CN113552338A

CN113552338A - Allergen specificity IgE antibody detection kit and preparation method thereof

Info

Publication number: CN113552338A
Application number: CN202010337996.0A
Authority: CN
Inventors: 贾丽霞; 周延; 白彩明; 董山山; 张苗苗; 夏吉涵; 姜敏; 菅丽萍
Original assignee: Green Biomedical Technology Tianjin Co ltd
Current assignee: Green Biomedical Technology Tianjin Co ltd
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2021-10-26

Abstract

The invention belongs to the field of clinical examination diagnostics, and relates to an allergen specificity IgE antibody detection kit and a preparation method thereof. The reagent strip comprises a membrane strip, a sample pad, a colloidal gold pad, a water absorption pad and a PVC plate; the membrane strip is provided with at least one detection line, and the detection line is coated with allergen extracting solution; the coating concentration of the allergen extract is 0.5-5 mg/mL. The colloidal gold reagent strip and the reagent kit can be used for detecting finger tip blood and whole blood, and realize the bedside detection of the allergen in the real sense; the patient can be diagnosed in time, and even if the patient himself can buy the reagent for self-checking, the early discovery and early treatment of more allergic disease patients are possible.

Description

Allergen specificity IgE antibody detection kit and preparation method thereof

Technical Field

The invention belongs to the field of clinical examination diagnostics, and relates to an allergen specificity IgE antibody detection kit and a preparation method thereof.

Background

The World Health Organization (WHO) considers allergic diseases (allergic asthma, rhinitis, rash, etc.) as a major health problem in the world today and ranks them as the fourth leading chronic disease in the world in 2010. The total incidence of allergic diseases worldwide is about 10-20%. The curative effect of the allergen vaccine as a medicine for diagnosing and treating allergic diseases is written into related guidelines by WHO, and the allergen vaccine enters the industrialized, large-scale and high-speed development stage in developed countries.

Among the respiratory allergic diseases in china, asthma (9.2%) and sinusitis (13.3%) and non-specific dermatitis (16.4%) are the most common. In the urban population, 2/3 patients with allergic rhinitis need to visit a hospital, and 40% of them need to be screened for allergens.

The progression of allergic disease is a chronic protracted process: the early stage of the disease is often manifested as allergic dermatitis, which further develops into intractable allergic rhinitis and finally develops into allergic asthma, and the whole course of the disease can reach more than ten years. Therefore, in order to avoid exposure to allergens as early as possible and provide a basis for symptomatic treatment, early diagnosis of allergic diseases and early detection of allergens are now advocated. The method can obviously reduce and delay the incidence rate of allergic asthma, relieve the pain of patients and reduce the medical burden of the country.

The diagnosis of allergic disease allergens is a systematic exclusion screening test, and often, in order to determine the allergens of a patient, screening of up to tens of suspected allergens is required. Allergen screening methods are divided into in vivo and in vitro tests.

In vivo tests generally include an intradermal test and a prick test. Initial screening in vivo tests (e.g., intradermal tests) can cause unnecessary pain to patients, especially infants, and can cause systemic severe adverse reactions such as anaphylactic shock in some patients with hypersensitivity.

The traditional methods for in vitro tests mainly include immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, fluoroimmunoassay, and the like. With the demand of point-of-care clinical diagnosis (POCT), point-of-care clinical decision, general disease screening and home disease self-examination (Homelab), rapid diagnosis methods using nitrocellulose membranes as carriers have been rapidly and widely developed in recent years. Among them, colloidal gold immunochromatography technology developed in the nineties is the most rapid and widely used. The colloidal gold immunochromatographic assay technology is praised as a new revolution in the field of in vitro diagnostic reagents due to the characteristics of rapidness, simplicity, good stability, single-person detection, patient self-detection, familiarity, wide application range, systematization of products, less investment, quick response and the like.

At present, the approved allergen in-vitro diagnosis products in China generally need expensive supporting equipment and special technical personnel for operation, and the popularization of the allergen in-vitro detection method is greatly influenced. At present, only part of domestic three hospitals have professional allergen in-vitro detection capability, but most of primary hospitals, clinics, army field hospitals and the like cannot carry out allergen in-vitro diagnosis. Moreover, the sample to be tested can be only serum, and a certain time is required from the blood collection to the processing of the sample into serum. Even the colloidal gold assay currently on the market uses serum assays, which defeats the purpose of point-of-care assays.

Disclosure of Invention

The invention aims to provide a reagent strip for detecting allergen-specific IgE antibodies.

The invention also aims to provide a colloidal gold test reagent strip which can take fingertip blood and whole blood as allergen-specific IgE antibodies of test samples.

Another object of the present invention is to provide a method for preparing a colloidal gold assay reagent strip.

Aiming at the current market demand, it is necessary to develop an accurate, rapid and easily popularized allergen in-vitro diagnostic reagent.

In order to solve the above technical problems, in one aspect, the present invention provides a reagent strip for detecting an allergen-specific IgE antibody, comprising a membrane strip, a colloidal gold pad, a water-absorbing pad and a PVC plate; the membrane strip is provided with at least one detection line, and the detection line is coated with allergen extracting solution; the coating concentration of the allergen extract is 0.5-5 mg/mL.

In some embodiments, the PVC plate is provided with a colloidal gold pad, a membrane strip and a water absorption pad in sequence from the lower end to the upper end.

In some embodiments, the membrane strip is overlaid on the absorbent pad at the upper end, and the membrane strip is overlaid on the colloidal gold pad at the lower end.

