CN111759360A - Allergen patch test diagnosis kit and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine, and particularly relates to an allergen patch test diagnosis kit and a preparation method thereof, in particular to an inhalant and edible allergen patch test diagnosis kit, an allergen and a preparation method thereof. The invention adopts soft and elastic material as a patch test carrier, a test patch is arranged on the carrier, and allergen immersion liquid paste required to be detected is coated on the test patch to prepare a patch adhesive tape and a patch test diagnosis box. The specific concentration allergen immersion liquid in the invention comprises immersion liquids such as pollen allergen, insect allergen, feather allergen, animal hair allergen, grain allergen, egg allergen, meat allergen, aquatic allergen, milk allergen, fruit allergen, dried fruit and oil crop allergen, cigarette smoke allergen and the like, which are respectively prepared into immersion liquid pastes for later use. The result shows that the spot adhesive tape is convenient and safe to operate and use, has good reaction sensitivity and accuracy of the detected skin part, has shorter detection time, and is suitable for detecting contact skin allergy, such as allergic allergy or contact dermatitis, eczema and other etiological factors caused by contact of different reasons and frequently occurring on epidermis and mucosa.

Description

Allergen patch test diagnosis kit and preparation method thereof

Technical Field

The invention belongs to the field of biomedicine, relates to an allergen detection technology, and particularly relates to an allergen patch test diagnosis box and a preparation method thereof, in particular to an inhalant and ingestible allergen patch test diagnosis box, an allergen and a preparation method thereof.

Background

The prior art discloses allergies, i.e. allergic reactions where foreign antigens read as harmful objects such as bacteria, pollen, dust mites, cockroaches, dust etc. The allergic reaction activates macrophages in immune cells to release histamine and prostaglandin, so that the effects of microvascular dilatation, increased vascular permeability, itching, smooth muscle contraction, reflex action and the like are caused; the clinical symptoms mainly include allergic rhinitis, allergic asthma, allergic enterogastritis, eczema, urticaria, macula, thalamus, scratch, atopic dermatitis, wheezy skin, skin pruritus and other allergic skin diseases. Detection means in clinical practice include detection of allergen-specific IgE in serum, in vivo allergen detection methods of skin prick, etc., but the methods have disadvantages such as possibility of causing immune reaction in vivo, relatively much serum required for long detection time, etc.; the subject is not easy to accept.

The microfluidic chip technology (Microfluidics) and the protein chip (protein array) high-throughput protein characteristic analysis technology are reported, and specific detection equipment, a series of biochemical detection processes and the like are required.

Based on the basis and the current situation of the prior art, the inventor of the application intends to provide an allergen patch test diagnosis kit and a preparation method thereof, in particular to an inhalant and ingestible allergen patch test diagnosis kit, an allergen and a preparation method thereof.

Disclosure of Invention

The invention aims to provide a non-invasive allergen detection technology based on the basis and the current situation of the prior art, in particular to an allergen patch test diagnosis box and a preparation method thereof, and especially relates to an inhalant and ingestible allergen patch test diagnosis box and a preparation method thereof.

Generally, when skin or mucosa of an allergic patient contacts a sensitizing substance, the sensitizing substance enters the body through the skin or mucosa, and antigen is presented to T lymphocytes by antigen presenting cells, so that specific T lymphocytes are activated to induce an inflammatory reaction;

the invention adopts soft and elastic material as a patch test carrier, a test patch is arranged on the carrier, and allergen immersion liquid paste required to be detected is coated on the test patch to prepare a patch adhesive tape and a patch test diagnosis box.

In the embodiment of the invention, the patch test carrier and the patch adhesive tape are prepared from paper or cloth materials.

In the present invention, suspected sensitizers to be tested are prepared into allergen immersion liquid and immersion liquid paste with specific concentration, and the immersion liquid and immersion liquid paste are loaded on a test point and applied to the covered parts of human body, such as the back, the bent side of the forearm, etc., and after a certain period of time, whether the test object is a sensitizer is determined according to the existence of positive reaction.

In the present invention, the allergen immersion liquid of a specific concentration includes pollen allergens, insect allergens, feather allergens, animal hair allergens, grain allergens, egg allergens, meat allergens, aquatic allergens, milk allergens, fruit allergens, dried fruit and oil crop allergens, cigarette smoke allergen immersion liquid, and the like; respectively making into extract.

