CN107247149A - A kind of dermatophagoides pteronyssinus specific IgE half-quantitative detection kit and application thereof - Google Patents

Abstract

The invention discloses a semi-quantitative detection kit for house dust mite-specific IgE and its application. The test kit includes a fluorescent liquid for detection and an immunochromatographic test strip for semi-quantitative detection of house dust mite-specific IgE; wherein, the fluorescent liquid for detection is obtained by adding carboxyl-containing quantum dot nanospheres on the surface, respectively. Covalently coupled with goat anti-human sIgE, mouse anti-rabbit IgG, and prepared after mixing; the immunochromatography test strip includes sequentially connected sample pads, nitrocellulose membranes, water-absorbing pads, and an assembly platform for providing On the lower plastic backing, the nitrocellulose membrane has a test line coated with dust mite allergen protein and a quality control line coated with rabbit IgG, and the sample pad provides a place for the sample to be tested to be added. The semi-quantitative detection of house dust mite-specific IgE can be realized by using the kit established by the present invention, which has a good correlation with clinical symptoms, high detection sensitivity, easy to use, and is suitable for large-scale population allergen screening and family detection Wait for the environment.

Description

A semi-quantitative detection kit for house dust mite-specific IgE and its application

technical field

The invention relates to a kit for detecting house dust mite-specific IgE and a detection method thereof, in particular to a house dust mite-specific IgE semi-quantitative detection kit and its method based on fluorescent quantum dot immunochromatography test paper and image analysis technology Detection method. The invention belongs to the technical field of immune analysis.

Background technique

House dust mite is one of the most important inhalant allergens, and it is ubiquitous in indoors. House dust mite and farinae farinae are the most important two types of mites. Among them, House Dust mite (HDM) is Oblong, with a body length of about 240-280 μm, also known as European dust mite, is the hottest allergenic mite species in Eurasia. Surveys in Shanghai, Beijing, Guangzhou, Harbin and other places have shown that household dust mites are also the dominant mite species in our country, and they exist on beds, quilts, pillows, carpets, sofas, etc. in bedrooms. The dominant species in the south of the Yangtze River in my country has a lower density in the north.

House dust mite allergy means that after exposure to house dust mite allergens, the body can produce more dust mite-specific IgE antibodies, which can penetrate into mucosal tissues and associate with corresponding antigens on the surface of mast cells and basophils. Combined, making it a sensitized tissue. The occurrence, development and persistence of symptoms of most allergic asthma are closely related to dust mite allergy. Studies have shown that 80% of asthmatics have a positive dust mite prick test, while the positive rate of normal people is 5%-30%. Epidemiological surveys around the world have shown that dust mite is the most important allergen, and the occurrence, development, acute onset and persistence of most allergic asthma are closely related to dust mite allergy. Dust mites are also the most important allergens of allergic rhinitis. After the nasal cavity is stimulated by dust mites, there will be acute and delayed reactions, which can further develop into nasal polyps through long-term induction of nasal mucosal inflammation. Allergic rhinitis is a global health problem. It is common all over the world. Its global disease rate reaches 10%-25%, and the number of patients is still increasing. It can affect patients' daily life, study and work efficiency, and cause serious economic burden. In addition, dust mites are considered to be one of the most important allergens in atopic dermatitis, and the degree of allergy to dust mites is closely related to the severity of atopic dermatitis. Dust mites can cause atopic dermatitis in two ways: directly through the skin and through inhalation of dust mite allergies.

The traditional method for detecting house dust mite-specific IgE is immunoblotting, but this method takes a long time, has complicated steps, and requires special instruments to complete.