In some embodiments, the reagent strip comprises an antigen-coated NC membrane strip, a sample pad, a colloidal gold pad, a water absorbent pad, and a PVC plate; the sample pad, the water absorption pad and the colloidal gold pad are all attached to the PVC board; the membrane strip is attached to the center of the PVC plate; the upper end of the membrane strip is stacked with the water absorption pad, and the upper end of the colloidal gold pad is stacked at the lower end of the membrane strip; the membrane strip is provided with a detection line, and the detection line is coated with allergen extracting solution; the coating concentration of the allergen extract is 0.5-5 mg/mL.

In some embodiments, the test strip is further covered with a PE film.

In some embodiments, the PE film is covered on a film strip, a colloidal gold pad.

In some embodiments, the PE film is attached to the film strip at an upper edge 2-3mm above the gold gel pad, and below the PE film, the PE film is flush with the gold gel pad.

In some embodiments, the test strip further comprises a sample pad.

In some embodiments, the upper end of the sample pad is superposed on the lower end of the colloidal gold pad.

In some embodiments, the detection lines are at least two and spaced 2-3mm apart.

In some embodiments, the detection lines are 2-10.

In some embodiments, the detection lines are 3-8.

In some embodiments, the detection lines are 5.

In some embodiments, the allergens include inhalation group allergens and food group allergens. In some embodiments, the inhalation group allergens include, but are not limited to, one or more of house dust mites, cat fur dander, dog fur dander, artemisia pollen, ragweed pollen, humulus pollen, chenopodium pollen, bermuda pollen, poplar pollen, willow pollen, elm pollen, ash pollen, phoenix tree pollen, sabina pollen, birch pollen.

In some embodiments, the food group allergen includes one or more of egg, milk, peanut, soybean, tomato, wheat, sea crab, shrimp, sea fish, mango, apple, peach, pineapple, strawberry, sesame, cashew, pistachio, hazelnut, almond, walnut, but is not limited thereto.

In some embodiments, each of the detection lines is coated with one allergen source.

Studies have indicated that of 519 dust mite allergic patients, 58.6% were allergic to Derp1, 67.9% were allergic to Derp2, and 59.8% were allergic to Derp 23; of these 8.1% of patients were allergic to Derp23 only. If a mixture of Derp1 and Derp2 molecules is coated, then eventually 8.1% of patients will be diagnosed as negative (actually positive) for dust mite allergy. The present invention can diagnose 8.1% of patients as positive by using dust mite extract (all allergenic protein components are included). Therefore, the invention has obvious advantages by adopting the allergen raw material (extracting solution) for coating.

In some embodiments, the allergen extract is coated at a concentration of 0.5-2 mg/mL.

In some embodiments, the allergen extract is coated at a concentration of 0.5-1 mg/mL.

In some embodiments, the artemisia pollen, egg and soybean extract is coated at a concentration of 0.5-2 mg/mL.

In some embodiments, the artemisia pollen, egg and soybean extract is coated at a concentration of 0.5 mg/mL.

In some embodiments, the coating concentration of the house dust mite, cat dander and cockroach extract is 1-2 mg/mL.

In some embodiments, the coating concentration of the house dust mite, cat dander and cockroach extract is 1.5-2 mg/mL.

In some embodiments, the coating concentration of the house dust mite, cat dander and cockroach extract is 1.5 mg/mL.

In some embodiments, the dermatophagoides farinae allergen, dog dander allergen, and peanut allergen coating concentration is 1.5-2.0 mg/mL.

In some embodiments, the dermatophagoides farinae allergen, dog dander allergen, peanut allergen coating concentration is 2 mg/mL.

In some embodiments, the colloidal gold pad is a glass fiber membrane coated with a colloidal gold composite; wherein the colloidal gold compound is colloidal gold particles-mouse anti-human IgE antibody.

In some embodiments, the colloidal gold complex is prepared using a blocking solution CE210/CE 510.

In some embodiments, the membrane strip is further provided with a quality control line; the antibody coated on the quality control line is goat anti-mouse lgG.

The principle of the reagent strip is as follows: the membrane strip, e.g. TI zone, is coated with (inhalation group): allergens such as house dust mite, cat fur dander, dog fur dander, artemisia pollen, ragweed pollen, humulus pollen, chenopodium pollen, bermuda pollen, poplar pollen, willow pollen, elm pollen, ash wax pollen, phoenix tree pollen, sabina chinensis pollen, birch pollen and the like are taken as detection lines; goat anti-mouse IgG was used as the control line. The TF zone of the nitrocellulose membrane was coated with (food group): the method comprises the following steps of taking allergens (but not limited to allergens) such as eggs, milk, peanuts, soybeans, tomatoes, wheat, sea crabs, sea shrimps, sea fish, mangoes, apples, peaches, pineapples, strawberries, sesames, cashews, pistachios, hazelnuts, almonds, walnuts and the like as detection lines (sheep anti-mouse IgG is taken as a quality control line, 5 allergen raw materials can be coated on each T area, using a mouse anti-human IgE monoclonal antibody marked by colloidal gold as a color substance and adopting an indirect method principle, dripping a sample into a sample adding hole of a detection card in the detection process, moving the sample to a detection area and a quality control area, combining a specific IgE antibody (sIgE) in the sample and the allergens coated in the T area to form an allergen-sIgE complex, combining the mouse anti-human IgE antibody marked by the colloidal gold with the allergens in the allergen-sIgE complex after a development liquid is added, and thus forming a complex of the allergen antigen-sIgE-mouse anti-human-IgE-colloidal gold in the detection area, when the concentration of the sIgE in the sample is high enough, a color band appears in the corresponding detection line, and the higher the concentration of the sIgE is, the faster the color band appears in the detection area, and the darker the color is. When the gold-labeled antibody moves to the control line, a goat anti-mouse IgG-mouse anti-human IgE-colloidal gold compound is formed and develops color, the appearance of a color band indicates that the reaction system is effective, and if the control line has no color band, the reaction system fails.

In some embodiments, the concentration of goat anti-mouse lgG is 0.5-1.0 mg/mL.