The spot adhesive tape prepared by the invention is packaged into a box to prepare a spot test diagnosis box, and the packaging specifications are 10 parts per box, 50 parts per box, 100 parts per box and 200 parts per box. 100 per box, and storing at 4-30 ℃ in the dark.

The invention provides a preparation method of an allergen extracting solution, which comprises the following steps:

1. buffered saline solution (buffered saline solution pH:8)

The formula is as follows:

sodium chloride A.R. 5.0 g

Potassium dihydrogen phosphate A.R. 0.36 g

Disodium hydrogen phosphate A.R. 7.0 g

Crystalline phenol A.R. 4.0 g

1000 ml of distilled water

Mixing the above materials, heating in water bath, measuring pH to below 8 or above 8, adjusting with 10N sodium hydroxide or hydrochloric acid, and storing at room temperature.

Or the like, or, alternatively,

2. bicarbonate-saline solution [ bicarbonate salt solution's solution ] pH 8.2]

The formula is as follows:

sodium chloride A.R. 5.6 g

Sodium bicarbonate A.R. 275 g

Crystalline phenol A.R. 4.0 g

1000 ml of distilled water

After the medicine is completely dissolved, measuring and adjusting the pH value to 8.2, and storing at room temperature for later use.

The above extractive solution can be selected for use.

The present invention also provides a positive control, wherein,

hydrochloric acid to phosphoric acid of 0.6 to 1

Histamine hydrochloride contains histamine 111/184 ═ 0.6

Histamine phosphate histamine containing 111/325 ═ 0.34

Adding 28.5 ml of distilled water into each gram of histamine phosphate; 28.5 ml of glycerol

Negative control:

50% of normal saline and 50% of glycerol.

When the patch test diagnosis kit is used, the prepared allergen immersion paste is loaded on a detection point and is applied to a human body covering part, and is usually applied to the bent side of the back and the forearm, and whether a test object is a sensitizing substance is determined according to the existence of positive reaction after a certain time.

The using method comprises the following steps: test sites: normal skin on both sides of the upper back spine or on the flexor side of the forearm;

cleaning skin of the detected part (removing oil), and wiping; removing the protective paper of the spot adhesive tape, firmly and flatly sticking the spot adhesive tape containing the low molecular compound from bottom to top, and lightly pressing the spot adhesive tape with a palm to discharge air; patch test time: 12-24 hours; observation time: after 12-24 hours of application, firstly removing the spot adhesive tape, and observing the result after removing the spot adhesive tape for at least 30 minutes in order to avoid the reaction possibly caused by pressing the skin by a detection point.

And (5) judging a result:

(question mark) -suspicious reaction; only faint (unclear) erythema.

(+) -weak (no blister) positive reaction; erythema, infiltrates, possibly with small rashes.

(+) - (blister) -strong positive reaction; erythema, infiltration, papules non-small blisters.

(+) -extremely positive reaction; red and swollen with bulla.

(-) - -No reaction.

(IR) -different types of stimulus responses.

(NT) -was not tested.

The result shows that the spot adhesive tape is convenient to operate and use, safe, good in accuracy, capable of converting the allergen immersion paste with specific concentration into liquid under the influence of body temperature, good in liquid absorbability and capable of ensuring the sustained and effective release of a detected object, good in reaction sensitivity and accuracy of a detected skin part, short in detection time, and suitable for detecting contact skin allergy, such as allergic allergy or contact dermatitis, eczema and other etiological factors caused by contact of different reasons and mostly occurring on epidermis and mucosa.

The invention provides an allergen patch test diagnosis box and a preparation method thereof, in particular to an inhalant and edible allergen patch test diagnosis box and a preparation method thereof. The kit is suitable for detecting contact skin allergy, such as allergic allergy which is caused by contact of different reasons and is frequently generated on epidermis and mucosa, contact dermatitis, eczema and other causes, and has the advantages of convenient use, safety and good accuracy.