Immunoassay technology is a detection method based on the principle of specific reaction between antibodies and antigens for qualitative and quantitative analysis of the test object. The immunoassay of low-concentration antigens is still an important problem in the field of clinical diagnosis and inspection and quarantine. The accuracy and accuracy of low-concentration antigens Reliable detection can provide an important scientific basis for early diagnosis and timely treatment of diseases, and one of the methods to improve the limit of immune detection is to prepare highly sensitive immune probes. Fluorescent immunoassay technology uses fluorescent substances to label antibodies or antigen molecules, and detects the intensity changes of fluorescent signals after specific binding to the analyte to achieve qualitative and quantitative detection of the target analyte. As a new type of fluorescent nanocrystal, quantum dots (QDs) have some unique fluorescent properties, such as controllable fluorescence emission wavelength, narrow and symmetrical emission peak, wide excitation wavelength range, high quantum efficiency, and good photostability. , the above fluorescent properties of quantum dots provide a good choice for the preparation of highly sensitive immunofluorescent probes (see literature: J.Lei, H.Ju, Signal amplification using functional nanomaterials for biosensing, Chem.Soc.Rev.2012,41 , 2122-2134).

In view of this, the present invention proposes a method and kit for semi-quantitative detection of house dust mite sIgE based on fluorescent quantum dot immunochromatography test paper and image analysis technology. The method is convenient, fast, and does not require special detection instruments. It satisfies the current urgent need for the detection of house dust mite-specific IgE, and is especially suitable for environments such as large-scale population allergen screening and home testing.

Contents of the invention

The purpose of the present invention is to overcome the problems of time-consuming, complicated steps, special instruments and other problems in the current detection method of house dust mite sIgE, and provide a semi-quantitative detection of house dust mite sIgE based on fluorescent quantum dot immunochromatography test paper and image analysis technology methods and kits.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

Quantum dot nanospheres with carboxyl groups on the surface were covalently coupled with goat anti-human sIgE and mouse anti-rabbit IgG to prepare a detection fluorescent solution; dust mite antigen and rabbit IgG were immobilized on the nitrocellulose membrane as T line and C line, respectively. Then assemble with sample pad, absorbent pad and plastic backboard to get test strip. After the sample is mixed and reacted with the fluorescent solution, the fluorescent solution is added to the sample pad to react with the T-line and C-line through chromatography; when there is dust mite sIgE in the sample, the fluorescent quantum dots will be specifically captured by the dust mite antigen on the T-line , the fluorescence intensity of T-line quantum dots is proportional to the concentration of dust mite sIgE in the sample. Fluorescent test strips were photographed with a digital camera under ultraviolet light, and the images were separated by RGB color channels with ImageJ software, and gray-scale intensity integration was performed to achieve semi-quantitative detection of sIgE. The developed detection method can semi-quantitatively detect sIgE in the range of 0.2-10U/mL.

A semi-quantitative detection kit for house dust mite-specific IgE of the present invention, comprising a detection fluorescent liquid and an immunochromatographic test strip for semi-quantitative detection of house dust mite-specific IgE;

Wherein, the fluorescent detection liquid is prepared by covalently coupling quantum dot nanospheres with carboxyl groups on the surface with goat anti-human sIgE and mouse anti-rabbit IgG respectively, and mixing them to obtain the fluorescent detection liquid;

Wherein, the immunochromatographic test strip includes sequentially connected sample pads, nitrocellulose membranes, water-absorbing pads, and a plastic substrate underneath for providing an assembly platform. The detection line coated with original protein and the quality control line coated with rabbit IgG, the sample pad provides the position where the sample to be tested is added.

In the kit of the present invention, preferably, the detection fluorescent liquid is prepared according to the following method:

(1) Quantum dots are coupled with mouse anti-rabbit IgG antibody to prepare calibrated quantum dot fluorescent antibody markers:

A. Take 100 μl of quantum dot nanospheres, disperse them in 200 μl of phosphate buffer, centrifuge at 10,000 rpm for 10 minutes, carefully discard the supernatant, then add 200 μL of phosphate buffer, and disperse evenly to obtain a solution of quantum dot nanospheres;

B. Weigh 1.9 mg of EDC, dissolve in 1 mL of phosphate buffer, and mix well to obtain a 10 mM EDC solution;

C. Take 20 μl of EDC solution, add it to 200 μl of quantum dot nanosphere solution, mix well, and incubate at room temperature for 0.5 h to obtain activated quantum dot nanospheres;

