KR20210066725A - A kit for diagnosing allergen of canine and a method of diagnosing allergy using the same - Google Patents

Abstract

According to a dog anti-allergen immunoglobulin E antibody diagnostic kit and a method using the same, it is possible to determine whether a dog is allergen-positive easily, within a short time, and with high precision.

Description

A kit for diagnosing allergens in dogs and a method for diagnosing allergic diseases using the same {A kit for diagnosing allergen of canine and a method of diagnosing allergy using the same}

The present invention relates to a kit capable of diagnosing an allergen causing an allergic disease in dogs by checking the presence of an anti-allergen immunoglobulin E antibody in dogs, and a method for diagnosing an allergic disease using the same.

The number of dogs in domestic breeding is estimated at about 9.25 million, and the number of breeding households is steadily increasing every year. With the increase in the number of dog breeds, diseases of dogs are also increasing, and about 20-30% of dog diseases are caused by skin diseases caused by bacteria, fungi, atopy, and food allergies. These skin diseases are also caused by bacteria and fungi, but recently, the proportion of allergies and atopy is increasing due to changes in the domestic breeding environment. Although skin diseases occur throughout a dog's life cycle, it is becoming a chronic disease, especially in older dogs.

Among skin diseases, atopic dermatitis and food allergy refer to a sudden hypersensitivity reaction that occurs in the body according to the antigen-antibody reaction when a foreign substance enters the living body. Among the causes of allergic diseases, inhaled/contact allergens are known to be important causes of diseases such as otitis media, rhinitis, hair loss, and skin infections even in dogs.

Currently, it takes 1 to 2 weeks for the test results to come out. However, in reality, most dogs who cannot tolerate itching do not receive accurate treatment and response until the test results are available, so lesions and symptoms may develop due to self-injury and secondary infection. often aggravate. In order to treat and treat allergic symptoms, prompt examination and provision of test results at the veterinary hospital are essential, and this is the surest way to prevent the deterioration and expansion of skin lesions and reduce the pain of dogs.

Allergen diagnostic reagents for companion animals have not yet been developed in Korea, and most of them depend on imports. Currently, imported products IDEXX inspection and Alert Panel (AP) inspection are being used a lot. IDEXX is testing 93 types and AP is testing 72 types of allergens. The test takes about 1-2 weeks including the transfer period of the sample. In the case of allergies and atopic dermatitis, if an accurate diagnosis is not made on the removal of the cause of the skin disease from the feed or the surrounding environment, it is difficult to accurately prescribe and cope with the disease. Treatment tends to be prolonged and cure is difficult.

Currently, dog allergy diagnosis in Korea relies on imported products, so tests that contain allergens that occur frequently in the United States or other countries are mainly not allergens (inhalation/contact or food) commonly encountered by domestic companion animals. is being done For this reason, there are many cases where test results do not reach the expected target value of treatment, so diagnostic reagents for allergens with high reproducibility according to the domestic indoor environment and natural environment and diet are needed.

One aspect is a sample pad for dripping the analyte sample; Assay comprising a conjugate pad in which a detection label is conjugated to an anti-dog immunoglobulin E antibody or an anti-canine immunoglobulin G antibody and a absorbent-dried conjugate pad, an allergen-coated test line, and an immunoglobulin G antibody-coated control line strip; And to provide a kit for diagnosing the anti-allergen immunoglobulin E antibody in dogs comprising an absorbent pad that absorbs the assay sample that has passed through the assay strip.

Another aspect comprises the steps of dripping the analyte sample to the sample pad; passing the dripped assay sample through an assay strip; To provide a method for diagnosing an anti-allergen immunoglobulin E antibody in dogs, comprising the step of absorbing an assay sample that has passed through the assay strip by an absorbent pad.

One aspect provides a kit for diagnosing an anti-allergen immunoglobulin E antibody in a dog.

