Contact allergen reagent combination and use thereof

Abstract

The present invention provides a contact allergen reagent combination and its use. Specifically, it relates to the use of the contact allergen reagent combination for preparing a kit for diagnosing potential allergens of skin allergies of a subject, wherein the allergen reagent combination includes at least five selected from the group consisting of nickel sulfate, potassium dichromate, disperse blue 106, cobalt chloride, aromatic mixture I, methylisothiazolinone, thimerosal, sodium lauryl sulfate, carbazine mixture, formaldehyde, iodopropynyl aldehyde and p-phenylenediamine. The kit of the present invention can quickly and accurately identify skin allergens of Chinese people, with a high detection rate.

Description

Technical Field

[0001] The invention relates to the medical field, and in particular to a contact allergen reagent combination and a use thereof. Background Art

[0002] The patch test is a simple and reliable method to determine the allergens of patients with dermatitis and eczema. When a patient develops an allergy due to skin or mucous membrane contact with an allergen, when the same allergen or a chemically similar substance with the same antigenicity comes into contact with any part of the body surface, skin inflammation changes will soon appear at the contact site. This is allergic contact dermatitis. The patch test uses this principle to artificially configure the suspected allergen into a certain concentration, place it in a special small room and apply it to the covered part of the human body (usually on the back and the extended side of the upper arm). After a certain period of time, whether the test substance is an allergen (i.e., a sensitizing substance) is determined based on whether there is a positive reaction. If the substance that causes the body's allergies can be identified through the test, targeted prevention and treatment can be taken.

[0003] The patch test is the gold standard for identifying contact allergens. It has a history of more than 100 years and has been used in China for more than 30 years. The antigen positivity rate of the patch test varies to a certain extent among different races and regions. The United States, Europe, Japan, etc. have established standard antigen series in various places. The systematic use of these antigen series has made researchers realize that geographical differences can lead to differences in the baseline of the antigen series. However, to date, there is no recognized patch test antigen series suitable for my country's national conditions, which greatly limits the promotion and application of this test in clinical practice, as well as the etiology diagnosis and treatment of clinical patients.

[0004] In the late 1980s and early 1990s, some Chinese dermatologists also realized the importance of antigen series for patch testing, and several baseline standard series were also developed. Among them, the Beijing Medical University series is basically modified according to the allergen series of the North American Contact Dermatitis Group (NACDG). It contains 19 allergens and 6 mixtures, and has been applied nationwide in China. During the same period, the standard series developed by Nanjing Medical University was called the Nanjing Medical University Standard Series. This series contains 20 allergens selected from the standard series of the International Contact Dermatitis Research Group (ICDRG), but currently this series is no longer used in the Chinese market. However, some dermatologists prefer to use the European standard series directly without any changes.

[0005] Although some of the above series have been used in China for many years and are claimed to be standard series, all of these "Chinese standard series" are based on foreign antigen series and are simply revised according to the experience of the developers, and are not supported by clinical research data in my country. Therefore, to date, China has no national standard method or allergen series for patch testing based on evidence-based medicine.

[0006] Therefore, there is an urgent need to provide an allergen reagent combination for Chinese skin allergies, which can be used to prepare reliable patch test kits with high detection rates, so as to improve the efficiency of skin allergen detection, enhance the diagnosis level of contact dermatitis in my country, better serve patients, reduce the incidence of contact dermatitis, and improve the quality of life of patients. Summary of the invention

[0007] The purpose of the present invention is to provide a contact allergen reagent combination for Chinese skin allergies, which is used for preparing a diagnostic skin allergen kit and a reliable kit with a high detection rate based on the contact allergen reagent combination.

[0008] The first aspect of the present invention provides a use of a contact allergen reagent combination for preparing a kit for diagnosing contact allergens that cause skin allergies in a subject, wherein the reagent combination comprises at least five of the following: nickel sulfate, potassium dichromate, disperse blue 106, cobalt chloride, aromatic mixture I, methylisothiazolinone, thimerosal, sodium lauryl sulfate, carbazine mixture, formaldehyde, iodopropynyl aldehyde and p-phenylenediamine.

[0009] In another preferred embodiment, the reagent combination includes 5, 6, 7, 8, 9 or 10, 11, 12 species selected from group A1.

[0010] In another preferred embodiment, the allergen combination further comprises one or more selected from group A2: octyl gallate, Kathon CG, paraben mixture, Span 83, Span 80, acetylsalicylic acid, aromatic mixture II and rosin.

[0011] In another preferred embodiment, the allergen combination further includes one or more selected from Group A3: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortisonol pivalate, dodecyl glycoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile.