Detailed Description

Example 1 preparation of pollen allergen steep

The material collecting and processing method comprises the following steps:

taking Mallotus japonicus, palm, Artemisia, rape, ragweed, humulus scandens, corn and the like, degreasing benzene, toluene and diethyl ether once for about 4-5 hours respectively, wherein the dosage of the diethyl ether is 2-3 times of that of the pollen;

the preparation method of the allergen immersion liquid comprises the following steps:

1) adding defatted pollen of Artemisia, ragweed, Sterculia Sprensis, palm, Artemisia, rape, ragweed, herba Humuli Scandentis, and semen Maydis into bicarbonate-saline extractive solution at a ratio of 1:25 (1 g pollen plus 25 ml extractive solution), extracting the rest pollen with bicarbonate-saline extractive solution at a ratio of 1:20 (1 g pollen plus 20 ml extractive solution), and shaking or stirring for 2 hr every day during 72 hr extraction in refrigerator;

2) after extraction, filtering the clear liquid at normal pressure; the pollen extract material is viscous, and can be separated by centrifugation and filtered under normal pressure, or filtered by Buchner funnel;

3) the pH was measured and corrected to 7;

4) after the sterilization and filtration by a sterilization Chua's filter, subpackaging the mixture into sterilized vaccine bottles under the aseptic operation;

5) 1 ml of sample is extracted from each bottle for bacterial and mould sterilization examination;

6) injecting 0.5 ml pollen allergen stock solution into abdominal cavity of 5 mice per mouse, and feeding for one week without death;

7) extracting 2 ml of stock solution under aseptic operation for protein nitrogen determination;

8) labeling, applying batch number, filling the preparation record sheet, and refrigerating in a refrigerator at-4-6 deg.C for use.

Example 2 preparation of insect allergen immersion liquid

Taking cockroaches, moths, houseflies, mosquitoes and the like;

the material processing method comprises the following steps:

1) inactivating materials such as cockroach, moth, housefly, mosquito, etc., naturally drying at room temperature, and then primarily smashing by using a tissue mashing machine;

2) adding acetone to degrease for 4 hr, pouring out acetone with grease, taking out material, breaking with tissue triturator after residual acetone is completely volatilized in toxic gas cabinet, sieving with No. 3 sieve or No. 4 sieve to obtain powder, and degreasing with diethyl ether for 2 times for 6-7 hr;

3) putting the degreasing material into a glass bottle for sealed storage without being affected with damp;

preparing allergen steep

1) Adding bicarbonate-saline extracting solution into various insect degreasing materials according to the proportion of 1:50 (1 g of degreasing insect dry powder and 50 ml of extracting solution):

2) extracting in refrigerator for 72 hr, and shaking or stirring for 2 hr to dilute;

3) after filtering the clarified liquid at normal pressure, measuring and correcting the pH to 7;

4) sterilizing, filtering, and subpackaging in vaccine bottles;

5) 1 ml of sample is taken from each bottle and inoculated into broth and Sabouraud's medium respectively for bacteria and mould examination;

6) all kinds of insect allergen immersion liquid should be used for toxicity test of mice before being applied to clinic for the first time, and moth and cockroach materials should be used for toxicity test again after changing batch number each time;

7) extracting sample, and performing protein nitrogen determination

8, labeling, filling and writing the preparation record sheet, and placing the prepared record sheet in a refrigerator for refrigeration for later use.

Example 3 preparation of feather allergen steep

Taking chicken feather, duck feather, pigeon feather and the like;

method for collecting and treating materials

1) Directly collecting clear water from the bodies of poultry or pigeons to wash away dirt on the feathers;

2) shearing the material as much as possible by using scissors;

3) defatting with diethyl ether for 1 time, about 4 hours;

preparing allergen steep

1) Adding the extracted solution of bicarbonate-saline into the degreased and cut feather according to the proportion of 1:15 (1 g of material is added with 15 ml of extracting solution);

2) placing into refrigerator for extraction for 72 hr, and stirring for 2 hr every day during extraction process;

3) wetting multiple layers of clean gauze with distilled water, squeezing out juice, filtering under normal pressure, clarifying liquid, and measuring and correcting pH to 7;

4) sterilizing, filtering, and subpackaging in sterile vaccine bottles;

5) extracting a sample, and carrying out sterilization examination on bacteria and mould;

6) the first time is used for a toxicity test of mice before clinic;

7) extracting a sample, and performing protein nitrogen determination;

8, sticking a label, marking a batch number, filling and writing a preparation record sheet, and putting the prepared record sheet into a refrigerator with the temperature of 4-6 ℃ above zero for storage.

