Method for detecting allergen-specific antibody in serum

An allergen, specific technology, applied in the field of diagnosis, can solve the problem of low sensitivity and accuracy, and achieve the effect of improving sensitivity

Inactive Publication Date: 2011-11-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention provides a method for detecting mite allergen-specific antibodies in serum, which solves the problem of low sensitivity and accuracy of traditional methods

Method used

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  • Method for detecting allergen-specific antibody in serum
  • Method for detecting allergen-specific antibody in serum
  • Method for detecting allergen-specific antibody in serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 5mmol FeCl 3 .6H 2 0, 3mmol citric acid and 20mmol urea were dissolved in 20mL ethylene glycol solvent, the mixed solution was placed in a 30mL hydrothermal kettle, and heated at 200°C for 6 hours, and the black precipitate was washed 3 times with ethanol and deionized water respectively, and dissolved In deionized water and adding 0.02% (w / v) sodium azide for long-term preservation, the prepared figure 1 The particle size shown is about 200nm, the surface small molecule carboxyl group modified hydrophilic monodisperse Fe 3 o 4 magnetic microspheres.

Embodiment 2

[0022] Immunomagnetic microspheres were prepared by coupling goat anti-human IgE antibody.

[0023] (1) Using 25mM MES (pH=6) as the buffer, take 9 μL, 15 μL, 30 μL, 90 μL of magnetic microspheres (concentration: 10 mg / mL), wash twice, and rotate for 10 min;

[0024] (2) Use freshly prepared EDC and NHS solutions as cross-linking agents, the concentration of EDC and NHS is 50mg / mL, and the solvent is 25mM MES (pH=6). Add 20 μL of cross-linking agent to each mg magnetic microsphere, and incubate with tilt rotation for 30 min at room temperature to activate the magnetic microsphere;

[0025] (3) Wash the activated magnetic microspheres twice with MES buffer, add 24 μL of goat anti-human IgE antibody (2.5 mg / mL), continue to add 6 μL of MES, vortex and incubate at room temperature for 1-2 hours;

[0026] (4) After incubation, magnetically separate, remove the supernatant and put it in a new tube (reserved for measuring the coupling rate); coupling rate (%) = (concentration of mo...

Embodiment 3

[0032] The 200nm immunomagnetic microspheres (15:1) prepared in Example 2 were used to capture the specific IgE antibody in the serum sample, and the detection effect of the method using the immunomagnetic microspheres on mite-specific sIgE was analyzed. The specific operation is as follows:

[0033] Take the immunomagnetic microspheres in a clean centrifuge tube according to the demand, add the buffer solution according to the volume ratio of 1:10 (10 μL immunomagnetic microspheres plus 100 μL activation buffer), and mix well. Magnetically separate and remove buffer. Wash again in this way, and then add activation buffer to make up to the original demand.

[0034] The serum samples containing mite-specific IgE (sIgE) to be tested were diluted 1-fold, 3-fold, 5-fold, 10-fold, 100-fold, and 1000-fold respectively for later use. Add 2 μL of activated immunomagnetic microspheres (concentration: 5 mg / mL) to each centrifuge tube, then add 100 μL of the above-mentioned serum sampl...

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Abstract

The invention discloses a method for detecting allergen-specific antibodies in serum. The method comprises the following steps: coupling anti-human IgE antibodies with carboxyl surface modified magnetic microballoons to obtain immunomagnetic microspheres; incubating the immunomagnetic microspheres with serum to be measured so as to enable the immunomagnetic microspheres to bind to IgE in the serum to be measured; carrying out magnetic separation to obtain immunomagnetic microsphere-IgE conjugates, dissolving deposition of the magnetic separation in a buffer solution, adding the mixed solution into apertures of an enzyme label plate which is coated with allergen, and carrying out detection by the ELISA adsorption method after incubation and plate washing. According to the invention, magnetic microballoons are coupled with anti-human IgE antibodies to prepare immunomagnetic microspheres which are mixed with serum to be measured for incubation and are bond to all the IgE in serum; the immunomagnetic microspheres are enriched and IgE is separated; the ELISA adsorption method is employed to detect whether there is specific IgE bond to allergen in serum. The method provided in the invention enables all the IgE to be separated from serum through immunomagnetic microspheres, impurities to be removed, and sensitivity, specificity and accuracy of ELISA adsorption detection to be improved.

Description

technical field [0001] The invention relates to the technical field of diagnosis, in particular to a method for detecting allergen-specific antibodies in serum. Background technique [0002] The incidence of allergic diseases is increasing year by year in modern society. About 5-10% of people in my country and 5-25% of people in developed countries such as the United States and Europe suffer from allergic diseases. Anaphylaxis, also known as allergic reaction, is a common immune disease in humans. But it will cause a series of clinical reactions, involving gastrointestinal tract, skin, respiratory tract and even more dangerous other reactions, so it should be paid enough attention. The antigens that induce allergic reactions are called allergens. There are more than 2,000 common antigenic substances that cause allergic reactions, and nearly 20,000 kinds are recorded in medical literature. Allergens can be divided into 3 categories according to the way they cause allergies:...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/68G01N33/531
Inventor 高其康吴善东楼兵干赵铖铖
Owner ZHEJIANG UNIV
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