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Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection

A comprehensive detection and injection technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inability to divide components or add immune enhancers, missing components with low content, poor fixation of haptens, etc.

Inactive Publication Date: 2013-12-11
HUNAN UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, some people propose to separate and fix the components of traditional Chinese medicine injections, and then judge with the antibody ELISA. A small molecule hapten is not easy to fix, and will be washed out during solid-phase ELISA. It is difficult to achieve the goal; at the same time, in addition to reacting with IgE in the blood, the allergens of traditional Chinese medicine injections also react with other IgG, IgA, IgM, and IgD, and the latter four are not sensitizing immunoglobulins. Therefore, there will be other interference
Now it has been changed to a single component-by-component analysis method, which aims to separate the individual components in the traditional Chinese medicine injection, and then test them one by one in a single-component mode. This is allowed for animal experiments on the allergies of traditional Chinese medicine injections, but it is not possible to test the effects of traditional Chinese medicine injections on humans. Allergen research, because Chinese medicine injections are used as a whole without adjuvants, and the components cannot be divided or added with immune enhancers for research. Such experimental results are inconsistent with the real situation of clinical use of Chinese medicine injections, so this method is also acceptable. unreasonable
In addition, if it is the sensitization caused by the metabolites of traditional Chinese medicine injection, the existing methods cannot be determined, and there may be omissions in the detection of ingredients with low content

Method used

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  • Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection
  • Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection
  • Comprehensive detection method for allergen in traditional Chinese medicine (TCM) injection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: Separating the chemical components of Shuanghuanglian injection

[0072] Shuanghuanglian injection was separated by solvent, acid-base, high-performance chromatography and other separation techniques. The separated components include chlorogenic acid, isochlorogenic acid, neochlorogenic acid, baicalein, baicalein, baicalin, wogonin, wogonin, Chrysin, dry layer paper, dry layer paper A glycoside, forsythin, forsythinin abiesinol, abiesinol-β-D-glucoside, forsythiaside A, forsythiaside C , Forsythiaside D, oleanolic acid, betulinic acid, ursolic acid, rutin, etc., and at the same time, the macromolecular polysaccharides and proteins that were removed during the preparation of Shuanghuanglian injection were separated.

Embodiment 2

[0073] Embodiment 2: the preparation of the whole antigen of known component

[0074] 1. Preparation of sugar or sugar-containing compound antigen

[0075] Accurately weigh 0.01 mmol of any sugar or glycosyl-containing compound in Example 1, add 2 ml of pyridine to dissolve, and obtain liquid A. Dissolve 0.015mmol sodium periodate in 2ml water to form B solution. Add solution B gradually to solution A, stir at room temperature for 0.5h, add 1μl of ethylene glycol to terminate the reaction; accurately weigh 65mg (0.001mmol) of bovine serum albumin (BSA), add 0.01mol / L, pH7.4PBS 5ml , this solution is called solution C.

[0076] Add the reacted solution drop by drop into solution C, stir while adding, stir at room temperature for 3h, then add 1ml of fresh aqueous solution of 0.01M sodium borohydride, stir at room temperature for 1h, after the reaction is completed, use 0.01M , pH7.4PBS dialysis for 3 days, changing the solution 3 times a day. Finally, the dialyzed solution w...

Embodiment 3

[0089] Embodiment 3: Preparation of IgE antibody

[0090] The whole antigens of the components of the Shuanghuanglian injection prepared in Example 2 were injected into New Zealand white rabbits for immunization. Take the antigens of baicalin, forsythin, forsythiaside A, forsythiaside C, forsythiaside D, and chlorogenic acid as examples. Select 21 healthy male New Zealand white rabbits and divide them into 7 groups with 3 rabbits in each group. One group was injected with normal saline as a control, and the other six groups were immunized with the above antigens respectively. Take concentration 1mg.ml -1 The above-mentioned antigen solution (dissolved with physiological saline) 2ml, add the same volume of complete Freund's adjuvant to fully emulsify and carry out the basic immunization, that is, inject 2ml (1mg) at multiple points on the back of the rabbit. After 20 days, booster immunization was carried out with the above antigen solution plus an equal volume of Freund's i...

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Abstract

The invention discloses a comprehensive detection method for an allergen in a TCM injection, and relates to the field of immunology and science of TCM. According to the invention, the method is based on combined ELISA (enzyme linked immunosorbent assay) principles and fingerprint chromatogram variance analysis, uses an immunoreaction of embedding antigens, IgE antibodies or anti-IgE antibodies with solid carriers and respectively carries out labeled immunoreaction between TCM injection component antigens and labeled anti-IgE antibodies, the embedded IgE antibodies and labeled antigens, the embedded IgE antibodies and known synthetic antigens, the embedded IgE antibodies and enzyme labeled antibodies, the embedded IgE antibodies and IgE antibodies in a blood sample captured by the embedded IgE antibodies, the labeled antigens or antigens and labeled antibodies; the captured IgE antibodies are combined with TCM injection components, and fingerprint chromatogram variance analysis is carried out by using GC / MS and HPLC / MS; specific IgE and corresponding allergens in human and animal blood samples allergic to the TCM injection are determined based on comprehensive consideration of information of a plurality of aspects. The detection method for the TCM injection provided in the invention has the characteristics of high specificity, high sensitivity, high efficiency and easy and convenient operation, is applicable to simultaneous detection of a plurality of components and can effectively prevent omission.

Description

technical field [0001] The invention relates to the fields of immunology and traditional Chinese medicine, in particular to a rapid detection method for allergens of traditional Chinese medicine injections. Background technique [0002] Chinese medicine injection is a new dosage form of modern Chinese medicine unique to my country, and has been widely used clinically in my country. With the increasing clinical application of traditional Chinese medicine injections, there are more and more reports on adverse reaction (ADR) and fatal cases of traditional Chinese medicine injections, and allergic reactions are one of the common adverse reactions. [0003] Allergic reactions caused by traditional Chinese medicine injections are mainly divided into type I (immediate type), type II (cytotoxic type), type III (immune complex type), and type IV (delayed type) allergic reactions. Among them, type I allergic reaction mediated by IgE is the most common type of allergic reaction caused...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543
Inventor 贺福元邓凯文邱云陈锋唐昱曾姣丽刘文龙石继连戴儒文
Owner HUNAN UNIV OF CHINESE MEDICINE
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