The canine anti-allergen immunoglobulin E antibody diagnostic kit includes: a sample pad for dripping an assay sample; Assay comprising a conjugate pad in which a detection label is conjugated to an anti-dog immunoglobulin E antibody or an anti-canine immunoglobulin G antibody and a absorbent-dried conjugate pad, an allergen-coated test line, and an immunoglobulin G antibody-coated control line strip; and an absorption pad for absorbing the analyte sample that has passed through the analysis strip.

The sample pad, assay strip, and absorbent pad in the kit may be sequentially arranged, and each may be overlapped in some end regions.

As used herein, the term “allergen” refers to an antigen that causes allergic diseases, and the allergen may be an inhalation/contact allergen, a food allergen, or a combination thereof.

The inhaled/contact allergens are North American (USA) house dust mite (D. farinae), European house dust mite (D. pteronyssinus), tropical mite (Blomiatropicalis), coarse leg mite (Acarus siro), long hair mite (Tyrophagus putrescentiae) , Blackant, Tick, Flea, Mosquito, Cockroach, Candida albican, Aspergillus fumigatus, Cladosporium herbarum, Alternaria Alternaria alternate, Penicillium notatum, Staphylococcus aureus, M. pachydermatis, Mixed Feathers, Cow Epithelia, Wool, Cat hair (Cat Hair), Eucalyptus (Eucalyptus Globulus), Poa pratensis, Amaranthus retroflexus, Rumex acetosella, Ragweed, Acacia, Umbrella grass (Cyndon dactylon), And it may be one selected from the group consisting of mulberry (Paper mulberry).

The food allergen is Whole Egg, Cheese, Milk, Yeast baker's, Yeast brewer's, Flax, Wheat white, Soy. bean), Peanut, Rice, Corn, Carrot, Potato, Potato sweet, Pumpkin, Broccoli, Cabbage, Pea (Pea), Tomato, Capsicum annuum, Seaweed, Chicken meat, Duck, Lamb, Beef, Turkey, Pork, Mackerel, Crab, Shrimp, Sardine, Anchovy, Cod fish, Salmon, Tuna, Watermelon ), melon (Cantaloupe), pear (Pear), tangerine (Mandarine), orange (Orange), apple (Apple), banana (Banana), and may be one selected from the group consisting of guava (Guava).

As used herein, the term "immunoglobulin E antibody (IgE: 　immunoglobulin 　 E)" is one of the immunoglobulin classes, and is an antibody composed of two H ε chains and two L chains. Since IgE has affinity for mast cells and basophils in the blood, the IgE antibody attached to them and the allergen corresponding thereto may react to cause an inflammatory reaction. According to one embodiment, since the dog with allergic disease has anti-allergen immunoglobulin E antibody, whereas the normal dog does not have anti-allergen immunoglobulin E antibody, the anti-allergen immunoglobulin E antibody is assayed strip. It can be coated with the inspection line of

As used herein, the term "immunoglobulin G antibody (IgG: 　immunoglobulin 　 G)" is one of the globulin class, and is an antibody composed of two Hγ chains and two L chains. IgG is the main antibody in blood and extracellular fluid, and plasma cells synthesize and secrete IgG. According to one embodiment, in order to check whether the kit is defective, the immunoglobulin G antibody may be coated with a control line of the assay strip.

As used herein, the term “diagnosis” refers to confirming the presence or characteristics of a pathological condition, and may mean including determining whether an allergen exists, whether or not an allergic disease occurs or is likely to occur.

As used herein, the term "analytical sample" may include whole blood, serum, plasma, saliva, urine, sputum, lymph, cells, tissues, etc. isolated from dogs, for example, blood, plasma, isolated from dogs; serum, or a combination thereof.

The sample pad is a pad on which an assay sample to be checked for the presence of anti-allergen immunoglobulin E antibody is loaded, and the material and size thereof may be arbitrarily determined by a person skilled in the art. In one embodiment, the sample pad may be a glass fiber membrane having a size of 0.1 to 1 x 1 to 3 cm.