Example 4 preparation of animal Hair allergen steep

Taking cat hair, dog hair and the like;

the material collecting and processing method comprises the following steps:

1) the animal skin scraps can be collected by a comb, a brush and other tools;

2) shearing the material as much as possible by using scissors;

3) defatting with diethyl ether for 1 time for about 4 hours;

preparing allergen steep

1) The degreased animal hair and dandruff are extracted by adding bicarbonate-saline according to the proportion of 1:15 (1 g material and 15 ml extract)

Liquid for treating urinary tract infection

2) Extracting in refrigerator for 72 hr while stirring for 2 hr

3) Squeezing juice with multi-layer gauze, filtering clarified liquid at normal pressure, and measuring and correcting pH to 7;

4) sterilizing, filtering and subpackaging in vaccine bottles;

5) extracting a sample to perform bacteria and mould sterilization inspection;

6) the first time is used for a toxicity test of mice before clinic;

7) extracting a sample to perform protein nitrogen determination;

8) placing the labeling label with batch number filling preparation record sheet into a refrigerator at-4-6 deg.C for storage

Example 5 preparation of cereal allergen infusions

Taking corn, wheat, barley, millet, rice and the like;

the material collecting and processing method comprises the following steps:

1) the grains can be pulverized by electric pulverizer and sieved with No. 3 or No. 4 sieve, or can be directly processed into commercially available flour

2) Defatting with diethyl ether for 1 time for about 4 hours;

preparing an allergen immersion liquid:

1) adding the buffered saline solution into various defatted powdery cereals at a ratio of 1:10 (1 g material and 10 ml extract);

2) extracting in refrigerator for 48 hr while shaking or stirring for 2 hr;

3) because the liquid is viscous, firstly using a Buchner funnel and filter paper to filter, and then filtering at normal pressure;

4) measuring and correcting the pH to 7;

5) sterilizing, filtering and subpackaging in vaccine bottles;

6) extracting a sample, and performing bacterial sterilization inspection and protein nitrogen determination;

7) labeling, marking a batch number, filling a preparation record sheet, and putting into a refrigerator with the temperature of 4-6 ℃ below zero for storage;

example 6 preparation of egg allergen infusions

The material collecting and processing method comprises the following steps:

taking the eggs and the like, wherein the eggs are taken,

1) washing fresh egg with water, making small holes at two ends of the egg to make the egg protein flow downwards, collecting with measuring cylinder, and recording liquid amount, wherein if drainage is not smooth, 5 ml or 10 ml empty needle (with needle removed) can be used to extract protein;

2) the yolk allergen preparation method comprises removing egg white from yolk, and making yolk into powder, wherein the yolk is placed in a triangular flask, thoroughly crushed, and then added with an appropriate amount of acetone for defatting for about 2-3 hr, the flask is continuously shaken during acetone soaking process, after defatting is completed, acetone with lipid is poured off, and after residual acetone in the yolk is volatilized, the yolk is crushed by a crusher, and finally, the yolk is defatted with diethyl ether for 1 time for about 4 hr, and stored for later use;

preparing an allergen immersion liquid:

1) adding the egg white into the buffered saline extract according to the proportion of 1:20 (adding 20 ml of the extract into 1 ml of egg white) for dilution without extraction, and adding the yolk defatted dry powder into the buffered saline extract according to the proportion of 1:50 for extraction for 48 hours;

2) the albumin solution is difficult to be viscous and filtered, and the reduced pressure filtration is preferably used;

3) measuring and correcting the pH to 6.5, egg products are prone to floc during storage, and thus lowering the pH helps to retard the formation of precipitates

The rest steps are the same as the previous steps.

Example 7 preparation of meat allergen infusion

Taking beef, chicken and the like;

the material collecting and processing method comprises the following steps:

1) cleaning fresh lean meat with clear water, removing fat and connective tissue, and mincing with knife or breaker;

2) alternately using acetone, toluene or xylene, repeatedly defatting with diethyl ether, naturally drying, crushing, sieving, and making into powder

Sieving with No. 3 or No. 4 sieve to remove fat;

preparing allergen steep

1) Adding the degreasing powder of various meats into the buffer saline extracting solution according to the proportion of 1:25 (1 g of material is added with 25 ml of extracting solution);

2) extracting in refrigerator for 48 hr while shaking or stirring for 2 hr;

3) filtering out precipitate with multilayer gauze, and filtering to clarify liquid at normal pressure;

4, measuring and correcting the pH value to 6.5, wherein the pH value is adjusted to be low because the meat product is easy to precipitate in the refrigeration process;

the rest steps are the same as the previous steps.