In some embodiments, the membrane strip is selected from the group consisting of nitrocellulose membranes, cellulose acetate membranes, PVDF, nylon membranes, polytetrafluoroethylene membranes.

In some embodiments, the membrane strip is a nitrocellulose membrane.

In some embodiments, the method of preparing the allergen extract comprises the steps of:

(1) uniformly mixing the allergen raw material and a buffer solution;

(2) stirring the allergen raw material solution obtained in the step (1) at the rotation speed of 100-300 rpm for 18-24 hours at the temperature of 2-8 ℃;

(3) centrifuging the allergen raw material solution at 2-8 ℃ for 10-20 minutes at 5000-8000 rpm;

(4) centrifuging, taking out the supernatant, and filtering to obtain allergen material extractive solution.

In some embodiments, the buffer solution is prepared by: weighing 26.48g of disodium hydrogen phosphate, 1.68g of monopotassium phosphate and 2.92g of sodium chloride, adding ultrapure water to 1000mL, and uniformly mixing to obtain the sodium phosphate-potassium phosphate-sodium solid.

In some embodiments, the buffer is a phosphate buffer.

In some embodiments, the ratio of allergen raw material to buffer solution in step (1) is 1:20 to 1: 50.

In some embodiments, the mass ratio of the allergen raw material to the buffer solution is 1:25 to 1: 40.

In some embodiments, the mass ratio of allergen material to buffer solution is 1: 25.

In some embodiments, the step of filtering comprises: filtration was performed using a 0.8 μm filter and then a 0.22 μm filter.

In another aspect, the present invention provides a method for preparing the test strip, comprising the steps of:

(1) preparing an allergen extracting solution;

(2) preparing a membrane strip: coating the allergen extracting solution on a membrane strip to form a plurality of detection lines at intervals;

(3) preparing a colloidal gold pad;

(4) assembling the test strip: pasting the membrane strip in the step (2) on the center of the PVC board, and pasting the upper end of the water absorption pad on the upper end of the PVC board; the lower end of the water absorption pad presses the upper end of the membrane strip for 2-3 mm; the lower end of the film pressing strip at the upper end of the colloidal gold pad in the step (3) is 2-3mm, and the lower end of the colloidal gold pad protrudes 5-7mm out of the PVC plate; and (3) sticking the upper edge of the PE film to the nitrocellulose membrane by 3-4mm beyond the upper edge of the colloidal gold pad, and leveling the lower part of the PE film to the colloidal gold pad to obtain the finished product.

In some embodiments, the step of preparing the colloidal gold pad comprises:

(5) coupling the mouse anti-human IgE antibody to the colloidal gold particles to obtain a mouse anti-human IgE-colloidal gold compound;

(6) spraying mouse anti-human IgE-colloidal gold compound on glass cellulose membrane.

In some embodiments, the step of preparing the murine anti-human IgE-colloidal gold complex comprises:

(a) preparing colloidal gold particles;

(b) adjusting the pH value of the gold solution by using 0.15-0.2 mol/L potassium carbonate or hydrochloric acid;

(c) adding mouse anti-human IgE to the gold solution so that the concentration of the IgE is 1-5 mu g/ml;

(d) adding 1-2.5% of CE210/CE510 confining liquid, uniformly mixing and stirring;

(e) centrifuging 5000-50000 g for 20-30 min at the temperature of 2-8 ℃, and carefully removing supernatant after centrifugation is finished;

(f) the pellet is suspended in a buffer solution of CE210/CE510 so that the absorbance of the solution at 530nm is 1.0, and the solution is kept at 2-8 ℃ for later use.

In still another aspect, the present invention provides a detection kit for an allergen-specific IgE antibody, comprising an upper case and a lower case, the upper case being provided with a sample well; a blood filtering pad is placed on the sample hole on the back surface of the upper shell; a sample pad is placed below the blood filtering pad; an in-shell support is arranged in the upper shell; the in-shell support is provided with a test strip groove, and the test strip of the claims 1-6 is placed in the test strip groove; and buckling the lower shell onto the upper shell to form the reagent card.

In the detection reagent strip, the method or the detection kit, the sample used by the detection reagent is whole blood or serum.

In some embodiments, the whole blood comprises fingertip blood or venous blood.

Compared with the prior art, one embodiment of the invention has the beneficial effects that:

1. semi-quantitatively, accurately grading the allergy level of a patient, and enabling the sensitivity to reach 0.35 IU/ml.

2. The sample dosage is small, and 100ul of fingertip blood/serum can detect at least 10 allergens.

3. The time is short, and only 20min is needed from blood sampling to result.

4. The colloidal gold reagent strip of the invention does not need equipment or only needs small equipment, and is more convenient to popularize in local hospitals across the country. The colloidal gold reagent strip and the reagent kit can be used for detecting finger tip blood and whole blood, and realize the bedside detection of the allergen in the real sense; the patient can be diagnosed in time nearby, even if non-professional personnel (such as the patient) can purchase reagents for operation self-checking, the cost of the patient for hospitalization can be reduced, the pressure of the outpatient service of a medical institution is effectively relieved, the diagnosis compliance of the patient to the allergen is improved, and the possibility is provided for early discovery and early treatment of more patients with allergic diseases.

5. The invention separately coats the same allergen raw material extract, so that a doctor can very clearly determine what allergen a patient is allergic to clinically, the next medication of the doctor is facilitated, the patient also can clearly determine what allergy the patient is allergic to, and the contact can be avoided or reduced as much as possible in the future life; and the extract is used for coating (namely all allergenic protein components of the same allergen raw material are contained), so that the occurrence of false negative results can be reduced.

Drawings

FIG. 1 is a standard curve log-log graph.

