A method for detecting serum allergen-specific IGE based on microfluidic chip and protein chip

A protein chip and microfluidic chip technology, applied in the field of biomedicine, can solve the problems of immune response, failure to reach blood volume, limited blood volume, etc., to reduce usage, suitable for large-scale epidemiological analysis, and reduce usage The effect of volume and detection time

Active Publication Date: 2018-01-16
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 safety and accurate and reliable test results, and is known internationally as "The gold standard for allergen detection", however, the practice shows that the disadvantage of this method is that the detection requires a relatively large amount of serum, 40 μl of serum is required to detect a sample, especially for infants and young children, the most convenient way is usually to draw fingertip blood or earlobe For blood testing, due to the limited amount of blood taken, the blood volume required by the UniCAP method cannot be achieved.

Method used

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  • A method for detecting serum allergen-specific IGE based on microfluidic chip and protein chip
  • A method for detecting serum allergen-specific IGE based on microfluidic chip and protein chip
  • A method for detecting serum allergen-specific IGE based on microfluidic 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 group of negative BSA) diluted into 4 gradients, respectively 1.5mg / ml, 1mg / ml, 0.4mg / ml, 0.04mg / ml. Added 25% of Glycerol, take 40ul of each diluted sample into a 384-well plate, and centrifuge at 4000 rpm for 1min;

[0043] (2) Spotting with a spotting machine, spotting on a solid phase (glass) protein carrier, and repeating spotting 3 times for each sample;

[0044] (3) After spotting the samples, put them in the refrigerator at 4°C overnight, and store them at -80°C the next day for later use.

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, blow dry with nitrogen, and bake at 200°C for 30 minutes; Pour AZ-50XT glue (Microchem Company) on the center of the silicon wafer; use a glue spreader to spin coat at 3000 rpm for 1 minute to make the glue evenly distributed, let it stand for 10 minutes, and then dry it softly on a constant temperature heating plate. Place at 65°C, 95°C, and 65°C for 3 minutes, 6 minutes, and 3 minutes, respectively;

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

[0048] (3) Developing: Put the silicon wafer into the developing solution for developing, and the developing i...

Embodiment 3

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

[0054] The bonded microfluidic chip and protein chip were blocked with PBS containing 3% BSA for 1 hour, and 1 μl (1:35 dilution) of serum from an allergic patient was pumped into the reaction chamber of the microfluidic chip, and fixed on the solid phase glass carrier. The allergen will bind to the specific IgE antibody in the serum. After washing with PBS, add 1 μl of goat anti-human IgE antibody (1:300 dilution) and incubate for 1 hour, then wash with PBS, add cy3-labeled donkey anti-goat antibody for incubation ( 1:1000 dilution) for 1 hour, after washing with PBS, the microfluidic chip and the protein chip were separated, and the protein chip was 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 entire detection system is mainly the control part and data analysis (comp...

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Abstract

The invention belongs to the field of biomedicine and relates to a method for detecting serum allergen-specific IgE based on a microfluidic chip and a protein chip. The present invention utilizes microfluidic microfluidic and rapid analysis capabilities, high-throughput analytical capabilities of protein chips, and the principle of specific immune reactions combined with antigens and antibodies, and through the combination of microfluidic chips and protein chips, it can treat clinical serum of allergic patients. The samples were tested for allergen-specific IgE, and the results showed that this method can effectively reduce the amount of serum testing used by patients, and is suitable for patients with a small amount of peripheral blood such as infants or children, while reducing the amount of testing reagents used and testing Time, compared with the existing technology, has the characteristics of rapidity, high throughput, saving detection serum, high efficiency, portability, and low cost. Especially suitable for large-scale epidemiological analysis.

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 allergy, that is, an allergic reaction in which foreign antigens are interpreted as harmful objects (eg, bacteria, pollen, dust mites, cockroaches, dust, etc.). Allergic reactions activate macrophages in immune cells and release histamine and prostaglandins, which can cause a series of effects such as microvascular dilation, increased vascular permeability, itching, smooth muscle contraction and reflex action; The symptoms of clinical manifestations are mainly allergic rhinitis, allergic asthma, allergic gastroenteritis, eczema, urticaria, macule, papules, scratches, atopic dermatitis, wheal, skin itching and other allergic skin sick. Allergic asthma is a relatively stubborn dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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