Method for detecting mite allergen specific antibody in blood serum

An allergen-specific technology, applied in the field of diagnosis, can solve the problems of low sensitivity and accuracy, and achieve the effects of improving sensitivity, improving detection efficiency, and shortening detection time

Inactive Publication Date: 2012-01-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 mite allergen specific antibody in blood serum
  • Method for detecting mite allergen specific antibody in blood serum
  • Method for detecting mite allergen specific antibody in blood serum

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Sodium oleate-coated Fe was prepared using a conventional chemical co-precipitation method 3 o 4 Magnetic fluid: prepare 0.12mol / L ferrous chloride (FeCl 2 .4H 2 O) and 0.2mol / L ferric chloride (FeCl 3 .6H 2 O) 25 mL each, transferred to a 250 mL three-neck flask, stirred under nitrogen protection, and the stirring speed was 500 rpm. After raising the temperature of the above mixture to 55°C for 15 minutes, quickly add 3mol / L NaOH to adjust the pH value to 10-12. The temperature of the mixture was raised to 65° C., and the stirring was maintained for 40 min. Then slowly add 4% sodium oleate 50mL, raise the temperature to 90°C, continue to stir for 30min, and cool to room temperature to obtain Fe coated with sodium oleate. 3 o 4 ferrofluid. Fe of the ferrofluid 3 o 4 The particles are superparamagnetic, with an average particle size of 30-50nm.

[0020] Preparation of carboxyl-modified magnetic microspheres by dispersion polymerization: take 4mL of the above-m...

Embodiment 2

[0022] Coupling mite allergen protein to prepare immunomagnetic microspheres.

[0023] (1) Using 25mM MES (pH=6) as a buffer solution, wash the magnetic microspheres (with a concentration of 10mg / mL) twice. Dissolve the mite allergen protein with the above-mentioned buffer solution;

[0024] (2) Use freshly prepared EDC and NHS as the cross-linking agent, the concentration of EDC and NHS is 50 mg / mL, add a certain volume of cross-linking agent to the washed magnetic microspheres, and add 20 μL of cross-linking agent to each mg of magnetic microspheres. Combined agent, incubated with tilt rotation at room temperature for 30min to activate the magnetic microspheres;

[0025] (3) The activated magnetic microspheres were washed twice with MES buffer, then an appropriate amount of mite allergen protein was added (the mass ratio of magnetic microspheres to mite allergen protein was 15:1), vortexed and incubated at room temperature for 1 -2h;

[0026] (4) Wash the magnetic microsp...

Embodiment 3

[0030] (1) Cleaning and activation of immunomagnetic microspheres: Take the immunomagnetic microspheres prepared and preserved above in a clean centrifuge tube, add PBS activation buffer containing 0.1% BSA in a volume ratio of 1:10, and mix well. Magnetically separate and remove buffer. Wash once more in this way, and then add the above-mentioned activation buffer to make up to the original demand.

[0031] (2) Sample incubation: Take 0.2 μL, 0.5 μL, 1 μL, 2 μL, and 4 μL of the above-mentioned cleaned and activated immunomagnetic microspheres (10 mg / mL), mix each with 100 μL of standard mite serum samples in each well of the microplate, and gently Shake gently and incubate with shaking for 15-20 minutes at room temperature.

[0032](3) Plate washing: After the incubation, magnetically separate, keep the supernatant in a new centrifuge tube for detection, add 200 μL of PBST buffer to each well to wash the plate, remove the supernatant after magnetic separation, and wash 3 tim...

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Abstract

The invention discloses a method for detecting mite allergen specific antibody in blood serum. The method comprises the following steps of: coupling mite allergen with surface carboxyl modified magnetic microspheres to prepare immunomagnetic microspheres; and mixing and incubating the blood serum to be detected and the immunomagnetic microspheres in holes of an elisa (enzyme-linked immunosorbent assay) plate to combine mite allergen with mite allergen specificity IgE in the blood serum, and detecting by adopting an enzyme-linked immunosorbent method after the elisa plate is washed. In the method disclosed by the invention, a mite allergen is directly coupled onto the surfaces of magnetic microspheres, thus all the impurities except the mite allergen specific antibody in the blood serum can be conveniently removed; and targeted mite allergen specificity antibody detection is carried out, detection time is shortened, and sensitivity, specificity and accuracy of detection are improved. The mite allergen immunomagnetic microspheres prepared by the invention can be used in standard automatic detection to improve detection efficiency.

Description

technical field [0001] The invention relates to the technical field of diagnosis, in particular to a method for detecting mite 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 aller...

Claims

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Application Information

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