Example 8 preparation of an aquatic allergen infusion

Taking hairtail, sea crab, sea shrimp and the like;

the material collecting and processing method comprises the following steps:

1, cleaning fresh materials with water, scraping off scales and epidermal tissues, removing internal organs, and removing shells of shrimps, crabs and shellfish;

2) chopping with a knife or a crusher, repeatedly defatting and naturally drying with acetone, toluene or xylene and diethyl ether alternately,

crushing to obtain powder with no fat removed by No. 3 or No. 4 sieve;

preparing an allergen immersion liquid:

1) the degreasing materials of meat and aquatic products are prepared according to the following steps of 1:25 portions (1 g material plus 25 ml extract) are extracted with buffered saline

Taking liquid;

the rest steps are the same as the previous steps.

EXAMPLE 9 preparation of milk allergen infusions

Taking milk and the like;

the material collecting and processing method comprises the following steps:

1) using the unprocessed, modified and pasteurized fresh emulsion, centrifugation (2400-

At the moment, the fat in the emulsion floats on the surface of the centrifugal tube, and the fat layer is removed by a small medicine spoon;

2) adding 5 ml of 1% rennet or lactone sheet (preparation method of 1% rennet or lactone sheet; weighing) into 400 ml of degreased emulsion

Adding 0.5 g rennin or lactone tablet into 50 ml distilled water, dissolving completely, stirring or shaking, and placing at 37 deg.C

Whey was separated from the coagulated, acidified milk in a water bath for 30 minutes;

3) filtering with multi-layer gauze to obtain whey containing albumin, and casein as precipitate in the gauze;

4) adding acetone into whey filtrate containing albumin, placing into refrigerator (at room temperature for 24 hr), centrifuging for 15 min to remove acetone, volatilizing residual acetone, grinding into powder,

finally, degreasing for 1 time by using ether for about 3-4 hours, and hermetically storing for later use;

5) the casein contains much fat, and is subjected to alternate defatting with acetone and diethyl ether for 3-4 times and once pulverizing each time

Then the mixture becomes powder which passes through a No. 3 or No. 4 sieve;

preparing allergen steep

The whey filtrate was used as prepared according to 1: diluting with 2% (1 ml of whey and 2 ml of extractive solution) in buffered saline

Without extraction, the preparation method comprises mixing lactalbumin and casein degreasing powder at a ratio of 1:50 (1 g powder material and 50 ml)

Extract) adding buffered saline extract, extracting in refrigerator for 48 hr, and shaking or stirring for 2 hr per day during extraction;

the rest steps are the same as the previous steps.

Example 10 preparation of fruit allergen infusion

Taking apples, strawberries, bananas, mangoes and the like;

the material collecting and processing method comprises the following steps:

cleaning fresh fruits, removing skin and core, and mashing into jam with tissue mashing machine;

juicy fruits can also be prepared by squeezing juice of the juicy fruits;

preparing an allergen immersion liquid:

extracting the crushed jam with 1:1 buffer saline solution for 48 hr, and extracting the juice with 2:1(2 ml juice and 1 ml juice)

Lifting the extract) is added into the juice extract to be diluted;

the rest steps are the same as the previous steps.

EXAMPLE 11 preparation of allergen extracts from dried fruits and oil crops

Taking peanuts, soybeans and the like;

the material collecting and processing method comprises the following steps:

1) taking raw and unprocessed materials, and a person with the skin needs to remove the skin and break the skin by a breaker;

2) alternately defatting with toluene, acetone and diethyl ether, and crushing to obtain defatting dry powder passing through No. 3 or No. 4 sieve to obtain allergen extract;

preparing an allergen immersion liquid:

1) the degreasing powder is prepared by mixing 1:25 (1 g material with 25 ml extract and buffer saline extract;

2) extracting in refrigerator for 48 hr while shaking or stirring for 2 hr;

3) filtering the clear liquid at normal pressure, and removing fat by adding diethyl ether into the filtrate with a separating funnel if the filtrate is not clear;

the rest steps are the same as the previous steps.

