Acrylic amide complete antigen preparation and its ELISA quantitative determination method

A technology of acrylamide enzyme and acrylamide, which is applied in the detection of acrylamide, the preparation of acrylamide whole antigen, and the quantitative detection of acrylamide ELISA, achieving the effects of good reproducibility, easy operation and high sensitivity

Inactive Publication Date: 2008-10-15
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no report on the preparation of acryl...

Method used

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  • Acrylic amide complete antigen preparation and its ELISA quantitative determination method
  • Acrylic amide complete antigen preparation and its ELISA quantitative determination method
  • Acrylic amide complete antigen preparation and its ELISA quantitative determination method

Examples

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Embodiment 1

[0048] Example 1: Preparation of Acrylamide Whole Antigen and Acquisition of Corresponding Polyclonal Antibody

[0049] 1) Synthesis of Acr-OVA whole antigen: 2mg NAS was sonicated in 1mL DMSO solvent; then 100μL of this solution was added dropwise to 1mL 0.01mol / L pH8.0 phosphate buffer solution (containing 1mg OVA), The reaction was stirred at 36°C for 2-3hr; finally, the obtained solution was desalted and freeze-dried to obtain about 1mg of the product, which was stored at -20°C.

[0050] 2) Immunization of animals: use the whole antigen obtained in step 1 to immunize rabbits according to the process in Table 1; then blood is collected, centrifuged at 4000rpm for 20min, serum is taken, and frozen at -20°C.

[0051] 3) Purification of polyclonal antibodies: take 1 mL of serum, dilute it 4 times with 0.06 mol / L, pH 4.8 HAc-NaAc buffer, add 120 μL octanoic acid dropwise, stir for 30 min; centrifuge at room temperature 12,000 rpm for 20 min, and take the supernatant Then add 0...

Embodiment 2

[0054] Embodiment 2: the minimum detection limit and the linear range of the detection method of the present invention

[0055] In a 96-well ELISA plate, coat each well with 100 μL of 50 μg / mL Acr-BSA, incubate overnight at 4°C or 2 hours at 37°C; Add 50 μL of 10 μg / mL purified antibody solution and 50 μL of serially diluted acrylamide standard solution (concentrations are 0, 0.1, 1, 10, 100, 1000 μg / mL), incubate at 37°C for 1 hour, and then each well successively Add 100 μL of biomarked secondary antibody solution and enzyme-labeled avidin solution of appropriate concentration, so that the final absorbance value is 0.5-1.5 (commercially purchased biomarked secondary antibodies need to be diluted 1:2000, and enzyme-labeled avidin needs to be diluted 1:2000). 1:400 dilution), after the same incubation process, the substrate was added to develop the color, and after 10-15 minutes, it was terminated with 2mol / L sulfuric acid. For each sample, 6 parallel sets were made at the sa...

Embodiment 3

[0057] Example 3: Comparison of the present invention with a standard high performance liquid chromatography (HPLC) method

[0058] Taking the commercially available French fries spiked extract (scaling amount is 100 μg / mL extract) as the measuring object, the concentration of the extract was measured respectively by the method of the present invention and HPLC.

[0059] 1) HPLC measurement

[0060] The operating conditions for the measurement by chromatography are as follows:

[0061] Column: C 18 Reversed-phase column (Dikma Technologies Diamonsil, 5μ, 150×4.6mm);

[0062] Mobile Phase: Methanol-H 2 O (5:95v / v), flow rate: 0.6mL / min;

[0063] Column temperature: 25°C

[0064] Detector: ultraviolet detector (UVD), the detection wavelength is 210nm;

[0065] Injector: 10 μL sample loop.

[0066] Using 0.1, 0.5, 1.0, 2.0, 5.0, 8.0, 10, 12, 15, 20, 30 μg / mL acrylamide standard solution as the working curve, using peak area integration and linear regression, determine the ...

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Abstract

The invention provides a method for preparing acrylamide complete antigen. The method is as follows: amine uncoupling reaction is directly carried out between N-acryloyloxy succinimide and the surface amino group of carrier protein molecule in a neutral or weakly alkaline phosphate buffer solution, thereby forming acrylamide complete antigen. Polyclonal antibody capable of realizing specificity identification of acrylamide is obtained through a complete antigen immune animal, so as to establish an enzyme immunoassay method suitable for measuring the content of acrylamide in an aqueous solution or food and to provide a corresponding reagent box. The method has simple operation and does not need other reaction reagent, thereby furthest maintaining the structural integrity of acrylamide; moreover, the method ensures that a coupling position is at a maximum distance from a feature group so as to fully expose the feature structure of acrylamide molecule. The established enzyme immunoassay method of acrylamide and the reagent box have strong specificity, high sensitivity and convenient operation, and are suitable to realize massive sample detection which has low cost and is quick and simple.

Description

technical field [0001] The invention relates to a detection method of acrylamide, in particular to a preparation method of acrylamide whole antigen and a corresponding acrylamide ELISA quantitative detection method, belonging to the field of compound analysis and detection. Background technique [0002] Acrylamide is a widely used organic synthetic monomer, which is widely used in the production of polyacrylamide and other copolymerized compounds, and is widely used in the synthesis of dyes and plastics and as a thickener in building materials and daily necessities. Polyacrylamide can be used as a coagulant to treat drinking water and sewage. The Food and Agriculture Organization of the United Nations and the World Health Organization Joint Expert Committee on Food Additives have conducted a systematic risk assessment of acrylamide in food. Its neurotoxicity, reproductive and developmental toxicity, and genotoxicity have all been confirmed, and it has been classified as Cate...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/53
Inventor 赵美萍周爽宓捷波
Owner PEKING UNIV
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