The assay strip is configured to detect the presence or absence of an anti-allergen immunoglobulin E antibody in an assay sample through an antigen-antibody reaction between the anti-allergen immunoglobulin E antibody and the allergen. In the assay strip, the conjugate of the conjugate pad mixed with the analyte sample may move from one end of the assay strip to the other through capillary action. The material and size of the assay strip are not particularly limited as long as the material and size are suitable for moving an appropriate amount of the assay sample by capillary action and observing the degree of reaction for detecting the immunoglobulin E antibody in the assay sample. A person skilled in the art may determine arbitrarily. According to one embodiment, the assay strip may have a rectangular shape having a size of, for example, 4 mm X 50-100 mm.

The assay strip may be a porous thin film material. For example, the porous thin film material may be nitrocellulose, cellulose, cellulose acetate, fiber glass, polyester, or polyethylene. In one embodiment, the assay strip may be a nitrocellulose membrane.

The porous thin film material may have pores of 5 to 15 um in diameter. When the diameter of the pores is less than 5 μm, when the analysis sample is absorbed from the surface of the analysis strip to the inside, absorption and movement speed due to capillary action are slowed, which is not practical. In addition, when the diameter of the pores exceeds 15 um, the movement speed of the analysis sample is too fast, a problem that inaccurate diagnosis may occur.

The immunoglobulin G antibody coated on the control line of the assay strip may be derived from a species selected from the group consisting of mouse, rat, guinea pig, rabbit, goat, and sheep. In addition, the immunoglobulin G antibody coated on the control line and the immunoglobulin G antibody of the conjugate absorbed and dried on the conjugate pad may be derived from the same species.

Each of the test line and the control line may be coated with an interval of about 10 mm from any one end of the assay strip. In addition, the allergen coated on the inspection line may be coated at an interval of 2 mm to 10 mm. In addition, the allergen coated on the test line and the immunoglobulin G antibody coated on the control line may be coated with an interval of 2 mm to 10 mm.

The conjugate pad of the assay strip may absorb and dry the conjugate by freeze drying, vacuum drying, or forced circulation of dry air. The detection labeling material of the conjugate may be a labeling reagent, an auxiliary specific binding member, and/or a component of a signaling system that allows the presence of an anti-allergen immunoglobulin E antibody to be externally identified through the naked eye or other instruments. . For example, the detection labeling material may be gold particles, latex particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, light emitting materials, or magnetic particles.

The absorbent pad is a pad having a function of absorbing the analyte sample that has passed through the analysis strip, and is used to rapidly absorb the analyte sample dripped on the sample pad to continue the flow of the fluid due to capillary action in the measurement system. will be. The material and size of the absorbent pad may be arbitrarily determined by a person skilled in the art. In one embodiment, the absorbent pad may be a cellulose membrane having a size of 0.1 to 1 x 1 to 5 cm.

According to the kit for diagnosing anti-allergen immunoglobulin E antibody, it can be used to diagnose allergens in dogs and allergic diseases by checking the presence of anti-allergen immunoglobulin E antibodies in an assay sample isolated from dogs.

Another aspect comprises the steps of dripping the analyte sample to the sample pad; passing the dripped assay sample through an assay strip; Provided is a method for diagnosing an anti-allergen immunoglobulin E antibody in dogs, comprising the step of absorbing an assay sample that has passed through the assay strip by an absorbent pad.

It is understood that any of the terms or elements mentioned in the kit as mentioned in the description of the method are as mentioned in the description of the kit above.

The method may further include the step of confirming whether the dog immunoglobulin E antibody and the allergen react with the antigen-antibody by confirming the reaction by the detection label from the analysis sample absorbed by the absorbent pad.

According to the method for diagnosing the anti-allergen immunoglobulin E antibody in dogs, the canine immunoglobulin E antibody and the antigen-antibody reaction of the allergen, which is an antigen thereof, is checked through the reaction with the detection label, thereby confirming the canine allergen and allergy caused by it. disease can be diagnosed.