D. Centrifuge the activated quantum dot nanospheres at 10000rpm for 10min, discard the supernatant, then add 200μl of 10mM, pH8.0 boric acid buffer, and disperse evenly by ultrasonic;

E. Take 50 μl of mouse anti-rabbit IgG antibody, add it to the activated quantum dot nanosphere solution, mix well, and then continue to incubate at room temperature for 1 hour;

F. Take 20 μl of 10w/w% BSA solution, add it to the nanosphere solution in step E, and continue to react for 1 hour;

G, the sealed quantum dot nanosphere solution, centrifuged at 10000rpm for 10min, then centrifuged and washed three times with PBST buffer, and finally dispersed in 100 μl of 10mM containing 1w/w% BSA, pH6.0 PBS buffer, ready for use;

(2) Quantum dots are coupled with goat anti-human sIgE to prepare quantum dot-anti-IgE fluorescent antibody markers;

A. Take 100 μl of quantum dot nanospheres, disperse them in 200 μl of 10 mM, pH 6.0 phosphate buffer, centrifuge at 10,000 rpm for 10 minutes, carefully discard the supernatant, then add 200 μL of phosphate buffer, and disperse evenly to obtain quantum dot nanospheres solution;

B. Weigh 1.9 mg of EDC, dissolve in 1 mL of phosphate buffer, and mix well to obtain a 10 mM EDC solution;

C. Take 20 μl of EDC solution, add it to 200 μl of quantum dot nanosphere solution, mix well, and incubate at room temperature for 0.5 h to obtain activated quantum dot nanospheres;

D. Centrifuge the activated quantum dot nanospheres at 10000rpm for 10min, discard the supernatant, then add 200μl of 10mM, pH8.0 boric acid buffer, and disperse evenly by ultrasonic;

E. Take 50 μl of goat anti-human sIgE antibody, add it into the activated quantum dot nanosphere solution, mix well, and then continue to incubate at room temperature for 1 hour;

F. Take 5 μl of 10w/w% BSA solution, add it to the nanosphere solution in step E, and continue to react for 1 hour;

G, the sealed quantum dot nanosphere solution, centrifuged at 10000rpm for 10min, then centrifuged and washed three times with PBST buffer, and finally dispersed in 100 μl of 10mM containing 1w/w% BSA, pH6.0 PBS buffer, ready for use;

(3) Mix the quantum dot nanosphere solutions covalently coupled with mouse anti-rabbit IgG and goat anti-human sIgE respectively obtained in step (2) and step (3) according to equal volumes to obtain the product.

In the kit of the present invention, preferably, the sample pad is obtained after being treated with a sample pad treatment solution, and the treatment solution contains 1.0w/w% BSA, 0.25v/v% Tween- 20. 0.01 M pH 8.0 sodium borate buffer with 1 w/w % sucrose and 0.1 w/w % NaN3.

In the kit of the present invention, preferably, the sample pad is treated by first soaking the sample pad in the sample pad treatment solution for 20-60 minutes, taking it out and drying it in a drying oven at 30-45°C for 9-15 hours, Instantly.

In the kit of the present invention, preferably, the kit also includes a fluorescent diluent, a calibrator, and a diluent of the calibrator, and the fluorescent diluent is 10 mM containing 1w/w% BSA, pH6 .0 PBS buffer, the calibrator is human anti-dust mite allergy protein specific IgE, and the diluent of the calibrator is 10mM, pH6.0 in PBS buffer.

When using the kit of the present invention for the detection of human anti-dust mite allergy protein-specific IgE, the following steps are included:

A. Take the detection fluorescent solution and dilute it with fluorescent diluent according to the volume of 40-120 times;

B. The diluted fluorescent solution is divided into 120μL each, sealed and stored at 2-8°C, and the calibration product is diluted with the calibration product diluent;

C. Take one piece of diluted fluorescent solution, then add 10 μL of calibrator, shake and mix gently;

D. Take 80 μL of the fluorescent solution after the reaction, add it to the sample pad of the immunochromatography test strip, and start timing;

E. After chromatographic reaction for 15 minutes, place the immunochromatographic test paper in a dark room, and take pictures to obtain fluorescent images under ultraviolet light;