FIG. 2A-FIG. 2B are the results of the determination of positive samples in comparative example 2, FIG. 2A is the use of CE210/CE510 to block the gold complex;

fig. 2B shows blocking of gold complexes using BSA.

FIGS. 3A-3B are graphs showing the results of negative sample determination in comparative example 2, and FIG. 3A is a graph showing the use of CE210/CE510 to block a gold complex;

fig. 3B shows blocking of gold complexes using BSA.

Fig. 4 shows the result of the detection of the fingertip blood sample by the reagent card a in comparative example 3.

Fig. 5 is a result of testing a fingertip blood sample of the comparative group 2 in the comparative example 3.

FIG. 6 shows the result of the measurement of the serum sample by the reagent card A in comparative example 3.

FIG. 7 shows the results of the serum samples tested in comparative example 2 of comparative example 3.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

The upper end or the lower end of the test strip is defined according to the flow direction of a sample, and when the test strip is contacted with the sample in the use process, the sample flows from the sample adding end to the direction of the water absorption pad through chromatography, the end of the water absorption pad is called the upper end, and the end of the sample adding end is called the lower end.

In the embodiment of the invention, the developing solution comprises the following components: 0.2301g of disodium hydrogen phosphate, 0.0456g of sodium dihydrogen phosphate and 0.9g of sodium chloride, and ultrapure water is added to the mixture to make the volume of the mixture to be 100 ml.

CE210/CE 510: purchased from JSR, Inc., and having a product number CE210/CE 510.

In the embodiment of the invention, the membrane strip is a nitrocellulose membrane.

Example 1 preparation of allergen raw material extract

1. Preparing an extraction buffer solution:

weighing 26.48g of disodium hydrogen phosphate, 1.68g of monopotassium phosphate and 2.92g of sodium chloride, adding ultrapure water to 1000ml, and uniformly mixing to obtain the product.

2. Extraction step

(1) Weighing 10g of allergen raw material, adding an extraction buffer solution according to the mass ratio of the raw material to the extracting solution of 1:20-1:50, and uniformly mixing; placing the mixed raw materials on a magnetic stirrer to stir and extract for 24 hours (the rotating speed is 100-300 r/min) at the temperature of 2-8 ℃;

(2) evenly distributing the raw materials into a centrifugal tube, and putting the centrifugal tube into a centrifugal machine; under the condition of 4 ℃, the rotating speed of a centrifugal machine is adjusted to be 5000-8000 revolutions/min, and the centrifugal machine is centrifuged for 10-20 min;

(3) after the centrifugation is finished, taking out the supernatant, and performing coarse filtration by using a 0.8um filter membrane; and finally filtering with a 0.22um filter membrane to obtain filtrate, namely the coated allergen raw material extracting solution.

(4) The protein content of the allergen raw material extract was determined using the Broadford method, in which samples with lower concentrations were concentrated.

Example 2 test strip for allergen-specific IgE antibody detection

The test strip comprises a membrane strip (an allergen-coated NC membrane), a sample pad, a colloidal gold pad, a water absorption pad and a PVC large plate. The sample pad, the colloidal gold pad, the membrane strip and the water absorption pad are sequentially attached to the center of the PVC large plate from the lower end to the upper end. The membrane strip is pasted in the middle of a PVC large plate, absorbent paper is pasted above the large plate, the upper side of the absorbent paper is flush with the upper edge of the large plate, and the lower edge of the absorbent paper presses the nitrocellulose membrane for 2 mm; sticking a colloidal gold pad below the large plate, pressing a nitrocellulose membrane for 3mm above the colloidal gold pad, stacking the upper end of a sample pad below the colloidal gold pad, and enabling the lower end of the sample pad to be flush with the PVC large plate; the upper edge of the PE film exceeds the upper edge of the colloidal gold pad by 3mm and is attached to the nitrocellulose membrane, and the lower part of the PE film is flush with the colloidal gold pad.

The membrane strip is provided with 5 detection lines, and the detection lines are coated with food group allergen extracting solution; the allergen is egg allergen, milk allergen, peanut allergen, soybean allergen, and tomato allergen.

Example 3 test strip for allergen-specific IgE antibody detection

The test strip comprises a membrane strip (an allergen-coated NC membrane), a sample pad, a colloidal gold pad, a water absorption pad and a PVC large plate. The sample pad, the colloidal gold pad, the membrane strip and the water absorption pad are sequentially attached to the center of the PVC large plate from the lower end to the upper end. The membrane strip is pasted in the middle of a PVC large plate, absorbent paper is pasted above the large plate, the distance between the upper edge of the absorbent paper and the upper edge of the large plate is 3mm, and the lower edge of the absorbent paper presses the nitrocellulose membrane for 2 mm; sticking a colloidal gold pad below the large plate, pressing a nitrocellulose membrane for 3mm above the colloidal gold pad, stacking the upper end of a sample pad below the colloidal gold pad, and enabling the lower end of the sample pad to be flush with the PVC large plate; the upper edge of the PE film exceeds the upper edge of the colloidal gold pad by 3mm and is attached to the nitrocellulose membrane, and the lower part of the PE film is flush with the colloidal gold pad.

The membrane strip is provided with 6 detection lines, and the detection lines are coated with an inhalation group allergen extracting solution; the allergen is house dust mite allergen, cat dander allergen, dog dander allergen, and Artemisia flower allergen.