According to one embodiment, since the anti-allergen immunoglobulin E antibody is not present in the analyte sample of a normal dog not having an allergic disease, it does not bind to the anti-dog immunoglobulin E antibody-detecting label material conjugate of the conjugate pad, and the test line It does not bind to allergens. Accordingly, the anti-dog immunoglobulin E antibody-detecting label material conjugate that has passed through the assay strip without binding to the allergen of the test line is detected by binding to the control line coated with the immunoglobulin G antibody to develop color. The conjugate detected in the control line appeared regardless of the amount of the anti-allergen immunoglobulin E antibody in the assay sample, indicating that the test using the kit was normally performed.

In contrast, since anti-allergen immunoglobulin E antibody is present in the analyte sample of a dog with allergic disease, it binds to the anti-dog immunoglobulin E antibody-detection label conjugate of the conjugate pad, and they move on the assay strip to form the test line. It also binds to an allergen and shows a reaction by a detection labeling substance. Therefore, it is possible to determine whether the anti-allergen immunoglobulin E antibody is present in the analysis sample by comparing the relative color development of the test line in a state in which the control line is colored.

An anti-allergen immunoglobulin E antibody diagnostic kit for canine according to an aspect and a method for diagnosing an allergic disease in dogs using the same can determine an allergen that is a cause of an allergic disease from a dog in a simple and short time on the spot.

In addition, since the anti-allergen immunoglobulin E antibody diagnostic kit for dogs according to an aspect includes not only a test line but also a control line, it is possible to check whether the assay strip itself is defective, so that it can be easily determined with high precision.

1 is a diagram showing a plan view of an assay strip of a dog anti-allergen antibody diagnostic kit.
2 is a schematic diagram showing the principle of color development of an assay strip of a dog anti-allergen antibody diagnostic kit.
3 is a view showing the results of confirming the performance of the dog anti-allergen antibody diagnostic kit using negative sera of normal dogs and positive sera of dogs positive for 5 types of inhalation/contact allergens (1: inhalation/contact allergen group) Normal dog sera for 1, 2: Dog sera positive for 2 allergens including European house dust mite and American house dust mite for inhalation/contact allergen group 1, 3: normal for inhalation/contact allergen group 2 Dog sera, 4: Dog sera positive for 3 allergens including Candida mold, yeast mold, Malassezia for inhalation/contact allergen group 2).
4 is a view showing the results of confirming the performance of the anti-allergen antibody diagnostic kit for dogs using negative sera of normal dogs and positive sera of dogs that are positive for five food allergens (1: negative for food allergen group 1 in normal dogs) Serum, 2: Sera of dogs positive for two allergens including wheat flour and corn for food allergen group 1, 3: negative sera of normal dogs for food allergen group 2, 4: eggs, chicken, and chicken for food allergen group 2, sera of dogs positive for three allergens, including mackerel).

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of allergen antigen

Allergen antigens were used after purchasing crude antigens and partially purified through defatting, extraction, and dialysis processes. Specifically, the degreasing process of the crude allergen was performed as follows. Ether was added in an amount sufficient to submerge the allergen crude antigen, mixed well, and incubated at room temperature for 5 minutes. After that, the ether was carefully decanted, and ether was added again. After repeating this process three times, the ether was carefully decanted and dried until the ether residue was completely evaporated. After that, the extraction process was performed as follows. Phosphate-buffered saline (PBS, pH 8.0) was mixed with the sample in a ratio of 10:1 (v/w), extracted using a homogenizer on ice, and then incubated at 4°C for 48 hours. Thereafter, centrifugation was performed at 4° C. and 15000 rpm for 30 minutes, followed by filtration using a 0.45 um membrane filter. After the dialysis process, the sample was dialyzed using 0.1x PBS (pH 7.5) for 24 hours using a dialysis membrane having a molecular weight cut-off of 1,000. After dialysis, the allergen antigen solution was centrifuged at 4° C. and 15000 rpm for 1 hour, and then filtered using a 0.45 um membrane filter. Proteins were quantified using the Bradford assay, aliquots, and stored at -70°C.