EXAMPLE 12 preparation of cigarette Smoke allergen infusion

The material collecting and processing method comprises the following steps:

1) selecting commercially available cigarettes of upper, middle and lower qualities for mixed use;

2) 2 cigarette holders or small glass tubes with the thickness similar to cigarettes, a suction bottle (500 or 1000 ml) and rubber tubes, hemostats and the like are prepared. Connecting the other end of the rubber tube connecting the cigarette holder or the small glass tube and a rubber tube with the length of about 5 cm to a bent glass tube on the bottle stopper of the first aspirator, wherein the inner end of the inlet glass tube is directly inserted into the bottle bottom through the bottle stopper;

preparing an allergen immersion liquid:

1) adding buffered saline solution at a ratio of 1:2 (2 ml of solution in 1 cigarette), and simultaneously placing the solution into two bottles, wherein the amount of the solution in the first bottle is more than that in the second bottle, for example, if the first bottle is added into 100 ml of the second bottle, 60 ml of the solution is added;

2) the cigarettes are arranged on a cigarette holder or a small glass tube, a vacuum pump is started to ignite the cigarette smoke, the vacuum pump is closed when every cigarette is burnt out in the extracting solution in the bottle or the vacuum pump is started to ignite the next cigarette smoke after the cigarette smoke which is in the extracting solution and is to be soared in the rubber tube is clamped by hemostatic forceps is stabilized, and the vacuum pump is started to ignite the next cigarette until all the required cigarettes are burnt out;

3) pouring the extracting solutions in the two bottles together, uniformly mixing, and filtering under normal pressure;

4 measuring and correcting the pH to 7;

and 5, sterilizing and filtering. Subpackaging in vaccine bottles;

6, extracting 1 ml of sample from each bottle for respectively carrying out bacterial and mould sterilization examination;

7) the first time is used for a toxicity test of mice before clinic;

8) extracting a sample for protein nitrogen determination;

9) after all the instruments are used, the instruments are soaked in 95% alcohol and then scrubbed by soapy water, and the tobacco tar on the utensils is removed.

Example 13 preparation of House dust allergen immersion liquid

Taking bedroom dust, cotton dust of textile factories and the like;

the material collecting and processing method comprises the following steps:

1) the mixture is lightly brushed in a clean vessel by a brush and a small broom, and is preferably collected by a small dust collector. The collection should be carried out by people who are not allergic to house dust or industrial dust instead to prevent diseases. Preferably, the medical staff collects the materials by themselves to ensure the quality of the materials, and if the materials are collected by patients and family members of the patients, the names, medical record numbers and collection places of the patients are noted on the material packages; particularly, no matter people collect the materials, the materials with oil and wax can not be collected, and particularly, the materials in places where toxic substances are placed are forbidden to be collected;

2) the house dust collecting place is taken from dust accumulated on the top of a cabinet, furniture and in a mattress and a sofa for a long time, but not dust swept from the ground; if the cotton is collected in the cotton mill, the dust falling from the machine is taken; industrial dust in flour mills and textile mills can sweep materials deposited beside machines and on the ground in a workshop, and especially old dust deposited for a long time is collected;

the material processing method comprises the following steps:

1) after the material is taken back to the laboratory, it should be checked whether the material is satisfactory, for example, if it is found that the material has poor quality or has suspicious substances, it should be discarded;

2) drying the qualified materials at room temperature, and then sieving the materials through a No. 3 or No. 4 sieve to remove impurities;

3) sequentially degreasing dust and industrial dust with toluene, anhydrous ethanol or 95% ethanol and diethyl ether for about 4-5 hr, pouring out the solvent with grease, volatilizing the residual solvent, placing into a glass bottle, labeling, noting the name of the material, and storing in shade and dark place in a sealed manner for use without being affected with damp;

preparing an allergen immersion liquid:

1) the lipid-removed material was taken and added to the bicarbonate-saline extract at a ratio of 1:5 (1 g material plus 5 ml extract). Extracting in refrigerator for 72 hr, and shaking or stirring with electric stirrer for 2 hr every day during extraction;

2) after extraction, filtering with multilayer gauze, removing precipitate, vacuum filtering with Buchner funnel and multilayer filter paper to clarify liquid;

3) weighing and recording the amount of the filtrate, putting the filtrate into a dialysis sac for dialysis for 24 hours, using a bicarbonate-saline extracting solution as a solution for dialysis, replacing the solution once every 6 hours, weighing the filtrate amount again after the dialysis is finished, if the original amount is increased due to salting-out, pouring the filtrate into the dialysis sac again, and concentrating at room temperature until the original amount is reached;

4) measuring and correcting the pH to 7;

5) after the sterilization and filtration by a sterilization Chua's filter, subpackaging the mixture into sterilized vaccine bottles under the aseptic operation;

6) taking 1 ml of sample from each bottle, inoculating into culture medium (or broth culture medium) and Sabouraud's medium respectively under aseptic operation, and performing bacterial and mold sterilization examination;

7) after the sterilization check is negative, performing a mouse toxicity test, taking 5 mice with 17-22 g, injecting 0.5 ml house dust or dust allergen solution into each abdominal cavity, observing and feeding for a week, and applying the mice after no death;

8) extracting 2 ml of stock solution of the sample under aseptic operation, and carrying out protein nitrogen determination;

9) labeling, printing a batch number, filling a prepared record list, and refrigerating in a refrigerator at-4-6 ℃ for later use.

