effect by detecting the growth of bacteria on the surface of the agar, converting the titer from the diameter of the inhibition zone and the standard titer to determine its content, because this method has low sensitivity, Unable to accurately quantify, easily affected by interference factors, easy to pollute the environment and other shortcomings, it is not easy to be popularized and used by various testing institutions
The second is liquid chromatography. After investigation, a liquid chromatography detection method was established in 2010 and a patent was applied for. Due to the expensive instruments and consumables required for this method, the detection limit is low, and it takes a long time, it is not easy to be widely promoted.
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[0038] The present invention is further described with reference to examples.
[0039] Implementation steps of the inventive method:
[0040] 1) Construction of the detection method:
[0041] Preparation of coated antigen: Dilute the coated antigen 1000 times with 0.03 mol / L carbonate buffer solution with a pH value of 9.6, add 100 μl per well, incubate at 37°C for 2 hours, pour off the coating solution, and use Contains 0.05% 0Phosphate buffer of sodium azide, 0.1% gelatin and 0.05% Tween, i.e. 0.02M, pH 7.4, wash twice, 30 seconds each time, pat dry, then add 150 μl of blocking solution to each well, i.e. 0.1 % 0 Phosphate buffer solution of sodium azide and 10% (mass percentage) bovine serum albumin, incubate at 37°C for 2 hours, pour off the liquid in the well, dry and store in a vacuum-sealed aluminum film;
[0042] Preparation of nisin polyclonal antibody: Take 1.0 mg / ml nisin solution and an equal amount of Freund's adjuvant (self-prepared, liquid paraffin and lanol...
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The present invention provides an enzyme-linked immunoassay method for a food preservative nisin. According to the present invention, the nisin is adopted as antigen to carry out animal immunization to obtain polyclonal antibody; a conjugate of the nisin hapten and ovalbumin (OVA) is adopted as coating antigen; the nisin is adopted as a standard substance; a treatment method for food samples by the immunoassay is established; a carrier kit of the method is provided. The establishment of the immunoassay method comprises: treating the sample requiring detection, preparing the coating antigen, preparing the polyclonal antibody, carrying out a competing reaction, adding enzyme-labeled second antibody, carrying out a color reaction, and the like. The obtained immunoassay kit comprises: an antigen-coated enzyme label plate, a bottle of an enzyme-labeled second antibody working solution, a bottle of an antibody working solution, six bottles of nisin standard substance solutions, two bottles of substrate coloration solutions, a bottle of a stop buffer, a bottle of a concentrated cleaning solution and a bottle of a reconstituted solution.
Description
Technical field [0001] The present invention involves biotechnology, and the natural preservatives obtained by the lactic acid linase enzyme enzyme -linked immune detection method, especially suitable for the measurement of lactic acid spinthin content in dairy products and beverages and meat products. Background technique [0002] Lactinin (Nisin) is a polypeptide antibacterial substance made from lactic acid lactococcus or lactic acid minobacteris. It is a world -recognized safe natural biological food preservative and antibacterial agent.my country began to approve Nisin in 1990. So far, Nisin has been widely used in more than 50 countries and regions in the world. [0003] Nisin is a polypeptide composed of 34 amino acids, and its molecular weight is 3510. It belongs to Lantibiotics. The active parts contain a large amount of rare amino acids, including wool sulfurine, β-methyl woolen sulfurine, and useSome non-coded amino acids such as acid, β-methyl hydrogenidine.Nisin's ac...
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IPC IPC(8): G01N33/569G01N33/532
Inventor 孟茹王振宝
Owner COMPREHENSIVE TECH SERVICE CENT YILI ENTRY EXIT INSPECTION & QUARANTINE BUREAU