Example 2. Preparation of canine anti-allergen antibody diagnostic kit against allergens, and principle of operation thereof

FIG. 1 is a plan view of an assay strip of a dog anti-allergen antibody diagnostic kit according to an embodiment. As shown in FIG. 1 , the sample pad (glass fiber membrane, 0,5x2 cm) located at the bottom of the assay strip was washed with deionized water, wetted with 1% casein-PBS, and dried prior to treatment. At 0.5 cm from the upper end of the membrane, 4 to 16 μl of a mixed solution of 1% casein-PBS in the same volume as the goat anti-dog immunoglobulin antibody-gold particle conjugate was added and dried under reduced pressure at room temperature. On the thin-film paper (nitrocellulose membrane 0.5x5cm) in the middle, at 0.8cm from the lower end, 2-3 types of allergens [inhalation/contact allergen antigen group 1 (2 types, D. pteronyssinus (European house dust mite) ), D. farinae (American house dust mite)), group 2 (3 species, Candida albican (Candida fungus), Aspergillus fumigatus (lear mold), M. pachydermatis (Malassezia)), and food allergen group 1 (two species) , Wheat whole (wheat), Corn), Group 2 (3 types, whole egg, chicken meat, mackerel) 1 mg/mL each] and rabbit anti-goat immunoglobulin G antibody (0.5 mg/mL) were each immobilized at 0.5 cm intervals. At the time of immobilization of each allergen antigen and antibody, a microsyringe pump was used to eject a small amount of solution (total 0.75 μl) at a constant speed (2 μl/min) to form a thin line in the area where the antibody was immobilized. In addition, an absorbent pad (cellulose membrane, 0.5x3 cm) located on the upper part of the analysis strip was used to quickly absorb an excess of sample and to continue the flow of fluid due to capillary action in the measurement system. These four types of membranes were adhered to the plastic film using double-sided tape so that they overlapped by 0.2 cm in length.

The principle of color development of the assay strip constituting the dog anti-allergen antibody diagnostic kit according to one embodiment is shown in FIG. 2 . As shown in Figure 2, the principle of operation of the dog anti-allergen antibody diagnostic kit is that when 0.1 mL of a sample such as blood, plasma, or serum collected from a dog suffering from an allergic disease to be tested is dropped onto the sample pad, the dog in the sample The anti-allergen immunoglobulin E antibody moves to the conjugate pad conjugated with the specimen pad, and binds to the conjugate pad with the dried goat anti-dog immunoglobulin E antibody-gold particle conjugate. The anti-dog immunoglobulin E antibody-gold particle complex bound to the conjugate moves upward along the assay strip by capillary action while depositing the ends of the assay strip. The canine anti-allergen immunoglobulin E antibody binds to the allergen by an antigen-antibody reaction while passing through the inhalation/contact allergen or food allergen conjugate layer, which is the test line, and anti-immunity bound to the canine anti-allergen immunoglobulin E antibody The color development is exhibited by the globulin E antibody-grade particle conjugate.

Example 3. Performance test of dog anti-allergen antibody diagnostic kit against allergens

To test the performance of the dog anti-allergen immunoglobulin E antibody diagnostic kit prepared as shown in FIG. 1, the anti-allergen immunoglobulin E against the negative sera of a normal dog without antibody and the anti-allergen immunoglobulin E against the three allergens The diagnostic kit was tested using antibody-positive sera.

As a result, as shown in FIG. 3 , the negative serum showed only a chromogenic line caused by the control rabbit anti-goat immunoglobulin G antibody and goat anti-dog immunoglobulin E antibody-gold particle conjugate reaction (see 1 in FIG. 3 ). and 3), it was confirmed that the control line and the three allergen test lines were all colored in the positive sera for the three inhaled/contact allergen antigens (2 and 4 in FIG. 3).

As shown in FIG. 4 , as a result of confirming the performance of the dog anti-allergen antibody diagnostic kit using the negative sera of a normal dog and the positive sera of a dog positive for 5 food allergens, the negative sera for the food allergen was a single color line. On the other hand, it was confirmed that both the control line and the three allergen test lines were colored in the sera positive for the food allergen group (2 and 4 in FIGS. 4).