[0012] In another preferred embodiment, the allergen combination further includes one or more selected from Group A4: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin.

[0013] In another preferred embodiment, the allergen combination further comprises one or more selected from group A5: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, p-tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, thiol mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, iodoquinol and budesonide.

[0014] In another preferred embodiment, the allergen combination is selected from the following group:

[0015] (i) 12 allergens in Group A1;

[0016] (ii) 12 allergens of group A1 + 8 allergens of group A2;

[0017] (iii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3;

[0018] (iv) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4;

[0019] (v) 12 allergens of Group A1 + 8 allergens of Group A2 + 10 allergens of Group A3 + 10 allergens of Group A4 + one or more allergens of Group A5; and

[0020] (vi) 12 allergens of Group A1 + 8 allergens of Group A2 + 10 allergens of Group A3 + 10 allergens of Group A4 + 20 allergens of Group A5.

[0021] In another preferred embodiment, the subject is a human.

[0022] In another preferred embodiment, the subject is Chinese.

[0023] In another preferred embodiment, the subject is a patient with chronic eczema, contact dermatitis, or a skin disease associated with delayed-type allergy.

[0024] A second aspect of the present invention provides a kit for diagnosing potential allergens that cause skin allergies in a subject, characterized in that the kit comprises an allergen reagent combination, wherein the reagent combination comprises at least five selected from Group A1: nickel sulfate, potassium dichromate, disperse blue 106, cobalt chloride, aromatic mixture I, methylisothiazolinone, thimerosal, sodium lauryl sulfate, carbazine mixture, formaldehyde, iodopropynyl aldehyde and p-phenylenediamine.

[0025] In another preferred embodiment, the reagent combination includes 5, 6, 7, 8, 9 or 10, 11, 12 species selected from group A1.

[0026] In another preferred embodiment, the allergen combination further comprises one or more selected from group A2: octyl gallate, Kathon CG, paraben mixture, Span 83, Span 80, acetylsalicylic acid, aromatic mixture II and rosin.

[0027] In another preferred embodiment, the allergen combination further includes one or more selected from Group A3: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortisonol pivalate, dodecyl glycoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile.

[0028] In another preferred embodiment, the allergen combination further includes one or more selected from Group A4: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin.

[0029] In another preferred embodiment, the allergen combination further comprises one or more selected from group A5: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, p-tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, thiol mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, iodoquinol and budesonide.

[0030] In another preferred embodiment, the allergen combination is selected from the following group:

[0031] (i) 12 allergens in Group A1;

[0032] (ii) 12 allergens of group A1 + 8 allergens of group A2;

[0033] (iii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3;

[0034] (iv) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4;

[0035] (v) 12 allergens of Group A1 + 8 allergens of Group A2 + 10 allergens of Group A3 + 10 allergens of Group A4 + one or more allergens of Group A5; and

[0036] (vi) 12 allergens of Group A1 + 8 allergens of Group A2 + 10 allergens of Group A3 + 10 allergens of Group A4 + 20 allergens of Group A5.

[0037] In another preferred embodiment, the kit comprises one or more prefabricated spot testers loaded with the allergen.

[0038] In another preferred embodiment, the spot tester comprises:

[0039] A substrate, and n independent patch chambers (i.e., test chambers) arranged on the substrate, wherein n is a positive integer of 5-100, wherein the patch chambers are independently loaded with the same or different allergens.

[0040] In another preferred embodiment, the contact area (i.e., cross-sectional area) of the n patch chambers is 0.1-1.5 cm 2 , preferably 0.3-1.0cm 2 , more preferably, 0.5-0.8cm 2 .

[0041] In another preferred embodiment, the contact areas of the n patch chambers may be the same or different.

[0042] In another preferred embodiment, the patch test chamber is circular (eg, with a diameter of about 8 mm) or square (with a side length of about 8 mm).

[0043] In another preferred embodiment, the cross-section of the patch chamber is not particularly limited, and representative shapes include (but are not limited to): circle, rectangle, ellipse, regular hexagon, oval, etc.

[0044] In another preferred embodiment, preferably n is ≥8, preferably ≥10, and more preferably ≥20.

[0045] In another preferred embodiment, n is 10-80, preferably 20-70, more preferably 30-60.

[0046] In another preferred embodiment, each of the patch chambers is independently loaded with different allergens.

[0047] In another preferred embodiment, the distance between any two independent patch chambers is ≥ 0.5 cm, preferably ≥ 0.8 cm.

[0048] In another preferred embodiment, the substrate is a transparent or semi-permeable plastic.

[0049] In another preferred embodiment, the substrate and the patch chamber are made of non-allergenic materials.