Example 4 test strip for allergen-specific IgE antibody detection

The test strip comprises a membrane strip, a sample pad, a colloidal gold pad, a water absorption pad and a PVC large plate. The sample pad, the colloidal gold pad, the membrane strip and the water absorption pad are sequentially attached to the center of the PVC large plate from the lower end to the upper end. The membrane strip is pasted in the middle of a PVC large plate, absorbent paper is pasted above the large plate, the upper side of the absorbent paper is flush with the upper edge of the large plate, and the lower edge of the absorbent paper presses the nitrocellulose membrane for 2 mm; sticking a colloidal gold pad below the large plate, pressing a nitrocellulose membrane for 3mm above the colloidal gold pad, stacking the upper end of a sample pad below the colloidal gold pad, stacking a blood filtering pad at the lower end of the sample pad, and enabling the blood filtering pad to be flush with the PVC large plate; the upper edge of the PE film exceeds the upper edge of the colloidal gold pad by 3mm and is attached to the nitrocellulose membrane, and the lower part of the PE film is flush with the blood filtering pad.

The membrane strip is provided with 8 detection lines and 1 quality control line, and the detection lines are coated with an inhalation group allergen extracting solution; the allergen inhalation group allergens: bermuda pollen, poplar pollen, willow pollen, elm pollen, ash pollen, phoenix tree pollen, juniper pollen and birch pollen.

EXAMPLE 5 preparation of test strips

1. Preparation of film strips

The allergen extract extracted according to the method of example 1 (the mass ratio of raw materials to extract is 1:25) is coated on a membrane strip, so that five detection bands are formed on the membrane strip of a P1 test strip, namely 5 allergens of an inhalation group are coated, and simultaneously 1 quality control line is coated on a P1 test strip; the allergen material was coated to form five test strips on the P2 test strip, i.e. 5 allergens of the food group were coated, and 1 quality control line was further coated on the P2 test strip.

(1) The allergens (P1 test strip) in inhalation group comprise allergens such as dermatophagoides pteronyssinus, dermatophagoides farinae, cat fur dander, dog fur dander, artemisia pollen, ragweed pollen, humulus pollen, chenopodium pollen, bermuda pollen, poplar pollen, willow pollen, elm pollen, ash wax pollen, phoenix tree pollen, juniper pollen, birch pollen, etc.;

(2) the food group allergen (P2 test strip) comprises allergen such as egg, milk, peanut, soybean, tomato, wheat, sea crab, sea shrimp, sea fish, mango, apple, peach, pineapple, strawberry, sesame, cashew nut, pistachio nut, hazelnut, almond, walnut, etc.;

(3) the quality control line is goat anti-mouse IgG;

(4) adjusting the humidity of the coating room to be 40-60% and the temperature to be 18-26 ℃, adhering the nitrocellulose membrane to a PVC large plate under the environment, and balancing for 30-60 min;

(5) adjusting the protein concentration of the allergen in the inhalation group, the allergen in the food group and the goat anti-mouse IgG to be 0.5-2.0 mg/ml; setting the spraying amount of a striping instrument to be 1ul/cm, the spraying speed to be 100mm/s, respectively placing 6 pump pipes into respective raw material solution bottles, and starting the striping instrument to coat allergen raw materials onto the nitrocellulose membrane; 5 inhalation group detection lines and 1 quality control line are formed on the P1 test strip film, and 5 food group detection lines and 1 quality control line are formed on the P2 test strip film.

(6) Drying the coated P1 and P2 films in a drying room with the temperature of 35 +/-5 ℃ and the humidity of less than 30 percent for 2 hours; after finishing, placing the aluminum foil bag with the drying agent for standby.

2. Preparation of colloidal gold particle labeled mouse anti-human IgE antibody

(1) Preparation of colloidal gold particles

Preparing colloidal gold particles: adding 250mL of distilled water into a round-bottom flask, heating on an electric heating jacket, boiling, adding 0.5mL of 10% chloroauric acid solution, adding 0.75mL of 10% trisodium citrate solution when the solution is boiled, keeping the solution boiling for 10 minutes, and gradually cooling for later use to obtain the gold solution. The prepared gold solution was examined three times in succession with a uv-vis spectrophotometer (wavelength 530nm) and the results recorded: the absorbance values were 1.096, 1.097 and 1.096, respectively. The gold solution particles thus prepared were 40 nm. The change in appearance after standing at 37 ℃ is shown in Table 1.

TABLE 1 gold solution standing at 37 ℃ appearance Change

Number of days	Appearance of the product
		5	Is uniformly dispersed
10	Is uniformly dispersed
		15	Is uniformly dispersed

(2) Mouse anti-human IgE antibody and gold particle combination

Firstly, determining the optimal pH and the optimal antibody adding amount when the mouse anti-human IgE and gold particles are marked by using a sodium chloride titration method;

② marking step

I: adjusting the pH value of the gold solution to 8.0 by using 0.2mol/L potassium carbonate or 0.2mol/L hydrochloric acid;

II: adding a mouse anti-human IgE antibody into 100ml of gold solution to enable the labeling amount of the mouse anti-human IgE antibody to be 5 mu g/ml (5 ug of the mouse anti-human IgE antibody needs to be added into each ml of colloidal gold compound), and continuously stirring for 10-30 min;

III: adding 100ul of 2% CE210/CE510 solution, uniformly mixing, and continuously stirring for 10-30 min;

IV: centrifuging 5000-50000 g for 20-30 min at the temperature of 2-8 ℃, and carefully removing supernatant after centrifugation is finished;

v: the pellet is suspended in a buffer containing 0.01% to 0.05% (volume fraction) of the blocking solution CE210/CE510 (buffer solution prepared in example 1) and the solution is kept at 2 to 8 ℃ until the absorbance of the solution at 530nm is preferably 1.0, A530 nm.

(3) Preparation of colloidal gold pad

Spraying the marked mouse anti-human IgE-colloidal gold compound on a glass cellulose membrane by using a gold spraying instrument;

secondly, drying the sprayed glass cellulose membrane for 2 hours in a drying room with the temperature of 35 +/-5 ℃ and the humidity of less than 30 percent; after finishing, placing the aluminum foil bag with the drying agent for standby.

