Method for detecting Beta accompany glycinin and specific antibody and reagent kit thereof

A conglycinin and monoclonal antibody technology, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problem that there is no simple and fast β-conglycinin detection method, and the soybean antigen protein cannot be completely removed. It is necessary to repeat the preparation and other problems to achieve the effect of small coefficient of variation, low cost and high titer

Active Publication Date: 2009-05-27
北京同力兴科农业科技有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In previous reports, the SDS-PAGE method was used to detect the content of β-conglycinin. This method can only be used for qualitative analysis, and the results are not easy to repeat. At present, many laboratories use the indirect ELISA method of multiple antisera to analyze soybean β-conglycinin. protein content (Murphy and Resurreccion, 1984), this method has inevitable disadvantages: multiple antisera need to be prepared repeatedly, cannot be referenced between different laboratories, and the sensitivity and accuracy are low; while the reversed-phase high-performance liquid phase method ( RP-HPLC) to measure the content of soybean 7S components (mainly β-conglycinin) (Mujoo et al., 2003), although the sensitivity and accuracy are high, the cost is high
[0007] A number of studies have confirmed that the existing processing technology cannot completely remove the activity of soybean antigen protein, and there is currently no simple, fast, sensitive, specific and efficient detection method for β-conglycinin

Method used

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  • Method for detecting Beta accompany glycinin and specific antibody and reagent kit thereof
  • Method for detecting Beta accompany glycinin and specific antibody and reagent kit thereof
  • Method for detecting Beta accompany glycinin and specific antibody and reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1, Preparation and detection of β-conglycinin monoclonal antibody

[0028] 1. Preparation of β-conglycinin monoclonal antibody

[0029] (1) Preparation of immunogen

[0030] 1. Extraction and purification of β-conglycinin

[0031] It is prepared by combining fractional salting out and gel filtration. The specific process is as follows:

[0032] Dehulled soybeans were dehulled, filtered and extracted with ether to degrease, and the residual ether was removed by rotary evaporation to obtain defatted soybean powder; at room temperature, 0.03M Tris-HCl (pH8.0, 10mM2-ME ) buffer (the ratio of defatted soybean powder to Tris-HCl is 1g defatted soybean powder: 20ml Tris-HCl), dissolved, centrifuged at 10,000rpm / min for 30min, and collected the supernatant; HCl adjusted the pH to 6.4, 10,000rpm / min( Centrifuge at 4°C for 30min, collect the supernatant; adjust the pH to 4.8 with HCl, collect the precipitate after centrifuging at 15,000rpm / min (4°C) for 30min; dissolv...

Embodiment 2

[0098] Example 2, Preparation and detection of β-conglycinin polyclonal antibody

[0099] Step 1: Preparation of polyclonal antibodies

[0100] Take the antigenic protein prepared in Example 1 and make it 5 mg / mL with physiological saline, mix it with an equal amount of Freund's adjuvant, and use a vortex mixer to emulsify it completely. Healthy female New Zealand white rabbits were selected, and the first immunization was the medial epithelium of the foot, and the complete adjuvant emulsified antigen was injected, and two sites were selected, 0.5 mL per point; 14 days after the first immunization, 4 points were randomly selected on the back for booster immunization. Complete adjuvant emulsified antigen, 0.5mL / dot; booster immunization once every 10 days, three consecutive booster immunizations. 10 days after the last booster immunization, select a clear vein on the ear and inject 0.1 mL of epinephrine per mouse. After 0.5 hours, inject the purified antigen directly into the ...

Embodiment 3

[0105] Example 3. Detecting β-conglycinin in samples with β-conglycinin monoclonal antibody

[0106] The monoclonal antibody of the invention can be used to detect β-conglycinin in soybeans, soybean protein extracts, soybean products, soybean milk powder, and soybean meal feed.

[0107] Based on the above prepared and purified β-conglycinin samples, monoclonal antibodies, and polyclonal antibodies, a quantitative detection method for β-conglycinin double-antibody sandwich ELISA was established. Coat the ELISA plate with the purified β-conglycinin polyclonal antibody prepared in Example 2, add the samples to be tested in sequence, prepare the purified monoclonal antibody in Example 1, goat anti-mouse IgG / HRP enzyme-labeled secondary antibody, OPD bottom solution, stop solution, and then use a microplate reader to detect the absorbance value (OD492) of the solution in each well. The purified β-conglycinin prepared in Example 1 was used as a standard substance, and the samples w...

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Abstract

The invention discloses a method for detecting beta conglycinin, the monoclonal antibody special therefor, and the kit. The kit for detecting beta conglycinin comprises beta conglycinin monoclonal antibody or polyclonal antibody, wherein the monoclonal antibody or the polyclonal antibody is obtained with the conjugate of beta conglycinin hapten and carrier protein as immunogen; and the beta conglycinin hapten is the antigen epitope peptide on alpha subunit of beta conglycinin molecule. Experiment proves that the beta conglycinin monoclonal antibody prepared by the invention method has high potency, high affinity, and strong reaction specificity, and can highly bond with the beta conglycinin without cross reaction; and when the monoclonal antibody of the invention is used for detection, itis possible to obtain wide detection range (0.002 mu g / ml-1.000 mu g / ml), high sensitivity (0.002 mu g / ml), and small coefficient of variation. The hybridoma of the invention can stably produce monoclonal antibody, thus the detection method and the monoclonal antibody special therefor of the invention are applicable to the detection of beta conglycinin in grain food, animal feed, and the like.

Description

technical field [0001] The invention relates to a method for detecting β-conglycinin, a special antibody and a kit thereof. Background technique [0002] As a high-quality vegetable protein and oil source for humans and animals, soybean has extremely high nutritional value and is widely used. The protein in soybeans accounts for about 40% of soybean grains, but it contains various anti-nutritional factors such as trypsin inhibitors, lectins, isoflavones, and antigenic proteins. Among them, soybean antigenic protein is a kind of protein in soybean that can cause allergic reactions in humans and livestock, mainly including: soybean hydrophobin (named Gly ml in immunology), soybean shell protein (Gly m2), soybean inhibitory protein (Gly m3) , glycinin (glycinin), β-conglycinin (β-conglycinin), etc. [0003] Current studies have confirmed that glycinin and β-conglycinin are the most abundant and highly immunogenic soybean antigenic proteins, and they together account for 65-80...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577C12N5/10
Inventor 马曦李德发于贵平谯仕彦贺平丽朴香淑黄承飞
Owner 北京同力兴科农业科技有限公司
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