From the above results, it can be confirmed that the dog anti-allergen antibody diagnostic kit according to one embodiment can detect the anti-allergen immunoglobulin E antibody, thereby diagnosing the allergen, the cause of the allergic disease, in a simple and short time on the spot. .

Claims (12)

a sample pad for dripping the analyte sample;
Assay comprising a conjugate pad in which a detection label is conjugated to an anti-dog immunoglobulin E antibody or an anti-canine immunoglobulin G antibody and a absorbent-dried conjugate pad, an allergen-coated test line, and an immunoglobulin G antibody-coated control line strip; and
A kit for diagnosing canine anti-allergen immunoglobulin E antibody, comprising an absorbent pad for absorbing the assay sample passing through the assay strip. The kit according to claim 1, wherein the assay sample is blood, plasma, serum, or a combination thereof isolated from dogs. The kit according to claim 1, wherein the assay strip is made of a porous thin-film material. The kit according to claim 3, wherein the porous thin film material has pores having a diameter of 5 to 15 um. The kit according to claim 3, wherein the porous thin film material is nitrocellulose, cellulose, cellulose acetate, fiber glass, polyester, or polyethylene. The kit according to claim 1, wherein the detection labeling material of the conjugate is gold particles, latex particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, light emitting materials, or magnetic particles. The method according to claim 1, wherein the allergen coated on the test line is
North America (USA) house dust mite (D. farinae), European house dust mite (D. pteronyssinus), tropical mite (Blomiatropicalis), coarse leg mite (Acarus siro), long hair mite (Tyrophagus putrescentiae), ants (Blackant), mites (Tick), Flea, Mosquito, Cockroach, Candida albican, Aspergillus fumigatus, Cladosporium herbarum, Alternaria alternate, blue Penicillium notatum, Staphylococcus aureus, M. pachydermatis, Mixed Feathers, Cow Epithelia, Wool, Cat Hair, Eucalyptus Eucalyptus Globulus, Poa pratensis, Amaranthus retroflexus, Rumex acetosella, Ragweed, Acacia, Cyndon dactylon, and Paper mulberry an inhalation/contact allergen selected from the group consisting of;
Whole Egg, Cheese, Milk, Yeast baker's, Yeast brewer's, Flax, Wheat white, Soy bean, Peanut Peanut, Rice, Corn, Carrot, Potato, Potato sweet, Pumpkin, Broccoli, Cabbage, Pea, Tomato, Capsicum annuum, Seaweed, Chicken meat, Duck, Lamb, Beef, Turkey, Pork ), Macerel, Crab, Shrimp, Sardine, Anchovy, Cod fish, Salmon, Tuna, Watermelon, Melon ( a food allergen selected from the group consisting of Cantaloupe), Pear, Mandarine, Orange, Apple, Banana, and Guava; or
A kit that is a combination thereof. The kit according to claim 1, wherein the allergen coated on the test line is coated at an interval of 2 mm to 10 mm. The kit according to claim 1, wherein the immunoglobulin G antibody coated on the control line is derived from an individual selected from the group consisting of mouse, rat, guinea pig, rabbit, goat, and sheep. The kit according to claim 9, wherein the immunoglobulin G antibody coated on the control line and the immunoglobulin G antibody of the conjugate absorbed and dried on the conjugate pad are derived from the same species. dripping the analyte sample onto the sample pad;
passing the dripped assay sample through an assay strip;
A method for diagnosing an anti-allergen immunoglobulin E antibody in canine comprising the step of absorbing an assay sample that has passed through the assay strip by an absorbent pad,
The assay strip comprises a conjugate pad in which an anti-dog immunoglobulin E antibody or an anti-dog immunoglobulin G antibody and a detection label conjugated thereto are absorbed and dried, an allergen-coated test line, and an immunoglobulin G antibody-coated control line. A method comprising The method according to claim 11, wherein the method further comprises the step of confirming whether or not the antigen-antibody reaction of the dog immunoglobulin E antibody and the allergen by observing whether or not color is developed by the detection label from the analysis sample absorbed in the absorbent pad. Way.

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