(4) Test strip assembly

In the drying room, water-absorbing paper, colloidal gold pad, PE film, PVC large board with P1 (suction group) film strip stuck thereon, and PVC large board with P2 (food group) film strip stuck thereon were prepared. Pasting absorbent paper above the large plate, enabling the upper side of the absorbent paper to be flush with the upper edge of the large plate, and pressing the nitrocellulose membrane by 2mm at the lower edge of the absorbent paper; sticking a colloidal gold pad below the large plate, pressing a nitrocellulose membrane for 3mm above the colloidal gold pad, and protruding the large plate for 5mm below the colloidal gold pad; the upper edge of the PE film exceeds the upper edge of the colloidal gold pad by 3mm and is attached to the nitrocellulose membrane, and the lower part of the PE film is flush with the colloidal gold pad.

EXAMPLE 6 preparation of test cards

The test strip prepared in example 5 was assembled with a housing as follows:

cutting the assembled P1 (inhalation group) and P2 (food group) large plates into test strips with width of 5mm by a slitter; placing a blood filtering pad at the sample hole on the back of the upper clamping shell, and then placing the sample pad above the blood filtering pad to enable the sample pad to protrude 2mm from the PE film; the left side of the card shell is provided with a P2 (food group) membrane strip, and the right side is provided with a P1 (suction group) membrane strip; after all the materials are put, the lower clamping shell is buckled on the upper clamping shell, and the upper clamping shell and the lower clamping shell are tightly pressed, so that a complete detection card is formed.

Example 7 quality control

(1) Preparation of standard detection card membrane strip

Firstly, adjusting the protein concentration of a mouse anti-human IgE antibody to be 0.5-1.0 mg/ml for establishing a standard curve; adjusting the protein concentration of the goat anti-mouse IgG antibody to 0.5-1.0 mg/ml as a quality control line;

secondly, adjusting the humidity of the coating room to be 40-60% and the temperature to be 18-26 ℃, attaching the nitrocellulose membrane to a PVC large plate under the environment, and balancing for 30-60 min;

thirdly, setting the spraying amount of the film scratching instrument to be 1ul/cm, the spraying speed to be 100mm/s, respectively placing 2 pump pipes into respective raw material solution bottles, and starting the film scratching instrument to coat the raw materials on the nitrocellulose membrane; forming 1 quality control band and 1 mouse anti-human IgE antibody detection band on the standard card membrane strip;

fourthly, drying the coated film for 2 hours in a drying room with the temperature of 35 +/-5 ℃ and the humidity of less than 30 percent; after finishing, placing an aluminum foil bag with a drying agent for later use;

and fifthly, assembling the standard card membrane strip into the standard detection card according to the steps of preparing the colloidal gold-mouse anti-human IgE in the preparation process of the detection card, preparing the colloidal gold pad and assembling the test strip.

(2) Establishment of a Standard Curve

Firstly, diluting an IgE international standard substance to 0.35IU/ml, 0.7IU/ml, 3.5IU/ml, 17.5IU/ml, 50IU/ml and 100 IU/ml; adding 100ul of the sample into the sample hole of the standard detection card, and timing for 5 min; after the completion, 500ul of development solution is added into the development solution hole, and the time is timed for 15 min;

secondly, placing the standard detection card on a colloidal gold reading instrument, and reading a gray value; and simultaneously taking logarithms of the concentration value of the standard substance and the corresponding gray value, and establishing a standard curve by using double logarithms.

(3) Standard card preparation

Writing the information of the standard curve in the step (2) into a standard card through a card writer, and inserting the standard card into an instrument, so that the information of the standard curve can be generated; the sample tested will calculate the final concentration value according to the standard curve.

(4) Sample detection

Adding 100ul of fingertip blood, whole blood or serum to be detected into sample holes of a colloidal gold detection card, and timing for 5 min;

adding 0.5ml of development solution into the development solution hole, and timing for 15 min;

and thirdly, after the reaction time of the test card is over, the test card is placed in a colloidal gold card reader to read the numerical value, and the sample allergic concentration is automatically calculated and classified by the instrument according to the batch of kit standard curves.

(5) Results of Performance testing

1) Standard curve

Taking 100ul serum samples of 0.35IU/ml, 0.7IU/ml, 3.5IU/ml, 17.5IU/ml, 50IU/ml and 100IU/ml, respectively adding into the sample holes of the standard card prepared in the step (3), and timing for 5 min; after the completion, 0.5ml of development solution is added into the development solution hole, and the time is timed for 15 min; after the reaction time of the test card is over, the test card is put into a colloidal gold card reader to read the numerical value, and the result is shown in table 2; the results are shown in FIG. 1, after which a standard curve is constructed according to the log-log.

TABLE 2

Concentration (IU/ml)	Grey scale value
		0.35	1.96
0.7	3.21
		3.5	6.73
17.5	21.95
		50	53.18
100	95.06

2) Quality standard and test of test paper strip

The quality of the test strip prepared in the embodiment 5 of the invention is detected according to the allergen-specific IgE antibody detection kit of the national medical industry standard of the people's republic of China; the detection result shows that the detection negative coincidence rate, the positive coincidence rate, the detection limit and the repeatability of the semi-quantitative detection kit all accord with the industrial standard. The quality control lines in the test strips referred to in tables 3-5 are all goat anti-mouse IgG antibody (coating concentration 1 mg/ml).

TABLE 3 serum sample test results

TABLE 4 fingertip blood sample test results

TABLE 5 Whole blood sample test results

3) Stability test

Placing the same 3 batches of membrane strips in the step 2) at 37 ℃ for 6 days, and then verifying according to the same detection requirements, wherein the stability detection results of the serum samples are shown in tables 6-8; the results of the stability tests on the whole blood samples are shown in tables 9-11.