F. Use the calibrator with multiple dilutions, perform detection according to steps B-E, and establish a calibration curve;

G, the fluorescence image that obtains, adopt software ImageJ to carry out RGB color channel separation;

H. Using the red channel fluorescence picture, the image pixel intensity analysis is performed on the detection area of the chromatography test paper;

1. Carry out pixel intensity integration to the detection area and the control area of the chromatography test paper respectively, calculate the T/C ratio, detect the calibration curve and the calibration equation according to the concentration and the T/C value of the calibrator;

J. The human serum sample is detected by the steps of B-I, the T/C value is obtained, and the concentration of IgE in the serum is calculated by the calibration equation.

Furthermore, the present invention also proposes the use of the kit in the preparation and detection of human anti-dust mite allergy protein-specific IgE reagents.

Compared with the prior art, the present invention has the following advantages:

1. The present invention uses fluorescent quantum dot immunochromatography test paper and image analysis technology to detect house dust mite-specific IgE. The method has the advantages of simple operation, fast detection and no need for special equipment.

2, the present invention compares with the sIgE detection method of traditional ImmunoCap, the detection result to the serum of 56 routine positive patients and 40 routine healthy control groups shows that its positive coincidence rate is 92.9% (52/56), and the negative coincidence rate is 90.4% % (38/42). It shows that the detection results of quantum dot immunochromatography have a good correlation with clinical symptoms, and the detection sensitivity is high.

3. The method for detecting household dust mite sIgE established by the present invention has the characteristics of simplicity, rapidity, and convenience in use, and is especially suitable for large-scale population allergen screening and home detection environments.

Description of drawings

Figure 1 is the characterization of quantum dot nanospheres and antibody conjugates.

(A) Transmission electron micrograph of quantum dot nanospheres; (B) quantum dots in nanospheres under high-magnification transmission electron microscopy; (C) particle size and particle size distribution of quantum dot nanospheres and antibody conjugates; (D ) and (E) the comparison of the UV-Vis absorption and fluorescence spectra of quantum dot nanospheres and antibody conjugates respectively;

Fig. 2 is the structure (A) of quantum dot nanosphere immunochromatographic test paper and the positive (B) and negative (C) result schematic diagram of chromatographic test paper detection dust mite-specific IgE;

Fig. 3 is the RGB color fluorescent photo (A) and the blue (B) after RGB channel separation of the dust mite-specific IgE human serum (0, 0.2, 1.0, 5.0, 10, 20 U/mL) of the detection ratio dilution, Green (C) and red (D) channel photos.

Figure 4 is the pixel intensity distribution diagram (A) of the test strip detection area and the intensity distribution diagram (B) after baseline processing; the relationship between the T/C ratio and the IgE concentration calculated after integrating the signal intensities of the control and detection areas Relationship (C); normal (D) and exponential (E) calibration curves of dust mite-specific IgE detected by quantum dot immunochromatography.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

The methods used in the following examples are conventional methods unless otherwise specified.

The reagents and instruments used in the examples are as follows: Quantum dot nanospheres (QDNs, 610nm, particle size about 100nm), purchased from Shanghai Kundao Biological Company, 1-ethyl-3-[3-dimethylaminopropyl ] Carbodiimide hydrochloride (EDC), molecular weight 191.7, was purchased from Sigma-Aldrich Company, mouse anti-rabbit IgG was purchased from Wuhan Boster Company, bovine serum albumin (BSA) was purchased from Sigma-Aldrich Company, goat anti-human IgE Purchased from Sigma-Aldrich Company, nitrocellulose membrane (NC membrane) was purchased from Millipore Company, glass fiber sample pad was purchased from Shanghai Jinbiao Biology, absorbent pad was purchased from Shanghai Jinbiao Biology, adhesive backing (with pressure sensitive adhesive) PVC board) was purchased from Shanghai Jinbiao Biotech, and dust mite allergen protein (1 mg/mL) Dermatophagoides pteronyssinus allergen 1 (nDer p 1) was purchased from Indoor Biotechnologies. Sample pad treatment solution:

Sample pad treatment solution A: 0.01M pH 8.0 sodium borate buffer solution containing 1.0% BSA, 0.25% Tween-20 and 0.1% NaN3.

