Serum allergen specific IgE detection method based on micro-fluidic chip and protein chip

A protein chip and microfluidic chip technology, applied in the field of biomedicine, can solve the problems of inability to achieve blood volume, limited blood collection, immune response, etc., to achieve large-scale epidemiological analysis, reduce the amount of use and detection time. , the effect of reducing usage

Active Publication Date: 2016-01-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Skin pricking is currently a clinically used in vivo allergen detection method, but this method has disadvantages, such as, it may cause an immune response in the body, and the detection time is long, etc.; the UniCAP automatic in vitro diagnostic system developed by the Swedish Pharmacia Company is At present, the most advanced laboratory system for checking allergens in the world has been confirmed by the International Clinical Laboratory Standards Committee. This method has been confirmed by the World Health Organization for its high

Method used

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  • Serum allergen specific IgE detection method based on micro-fluidic chip and protein chip
  • Serum allergen specific IgE detection method based on micro-fluidic chip and protein chip
  • Serum allergen specific IgE detection method based on micro-fluidic chip and protein chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Preparation of protein chip

[0042] (1) Recombinant antigen (rDerf 2 , RBt5, rRcla, rPlaa1, rHumj, a set of negative BSA) diluted into 4 gradients, respectively 1.5mg / ml, 1mg / ml, 0.4mg / ml, 0.04mg / ml. 25% of each sample was added Take 40ul of glycerol and diluted sample into a 384-well plate, centrifuge at 4000 rpm for 1 min;

[0043] (2) Use the spotting machine to spot samples, spot them on the solid phase (glass) protein carrier, and repeat the spot 3 times for each sample;

[0044] (3) After spotting, place it in a refrigerator at 4°C overnight, and store it at -80°C the next day.

Embodiment 2

[0045] Example 2 Preparation of microfluidic chip

[0046] (1) Substrate preparation: Put the silicon wafer into Piranha solution (98% concentrated sulfuric acid: 30% hydrogen peroxide = 7:3) and boil for 15 minutes; rinse with deionized water and blow dry with nitrogen, and bake at 200°C for 30 minutes; Pour the AZ-50XT glue (Microchem Company) on the center of the silicon wafer; spin-coat at 3000 revolutions / min for 1 minute with a homogenizer to make the glue distribution more uniform, let it stand for 10 minutes, and then soft-bake on a constant temperature heating plate. Place at 65℃, 95℃, 65℃ for 3min, 6min, 3min respectively;

[0047] (2) Exposure: Place the designed silicon wafer templates of the fluid layer and the valve control layer on the baked substrates, expose them with an ultraviolet exposure machine, and then put them on the heating plate for baking;

[0048] (3) Development: Put the silicon wafer in a developer solution for development. The development is carried o...

Embodiment 3

[0053] Example 3 Serological detection of allergy patients based on microfluidic chip

[0054] The bonded microfluidic chip and protein chip were sealed with PBS containing 3% BSA for 1 hour. 1μl of serum from allergy patients (diluted 1:35) was pumped into the microfluidic chip reaction chamber and fixed on a solid glass carrier. The allergen will bind to the specific IgE antibody in the serum. After washing with PBS, add 1μl of goat anti-human IgE antibody (diluted at 1:300) and incubate for 1 hour, then wash with PBS, add cy3 labeled donkey anti-goat antibody and incubate ( 1:1000 dilution) for 1 hour, after washing with PBS, the microfluidic chip and protein chip are separated, and the protein chip is placed on the chip scanner to collect fluorescence signals for data analysis; one microfluidic chip can detect eight serums at the same time , The hardware part of the whole detection system is mainly the control part and data analysis (computer), operating system (integrated mi...

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Abstract

The invention belongs to the field of biomedicine, and relates to a serum allergen specific IgE detection method based on micro-fluidic chip and protein chip. Micro fluidic chip only need a trace amount of sample and is capable of quickly carrying out analysis; protein chip can perform high-flux analysis, and the provided method adopts the micro fluidic chip and protein chip and is developed on an antigen-antibody specific immunity response principle. The micro fluidic chip and protein chip are combined together to detect the allergen specific IgE in a clinical serum sample of an allergy sufferer. The results show that the provided method can effectively reduce the using amount of serum for detection. So the method is suitable for infants or children, who have little peripheral blood. Moreover, the using amount of detection reagents and detection time are both reduced. Compared with the prior art, the provided method has the characteristics of rapidness, high flux, reduced amount of serum, high efficiency, portability, and low cost. The method is especially suitable for analysis of large-scale epidemic disease.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and relates to a serum immunological detection method, in particular to a method for detecting serum allergen-specific IgE based on a microfluidic chip and a protein chip. Background technique [0002] The prior art discloses allergies, that is, allergic reactions caused by the interpretation of foreign antigens as harmful objects (such as bacteria, pollen, dust mites, cockroaches, dust, etc.). Allergies activate macrophages in immune cells and release histamine and prostaglandin. Histamine and prostaglandin will cause a series of effects such as microvascular expansion, increased vascular permeability, itching, smooth muscle contraction and reflex; The clinical symptoms are mainly allergic rhinitis, allergic asthma, allergic gastroenteritis and eczema, urticaria, macula, papulosis, scratch syndrome, atopic dermatitis, wind rash, skin itching and other allergic skin disease. Allergic asthma is a relativel...

Claims

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Application Information

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IPC IPC(8): G01N35/00G01N33/96
Inventor 隋国栋程训佳郑璐璐付永锋冯萌荆雯雯
Owner FUDAN UNIV
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