TABLE 6 serum sample test results (201905001 batches)

TABLE 7 serum sample test results (201905002 batches)

TABLE 8 serum sample test results (201905003 batches)

TABLE 9 Whole blood sample test results (201905001 batches)

TABLE 10 Whole blood sample test results (201905002 batches)

TABLE 11 Whole blood sample test results (201905003 batches)

Comparative example 1

Respectively coating raw material extract of house dust mite allergen, cat fur allergen, dog fur allergen, artemisia pollen allergen, egg allergen, milk allergen, peanut allergen and soybean allergen (the extraction method is example 1, the ratio of the raw materials to the extract is 1:25) with 0.5mg/ml, 1.0mg/ml, 1.5mg/ml and 2mg/ml, coating goat anti-mouse IgG antibody with three concentrations of 0.5mg/ml, 1mg/ml and 1.5mg/ml, drying and assembling into strips. The other conditions were the same as in example 5.

3 concentration-specific sera calibrated by Phaida UniCAP were used for detection, S1:0.35IU/ml, S2:1.0IU/ml, S3:2.0IU/ml and S4:4.0IU/ml, and the lowest detection limit was used as the assessment index.

The results are shown in tables 12-21.

TABLE 12 Dermatophagoides pteronyssinus allergen coating concentration selectivity test

TABLE 13 Dermatophagoides pteronyssinus allergen coating concentration selectivity test

TABLE 14 Cat dander allergen coating concentration selectivity test

TABLE 15 selectivity of dog fur dandruff allergen coating concentration test

TABLE 16 Artemisia pollen allergen concentration selectivity test

TABLE 17 egg allergen coating concentration selection test

TABLE 18 milk allergen coating concentration selection test

TABLE 19 peanut allergen coating concentration selectivity test

TABLE 20 Soy allergen coating concentration selectivity test

TABLE 21 sheep anti-mouse IgG antibody coating concentration selectivity test

From the above results, it can be seen that different allergen coating concentrations affect the final results of the assay. Wherein the coating concentration of Artemisia pollen allergen, egg allergen and soybean allergen is 0.5 mg/ml; the milk allergen coating concentration is 1.0 mg/ml; the coating concentration of house dust mite allergen, cat dander allergen and cockroach allergen is 1.5 mg/ml; the coating concentration of the dermatophagoides farinae allergen, the dog fur crumb allergen and the peanut allergen is 2.0 mg/ml; the coating concentration of the goat anti-mouse IgG antibody is 1 mg/ml.

Comparative example 2

Detecting a card A: the test card was prepared as in example 6, but only one test line (the specific allergen was an artemisia pollen allergen, and the protein coating concentrations of the allergen and goat anti-mouse IgG were 1.5mg/mL and 1mg/mL, respectively).

Comparative group 1: the difference from the detection card A is that the blocking solution CE210/CE510 is replaced by blocking solution BSA, and the rest conditions are the same.

(1) Noise to noise ratio test

Detecting the same positive sample: the test strip obtained in example 2 is shown in FIG. 2A, and the test strip obtained in comparative example 1 is shown in FIG. 2B. (Artemisia pollen allergen, blood of finger tip)

Detecting the same negative sample: the test strip obtained in example 2 is shown in FIG. 3A, and the test strip obtained in comparative example 1 is shown in FIG. 3B. (Artemisia pollen allergen, blood of finger tip)

The results are shown in Table 22:

TABLE 22

As a result: as can be seen from the data in Table 22, the overall detection noise is improved by 6.5 times compared with the conventional BSA after the CE210/CE510 is used as the blocking solution in the present invention.

(2) Sensitivity of the probe

One calibration positive sample was diluted to different concentrations and the colloidal gold complex assay results were blocked with different blocking solutions.

The results are shown in Table 23: (Artemisia pollen allergen, blood of finger tip)

TABLE 23

From the results in Table 23, it is clear that the sensitivity of the product of the present invention can reach 0.35IU/ml by using CE210/CE510 as a blocking solution; and if the traditional BSA is used as a blocking solution, the sensitivity can only reach 1IU/ml at most.

Comparative example 3

Detecting a card A: the test card prepared in example 6 (the specific allergen was an artemisia pollen allergen, and the protein coating concentrations of the allergen and goat anti-mouse IgG were 1.5mg/mL and 1mg/mL, respectively).

Comparative group 2: the allergen-specific IgE antibody detection kit (colloidal gold method) is commercially available at present (manufacturer: Beijing Xinhualian synergetic and pharmaceutical industry, LLC).

The two kinds of reagent cards were tested according to the respective instructions, and 100ul of the fingertip blood sample was added, and the results are shown in fig. 4 and 5, respectively.

Performing gradient dilution on a part of artemisia pollen allergic serum, respectively detecting a detection card A and a comparison group 2 reagent card according to respective operation instructions, and adding 100ul of serum sample; the detection results are shown in fig. 6, fig. 7, and table 24.

Watch 24

As can be seen from the results in Table 24, when the calibration serum samples were simultaneously tested using the test card A and the control group 2, the deviation between the test results of the present invention and the standard values was smaller, indicating that the actual values were more realistic; the deviation between the control group and the calibration result is larger, which indicates that the deviation is more different from the true value.

Claims

1. The reagent strip for detecting the allergen-specific IgE antibody is characterized by comprising a membrane strip, a colloidal gold pad, a water absorption pad and a PVC (polyvinyl chloride) plate; the membrane strip is provided with at least one detection line, and the detection line is coated with allergen extracting solution; the coating concentration of the allergen extract is 0.5-5 mg/mL.