Sample pad treatment solution B: 0.01M pH 8.0 sodium borate buffer containing 1.0% BSA, 0.25% Tween-20, 1% sucrose and 0.1% NaN3.

Sample pad treatment liquid C: 0.01M pH 8.0 sodium borate buffer containing 1.0% BSA, 0.25% Tween-20, 3% sucrose and 0.1% NaN3.

Example 1: Preparation of calibration quantum dot fluorescent antibody markers

A. Take 100 μl of quantum dot nanospheres, disperse them in 200 μl of phosphate buffer (10 mM, pH 6.0), centrifuge at 10,000 rpm for 10 min, carefully discard the supernatant, and then add 200 μL of phosphate buffer (10 mM, pH 6.0), Uniformly disperse to obtain quantum dot nanosphere solution;

B. Weigh 1.9 mg of EDC, dissolve in 1 mL of phosphate buffer, and mix well to obtain a 10 mM EDC solution;

C. Take 20 μl of EDC solution, add it to 200 μl of quantum dot nanosphere solution, mix well, and incubate at room temperature for 0.5 h;

D. The activated quantum dot nanospheres were centrifuged at 10,000rpm for 10min, the supernatant was discarded, and then 200μl of boric acid buffer (10mM, pH8.0) was added, and dispersed evenly by ultrasonication;

E. Take 50 μl of mouse anti-rabbit IgG, add it to the activated quantum dot nanosphere solution, mix well, and then continue to incubate at room temperature for 1 hour;

F. Take 20 μl of 10w/w% BSA solution, add it to the above nanosphere solution, and continue to react for 1 hour;

G, the sealed quantum dot nanosphere solution, centrifuged at 10000rpm for 10min, then centrifuged and washed three times with PBST buffer, and finally dispersed in 100 μl of 1w/w% BSA in PBS buffer (10mM, pH6.0) for use .

Embodiment 2: Preparation of quantum dot-anti-IgE fluorescent antibody marker

A. Take 100 μl of quantum dot nanospheres, disperse them in 200 μl of phosphate buffer (10 mM, pH6.0), centrifuge at 10,000 rpm for 10 minutes, carefully discard the supernatant, then add 200 μL of phosphate buffer, and disperse evenly to obtain quantum dot nanospheres. ball solution;

B. Weigh 1.9 mg of EDC, dissolve in 1 mL of phosphate buffer, and mix well to obtain a 10 mM EDC solution;

C. Take 20 μl of EDC solution, add it to 200 μl of quantum dot nanosphere solution, mix well, and incubate at room temperature for 0.5 h;

D. The activated quantum dot nanospheres were centrifuged at 10,000rpm for 10min, the supernatant was discarded, and then 200μl of boric acid buffer (10mM, pH8.0) was added, and dispersed evenly by ultrasonication;

E. Take 50 μl of goat anti-human IgE, add it to the activated quantum dot nanosphere solution, mix well, and then continue to incubate at room temperature for 1 hour;

F. Take 5 μl of 10w/w% BSA solution, add them to the above nanosphere solution respectively, and continue to react for 1 hour;

G, the sealed quantum dot nanosphere solution, centrifuged at 10000rpm for 10min, then centrifuged and washed three times with PBST buffer, and finally dispersed in 100 μl of PBS buffer (10mM, pH6.0) containing 1w/w%BSA, ready for use .

Figure 1A is a transmission electron micrograph of quantum dot nanospheres, and Figure 1B is a quantum dot in nanospheres under high-magnification transmission electron microscopy; Figure 1C is a particle size and particle size distribution diagram of quantum dot nanospheres and antibody conjugates; Figure 1D and Figure 1E are the comparison of the UV-Vis absorption and fluorescence spectra of quantum dot nanospheres and antibody conjugates, respectively.