Example 14 preparation of fungal allergen infusions

Taking penicillium, saccharomycetes and the like;

the laboratory collection and treatment method comprises the following steps:

1) taking the strain preserved in the laboratory, and identifying the strain to have pollution, variation or sterility under the slide of the slide. If the conditions are not met, the strain is inoculated into a liquid culture medium or a solid culture medium under aseptic operation, the liquid culture medium is used for inoculating filamentous sensitized fungi, and the solid culture medium can be used for inoculating a small number of strong toxigenic fungi and pathogenic fungi to prevent pollution;

2) culturing at room temperature for several days, wherein the culture days can be determined according to the growth speed of lawn, and spore growth is preferably up to peak, and during the growth process, if bacteria or other fungi are found to pollute the lawn, the lawn is discarded uniformly;

3) after the culture grows up, the flask can be shaken firstly to wet the spore layer, so that the spores are prevented from scattering around when poured out, then the lawn is poured into a No. 3 sieve together with the culture medium, the sieve is placed in a water pool, and the residual culture medium on the lawn is washed away by clear water;

4) removing water, and soaking the culture in 95% ethanol for 4 hr for inactivation;

5) if the culture is cultured in a solid medium, a small amount of toluene or acetone is poured into each bottle after the culture is grown, and the culture in the bottle can be killed and washed after 1-2 hours;

6) pouring out alcohol, spreading the inactivated thallus Porphyrae, air drying, naturally drying, pouring out the culture in solid culture medium together with toluene or acetone, naturally volatilizing, and collecting the culture;

7) crushing the dried culture in a tissue triturator to obtain powder;

8) methanol and diethyl ether are degreased once respectively for about 4-5 hours. Hermetically storing for later use;

preparing an allergen immersion liquid:

1) adding bicarbonate-saline solution into all fungi at a ratio of 1:25 (1 g dried powder of defatted fungi and 25 ml extractive solution);

2) extracting in refrigerator for 72 hr while shaking or stirring for 2 hr;

3) filtering at normal pressure to clarify the liquid;

4) measuring and correcting the pH to 7;

5) sterilizing with a sterilizing filter, and subpackaging in sterile vaccine bottles;

6) extracting 1 ml of sample from each bottle, respectively inoculating the samples into a meat soup culture medium and a Sha's culture medium, and carrying out sterilization examination on bacteria and mould;

7) before the first application of various fungi in clinic, all the fungi are used for mouse toxicity test;

8) extracting a sample to perform protein nitrogen determination;

9) labeling, applying batch number, filling preparation record, and storing in refrigerator at-4-6 deg.C.

Example 15 preparation of dust mite allergen immersion liquid

House dust mite (laboratory culture),

the material processing method comprises the following steps:

1) after acetone inactivation and cleaning, filtering out acetone by using a plurality of layers of gauze, taking mite-containing materials remained in the gauze, and performing smear microscopy to obtain a qualified product with the mite content of more than 80%;

2) adding diethyl ether into the qualified material after microscopic examination for degreasing once (about 4 hours), and then storing in a sealed manner for later use;

the preparation method of the allergen diffusion liquid comprises the following steps:

1) taking the dried powder of the dust mites to be removed, adding bicarbonate-saline extracting solution into the dried powder of the dust mites according to the proportion of 1:50 (1 g of materials is added with 50 ml of extracting solution);

2) extracting in refrigerator for 72 hr. In the extraction process, oscillating or stirring for 2 hours every day;

3) filtering the clarified liquid at normal pressure;

4) measuring and correcting the pH to 7;

5) sterilizing, filtering, and subpackaging in sterile vaccine bottles;

6) extracting a sample, and carrying out sterilization examination on bacteria and mould;

7) the first time is used for the preclinical test, and a mouse toxicity test is carried out;

8) aseptically extracting a sample, and carrying out protein nitrogen determination;

9) labeling, applying batch number, filling in preparation record, and refrigerating in a refrigerator at-4-6 deg.C.