2. The strip according to claim 1, wherein the colloidal gold pad, the membrane strip and the absorbent pad are sequentially laid on the PVC plate from bottom to top;

preferably, the upper end of the membrane strip is overlapped with the water absorption pad, and the lower end of the membrane strip is overlapped with the upper end of the colloidal gold pad;

further preferably, the detection reagent strip is also covered with a PE film;

or preferably, the PE film is covered on the film strip and the colloidal gold pad;

more preferably, the upper end of the PE film is 2-3mm above the colloidal gold pad, and the lower end of the PE film is flush with the lower end of the colloidal gold pad;

preferably, the test strip further comprises a sample pad;

more preferably, the upper end of the sample pad is stacked on the lower end of the colloidal gold pad.

3. The test reagent strip of claim 1 or 2, wherein the test lines are at least two and are spaced 2-3mm apart;

preferably, the detection lines are 2-10;

more preferably, the detection lines are 3-8;

still more preferably, the detection lines are 5.

4. The test strip of claim 1 or 2, wherein the allergens comprise inhalation group allergens and food group allergens;

preferably, the inhalation group allergens comprise one or more of mites, animal dander and pollen;

preferably, the inhalation group allergens comprise one or more of house dust mites, cat fur dander, dog fur dander, artemisia pollen, ragweed pollen, humulus pollen, chenopodium pollen, bermuda pollen, poplar pollen, willow pollen, elm pollen, ash pollen, phoenix tree pollen, sabina pollen, birch pollen;

or preferably, the food group allergen comprises one or more of egg, milk, peanut, soybean, tomato, wheat, sea crab, sea shrimp, sea fish, mango, apple, peach, pineapple, strawberry, sesame, cashew, pistachio, hazelnut, almond and walnut;

more preferably, each of the detection lines is coated with an allergen source;

or more preferably, the coating concentration of the allergen extract is 0.5-2 mg/mL;

still more preferably, the allergen extract is coated at a concentration of 0.5-1 mg/mL;

or more preferably, the coating concentration of the artemisia pollen, the egg and the soybean extracting solution is 0.5-2 mg/mL;

still more preferably, the coating concentration of the artemisia pollen, the egg and the soybean extract is 0.5 mg/mL;

or more preferably, the coating concentration of the house dust mite, the cat fur dander and the cockroach extracting solution is 1-2 mg/mL;

still more preferably, the coating concentration of the house dust mite, cat fur dander and cockroach extract is 1.5-2 mg/mL;

still more preferably, the coating concentrations of the house dust mite, the cat fur dander and the cockroach extract are all 1.5 mg/mL;

or more preferably, the concentrations of the dermatophagoides farinae allergen, the dog fur crumb allergen and the peanut allergy coating are all 1.5-2.0 mg/mL;

still more preferably, the concentrations of the dermatophagoides farinae allergen, the dog dander allergen and the peanut allergen coating are all 2 mg/mL.

5. The test strip of claim 1 or 2, wherein the colloidal gold pad is a glass fiber membrane coated with a colloidal gold complex;

preferably, wherein the colloidal gold complex is a colloidal gold particle-murine anti-human IgE antibody;

further preferably, the colloidal gold complex is prepared by using a confining liquid CE210/CE 510.

6. The test reagent strip of claim 1 or 2, wherein the membrane strip is further provided with a quality control line; the antibody coated on the quality control line is goat anti-mouse lgG;

preferably, the concentration of the goat anti-mouse lgG is 0.5-1.0 mg/mL;

or preferably, the membrane strip is selected from nitrocellulose membranes, cellulose acetate membranes, PVDF, nylon membranes, polytetrafluoroethylene membranes;

more preferably, the membrane strip is a nitrocellulose membrane.

7. The test strip of claim 1 or 2, wherein the allergen extract is prepared by a method comprising the steps of:

(1) uniformly mixing the allergen raw material and a buffer solution;

(4) centrifuging, taking out the supernatant, and filtering to obtain allergen raw material extract;

preferably, the mass ratio of the allergen raw material to the buffer solution in the step (1) is 1:20-1: 50;

more preferably, the mass ratio of the allergen raw material to the buffer solution is 1: 25-1: 40;

still more preferably, the mass ratio of the allergen raw material to the buffer solution is 1: 25;

or preferably, the step of filtering comprises: filtration was performed using a 0.8 μm filter and then a 0.22 μm filter.

8. A method of making a test strip according to any of claims 1-7 comprising the steps of:

(1) preparing an allergen extracting solution;

(3) preparing a colloidal gold pad;

(4) assembling the test strip;

preferably, the preparation of the colloidal gold pad comprises the steps of:

(6) spraying mouse anti-human IgE-colloidal gold compound on the glass cellulose membrane to obtain the mouse anti-human IgE-colloidal gold compound;

or preferably, the preparation step of the mouse anti-human IgE-colloidal gold complex comprises the following steps:

(a) preparing colloidal gold particles;

(b) adjusting the pH value of the gold solution to 7.0-8.0 by using 0.15-0.2 mol/LpH regulator;

(c) adding mouse anti-human IgE into the gold solution to enable the concentration of the IgE to be 1-5 mu g/ml;

(d) adding 1-2.5% of CE210/CE510 confining liquid to make the final concentration of the confining liquid be 0.01-0.1%, uniformly mixing and stirring;

9. A detection kit for allergen-specific IgE antibodies is characterized by comprising an upper shell and a lower shell, wherein the upper shell is provided with a sample hole; a blood filtering pad is placed on the sample hole on the back surface of the upper shell; a sample pad is placed below the blood filtering pad; an in-shell support is arranged in the upper shell; the in-shell support is provided with a test strip groove, and the test strip of the claims 1-6 is placed in the test strip groove; and buckling the lower shell onto the upper shell to form the reagent card.

10. The test strip of claims 1-7 or the method of claim 8 or the test kit of claim 9, wherein the sample for the test reagent is whole blood or serum;

preferably, the whole blood comprises fingertip blood or venous blood.