Embodiment 3: the assembly of chromatography test strip

A. Take 100 μl of dust mite allergen protein and dilute it with 100 μl of PBS buffer;

B. Spray 0.4-1.2mg/mL dust mite allergen protein and rabbit IgG (1mg/mL) on the NC film respectively by using Bio-Dot XYZ 3000 automatic film spotting instrument, as a test strip detection line (T line) With the quality control line (C line), dry overnight at 37°C.

C. The sample pad, the NC film sprayed with the detection line (T line) and the quality control line (C line), and the absorbent pad are sequentially connected and assembled on the top of the plastic substrate used to provide the assembly platform. The assembly diagram is shown in Figure 1. Show.

D. Treat the sample pad with three sample pad treatment solutions A, B, and C respectively. The treatment method is to soak the sample pad in the sample pad treatment solution for 40 minutes, take it out and dry it in a 37°C drying oven for 12 hours, and seal it for later use.

E. Add the same amount of in vitro incubations to the sample wells of test strips treated with different sample pad treatment solutions, and perform parallel detection under each condition according to the changes in the fluorescence intensity of the T and C lines of the test strips and the release of quantum dot fluorescent microspheres. 5 times.

Results: Judging from the changes in the fluorescence intensity of test strip T and C lines and the release of quantum dot fluorescent microspheres, the treatment effect of sample pad treatment solution B is better than that of sample pad treatment solution A and sample pad treatment solution C.

The assembly of embodiment 4 kits

A semi-quantitative detection kit for house dust mite-specific IgE includes the following reagents:

1. Fluorescence detection liquid: the quantum dot-anti-IgE fluorescent antibody marker prepared in Example 2 and the calibrated quantum dot fluorescent antibody marker prepared in Example 1 were obtained by mixing equal volumes;

2. House dust mite-specific IgE semi-quantitative detection test strip: prepared in Example 3;

3. Fluorescence diluent: PBS buffer (10mM, pH6.0) containing 1w/w% BSA;

4. Calibrator: human anti-dust mite allergy protein-specific IgE;

5. Calibrator diluent: PBS buffer (10mM, pH6.0).

Example 5: Application of the House dust mite-specific IgE semi-quantitative detection kit in the detection of dust mite-specific IgE in serum

1. Sample

20 serums from patients with allergic asthma.

2. Kit

Prepared in Example 4, wherein the calibrator is dust mite-specific IgE human serum (0, 0.2, 1.0, 5.0, 10, 20 U/mL) diluted in PBS buffer (10 mM, pH 6.0).

3. Method

A. Take 200 μL of detection fluorescence solution and dilute it with fluorescence diluent according to 40 times volume;

B. The diluted fluorescent solution is divided into 120 μL each, sealed and stored at 2-8°C;

C. Take one piece of diluted fluorescent solution, then add 10 μL of sample or calibrator, shake and mix gently;

D, get 80 μ L of fluorescent liquid after reaction, add in the sample hole of immunochromatography test paper (prepared in embodiment 3), start timing;

E. After chromatographic reaction for 15 minutes, place the immunochromatographic test paper in a dark room, and take pictures to obtain fluorescent images under ultraviolet light;

F. Use the calibrator with multiple dilutions, perform detection according to steps B-E, and establish a calibration curve;

G, the fluorescence image that obtains, adopt software ImageJ to carry out RGB color channel separation;

H. Using the red channel fluorescence picture, the image pixel intensity analysis is performed on the detection area of the chromatography test paper;

1. Carry out pixel intensity integration to the detection area and the control area of the chromatography test paper respectively, calculate the T/C ratio, detect the calibration curve and the calibration equation according to the concentration and the T/C value of the calibrator;

J. The collected human serum is detected by the steps of B-I, the T/C value is obtained, and the concentration of IgE in the serum is calculated by the calibration equation.

result:

The schematic diagram of positive (B) and negative (C) results of dust mite-specific IgE detected by chromatographic test paper is shown in Figure 2. Fig. 3 is the RGB color fluorescent photo (A) and the blue (B) after RGB channel separation of the dust mite-specific IgE human serum (0, 0.2, 1.0, 5.0, 10, 20 U/mL) of the detection ratio dilution, Green (C) and red (D) channel photos. Figure 4A is the pixel intensity distribution map of the detection area of the test strip, and Figure 4B is the intensity distribution map after baseline processing; Figure 4C is the relationship between the T/C ratio and the IgE concentration calculated after integrating the signal intensities of the control and detection areas ; Figure 4D is the normal calibration curve of dust mite-specific IgE detected by quantum dot immunochromatography; Figure 4E is the calibration curve of dust mite-specific IgE index detected by quantum dot immunochromatography.