EXAMPLE 16 preparation of fiber and cereal husk allergen infusions

Taking cotton wool, silk and the like;

the material collection method comprises the following steps:

1) the silk is prepared from unprocessed and modified materials, and is generally prepared from silkworm cocoons, and silk cottons and silk products are subjected to heat treatment and are not suitable for use;

2) the cotton wool needs to be selected with a new material to extract effective components in the fiber of the cotton wool, while old materials which are used for a long time contain a large amount of organic dust, various microorganisms, mites and the like, and become a mixture consisting of a plurality of components, and the results obtained by skin test of the leachate can not draw the conclusion of allergy to the materials, for example, if the allergen leachate is prepared from the old materials, the evidence needs to be taken, and absorbent cotton can not be used for extracting the cotton wool material;

preparing an allergen immersion liquid:

1) adding bicarbonate-saline solution into various fibers and cereal husks at a ratio of 1:15 (1 g material and 15 ml extract);

2) extracting in refrigerator for 72 hr while stirring for 2 hr each day;

3) squeezing out juice with multiple layers of gauze, filtering the clarified liquid at normal pressure, and measuring and correcting pH to 7;

4) sterilizing, filtering, and subpackaging in vaccine bottles;

5) extracting a sample, and carrying out sterilization examination on bacteria and mould;

6) the first time is used for the preclinical test, and a mouse toxicity test is carried out;

7) extracting a sample to perform protein nitrogen determination;

8) labeling, adding batch number, filling preparation record, and refrigerating in a refrigerator at-4-6 deg.C.

Claims (7)

1. An allergen patch test diagnosis box is characterized in that a flexible and elastic material is adopted as a patch test carrier, a test patch is arranged on the carrier, and an allergen immersion fluid paste to be detected is coated on the test patch to prepare a patch adhesive tape and the patch test diagnosis box.

2. The allergen patch test diagnosis kit according to claim 1, wherein the patch tape is made of paper or cloth material to prepare the patch test carrier and the patch tape.

3. The allergen patch test diagnosis kit according to claim 1, wherein the allergen immersion liquid paste is an allergen immersion liquid and an immersion liquid paste prepared by formulating a suspected sensitizing substance to be detected into a specific concentration.

4. The allergen patch test diagnostic kit according to claim 3, wherein the allergen immersion liquid of specific concentration comprises pollen allergen immersion liquid, insect allergen immersion liquid, feather allergen immersion liquid, animal hair allergen immersion liquid, grain allergen immersion liquid, egg allergen immersion liquid, meat allergen immersion liquid, aquatic allergen immersion liquid, milk allergen immersion liquid, fruit allergen immersion liquid, dried fruit and oil crop allergen immersion liquid, cigarette smoke allergen immersion liquid.

5. The allergen patch test diagnosis kit according to claim 3, wherein the allergen immersion liquid of a specific concentration is prepared as an immersion liquid paste for use.

6. The allergen patch test diagnosis kit according to claim 1, wherein the patch tape is packaged into a kit to make a patch test diagnosis kit, and the packaging specifications are 10 persons/kit, 50 persons/kit, 100 persons/kit, 200 persons/kit; 100 per box, and storing at 4-30 ℃ in the dark.

7. The allergen patch test diagnostic kit according to claim 1, wherein the allergen extract is prepared by a method comprising:

1) buffered saline solution (buffered saline solution pH:8)

The formula is as follows:

sodium chloride A.R. 5.0 g

Potassium dihydrogen phosphate A.R. 0.36 g

Disodium hydrogen phosphate A.R. 7.0 g

Crystalline phenol A.R. 4.0 g

1000 ml of distilled water

Mixing the above materials, heating in water bath, measuring pH to below 8 or above 8, adjusting with 10N sodium hydroxide or hydrochloric acid, and storing at room temperature;

or the like, or, alternatively,

2) bicarbonate-saline solution [ bicarbonate salt solution's solution ] pH 8.2]

The formula is as follows:

sodium chloride A.R. 5.6 g

Sodium bicarbonate A.R. 275 g

Crystalline phenol A.R. 4.0 g

1000 ml of distilled water

After the medicine is completely dissolved, measuring and adjusting the pH value to 8.2, and storing at room temperature for later use.