The calibration equation was used to calculate the concentration of IgE in the serum of patients with allergic asthma. It was found that among the 20 serum samples, 8 samples were between 0.35U/mL-3.0U/mL, and 8 samples were between 3U/mL-5.0U/mL. There are 5 serums, 2 serums between 5U/mL-10U/mL, and 5 serums between 0.2U/mL-0.35U/mL. The developed detection method can semi-quantitatively detect sIgE in the range of 0.2-10U/mL.

Embodiment 6 compliance test

In order to illustrate the accuracy of the test kit of the present invention, the present invention uses the test kit of the present invention and the sIgE detection method of traditional ImmunoCap to compare, and the serum of 56 routine positive patients and 40 routine healthy control groups is according to the method for embodiment 5 The test was carried out, and the test results showed that the positive coincidence rate was 92.9% (52/56), and the negative coincidence rate was 90.4% (38/42). It shows that the result of the detection kit of the present invention has good correlation with clinical symptoms, and the detection sensitivity is high.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the description only illustrates the principles of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (7)

1. a semi-quantitative detection kit for house dust mite-specific IgE, characterized in that the kit includes detection of fluorescent liquid and immunochromatographic test strips for the house dust mite-specific IgE semi-quantitative detection; Wherein, the fluorescent detection liquid is prepared by covalently coupling quantum dot nanospheres with carboxyl groups on the surface with goat anti-human sIgE and mouse anti-rabbit IgG respectively, and mixing them; Wherein, the immunochromatographic test strip includes sequentially connected sample pads, nitrocellulose membranes, water-absorbing pads, and a plastic substrate underneath for providing an assembly platform. The detection line coated with mite allergen protein and the quality control line coated with rabbit IgG, the sample pad provides the position where the sample to be tested is added. 2. test kit as claimed in claim 1, is characterized in that, described detection fluorescent liquid is prepared according to the following method: (1) Quantum dots are coupled with mouse anti-rabbit IgG antibody to prepare calibrated quantum dot fluorescent antibody markers: A. Take 100 μl of quantum dot nanospheres, disperse them in 200 μl of phosphate buffer, centrifuge at 10,000 rpm for 10 minutes, carefully discard the supernatant, then add 200 μL of phosphate buffer, and disperse evenly to obtain a solution of quantum dot nanospheres; B. Weigh 1.9 mg of EDC, dissolve in 1 mL of phosphate buffer, and mix well to obtain a 10 mM EDC solution; C. Take 20 μl of EDC solution, add it to 200 μl of quantum dot nanosphere solution, mix well, and incubate at room temperature for 0.5 h to obtain activated quantum dot nanospheres; D. Centrifuge the activated quantum dot nanospheres at 10000rpm for 10min, discard the supernatant, then add 200μl of 10mM, pH8.0 boric acid buffer, and disperse evenly by ultrasonic; E. Take 50 μl of mouse anti-rabbit IgG antibody, add it to the activated quantum dot nanosphere solution, mix well, and then continue to incubate at room temperature for 1 hour; F. Take 20 μl of 10w/w% BSA solution, add it to the nanosphere solution in step E, and continue to react for 1 hour; G, the sealed quantum dot nanosphere solution, centrifuged at 10000rpm for 10min, then centrifuged and washed three times with PBST buffer, and finally dispersed in 100 μl of 10mM containing 1w/w% BSA, pH6.0 PBS buffer, ready for use; (2) Quantum dots are coupled with goat anti-human sIgE to prepare quantum dot-anti-IgE fluorescent antibody markers; A. Take 100 μl of quantum dot nanospheres, disperse them in 200 μl of 10 mM, pH 6.0 phosphate buffer, centrifuge at 10,000 rpm for 10 minutes, carefully discard the supernatant, then add 200 μL of phosphate buffer, and disperse evenly to obtain quantum dot nanospheres solution; B. Weigh 1.9 mg of EDC, dissolve in 1 mL of phosphate buffer, and mix well to obtain a 10 mM EDC solution; C. Take 20 μl of EDC solution, add it to 200 μl of quantum dot nanosphere solution, mix well, and incubate at room temperature for 0.5 h to obtain activated quantum dot nanospheres; D. Centrifuge the activated quantum dot nanospheres at 10000rpm for 10min, discard the supernatant, then add 200μl of 10mM, pH8.0 boric acid buffer, and disperse evenly by ultrasonic; E. Take 50 μl of goat anti-human sIgE antibody, add it into the activated quantum dot nanosphere solution, mix well, and then continue to incubate at room temperature for 1 hour; F. Take 5 μl of 10w/w% BSA solution, add it to the nanosphere solution in step E, and continue to react for 1 hour; G, the sealed quantum dot nanosphere solution, centrifuged at 10000rpm for 10min, then centrifuged and washed three times with PBST buffer, and finally dispersed in 100 μl of 10mM containing 1w/w% BSA, pH6.0 PBS buffer, ready for use; (3) Mix the quantum dot nanosphere solutions covalently coupled with mouse anti-rabbit IgG and goat anti-human sIgE respectively obtained in step (2) and step (3) according to equal volumes to obtain the product. 3. The kit according to claim 1, wherein the sample pad is obtained after being treated with a sample pad treatment solution, and the treatment solution contains 1.0w/w% BSA, 0.25v/v 0.01 M pH 8.0 sodium borate buffer with % Tween-20, ₁ w/w % sucrose and 0.1 w/w % NaN3. 4. The kit according to claim 3, characterized in that, the processing method of the sample pad is to first soak the sample pad in the sample pad treatment liquid for 20-60min, take it out and dry it in a drying oven at 30-45°C for 9 -15h, that is. 5. test kit as claimed in claim 1, is characterized in that, also comprises fluorescent diluent, calibrator and calibrator diluent in the described test kit, and described fluorescent diluent is containing 1w/w%BSA 10 mM PBS buffer solution with pH 6.0, the calibrator is human anti-dust mite allergy protein specific IgE, and the diluent of the calibrator is 10 mM PBS buffer solution with pH 6.0. 6. kit as claimed in claim 5 is characterized in that, when being used for human anti-dust mite allergy protein specific IgE detection, comprises the following steps: A. Take the detection fluorescent solution and dilute it with fluorescent diluent according to the volume of 40-120 times; B. The diluted fluorescent solution is divided into 120μL each, sealed and stored at 2-8°C, and the calibration product is diluted with the calibration product diluent; C. Take one piece of diluted fluorescent solution, then add 10 μL of calibrator, shake and mix gently; D. Take 80 μL of the fluorescent solution after the reaction, add it to the sample pad of the immunochromatography test strip, and start timing; E. After chromatographic reaction for 15 minutes, place the immunochromatographic test paper in a dark room, and take pictures to obtain fluorescent images under ultraviolet light; F. Use the calibrator with multiple dilutions, perform detection according to steps B-E, and establish a calibration curve; G, the fluorescence image that obtains, adopt software ImageJ to carry out RGB color channel separation; H. Using the red channel fluorescence picture, the image pixel intensity analysis is performed on the detection area of the chromatography test paper; 1. Carry out pixel intensity integration to the detection area and the control area of the chromatography test paper respectively, calculate the T/C ratio, detect the calibration curve and the calibration equation according to the concentration and the T/C value of the calibrator; J. The human serum sample is detected by the steps of B-I, the T/C value is obtained, and the concentration of IgE in the serum is calculated by the calibration equation. 7. Use of the kit according to any one of claims 1-6 in the preparation and detection of human anti-dust mite allergy protein-specific IgE reagents.