[0050] In another preferred embodiment, the material of the substrate is selected from the following group: aluminum, polyethylene, polyurethane, PET, polycarbonate, or a combination thereof.

[0051] In another preferred embodiment, the substrate and the patch chamber are made of different materials.

[0052] In another preferred embodiment, the patch chamber contains or includes a material that can adsorb the allergen.

[0053] In another preferred embodiment, the thickness of the substrate (or base sheet) is not particularly limited, and is usually 1 micron to 500 microns.

[0054] In another preferred embodiment, the substrate is made of a breathable material.

[0055] In another preferred embodiment, the substrate is made of non-waterproof material or waterproof material.

[0056] In another preferred embodiment, the patch chamber is provided with anti-leakage protrusions around it.

[0057] In another preferred embodiment, an absorbent lining is fixed in the patch chamber for absorbing (or adhering to or fixing) the allergen (or the test agent containing the allergen).

[0058] In another preferred embodiment, the patch chamber is equipped with filter paper for absorbing liquid allergens.

[0059] In another preferred embodiment, the test agent is selected from the following group: solution, emulsion, cream, gel, powder or a combination thereof.

[0060] In another preferred embodiment, the test agent is prepared by mixing water or vaseline with the allergen.

[0061] In another preferred embodiment, the kit contains a blank spot tester not loaded with the allergen, and an allergen (or a test agent containing the allergen) for preparing or preparing the spot tester loaded with the allergen.

[0062] In another preferred embodiment, the kit further comprises a label or instructions.

[0063] In another preferred embodiment, the label or instructions record the judgment criteria, which are: (-) is a negative reaction; (±) is a suspicious reaction, with only weak erythema; (+) is a positive reaction, with erythema, infiltration, and small papules; (2+) is a strong positive reaction, with erythema, infiltration, papules and blisters; (3+) is an extremely positive reaction, with obvious erythema, severe infiltration (edema), and confluent blisters; the reaction exceeds the test area; IR is an irritation reaction.

[0064] In another preferred embodiment, the antigen combination is in contact with the subject's skin for 24-96 hours, preferably 24-48 hours, and more preferably 48 hours.

[0065] In another preferred embodiment, the allergen reagent contacts the back and the extended side of the upper arm of the subject.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0067] Attached photos

[0068] Figure 1 This is a flow chart for screening epidemiological validation literature for Example 2. DETAILED DESCRIPTION

[0069] After extensive and in-depth research, a large number of screenings and tests, the inventors have provided a contact allergen reagent combination and its use, as well as a kit based thereon. The allergens contained in the allergen reagent combination of the present invention have been obtained through clinical practice by the inventors and verified after further statistics of a large amount of data. The allergens set on the spot tester of the kit of the present invention are highly consistent with the allergens with the highest positive rate in China in the past 30 years, including various allergens with high positive rates or high positive rate indexes (the positive rate of most allergens is ≥2%), which is particularly suitable for the identification of skin allergens in Chinese. The present invention was completed on this basis.

[0070] Specifically, the inventors have prepared an allergen detection kit suitable for the Chinese population, which can be widely used in dermatology and allergy departments. It can be used as an auxiliary diagnostic method for contact dermatitis, eczema, exanthematous drug eruption, occupational skin diseases, cosmetic skin diseases and other skin diseases that may be associated with delayed allergic reactions. Different allergen combinations can be used for detection according to clinical case requirements. The kit has a high positive rate and can clarify the patient's contact allergens, thereby guiding clinical treatment and prevention, and has positive and significant significance for clinical diagnosis and treatment.

[0071] the term

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0073] As used herein, the terms "allergen" and "allergen" and "antigen" are used interchangeably to refer to substances that may cause a positive skin allergic reaction.

[0074] As used herein, the terms "positive rate", "positive reaction rate" or "allergy positive rate" are used interchangeably and refer to the rate at which a sample shows a skin allergic reaction to a specific allergen in a screening test including a specific allergen.

[0075] Contact allergen reagent combination

[0076] The present invention provides a set of contact allergen reagent combinations, which comprises at least five selected from Group A1: nickel sulfate, potassium dichromate, disperse blue 106, cobalt chloride, aromatic mixture I, methylisothiazolinone, thimerosal, sodium lauryl sulfate, carbazine mixture, formaldehyde, iodopropynyl aldehyde and p-phenylenediamine.

[0077] In another preferred embodiment, the reagent combination includes 5, 6, 7, 8, 9 or 10, 11, 12 species selected from group A1.

[0078] In another preferred embodiment, the allergen combination further comprises one or more selected from group A2: octyl gallate, Kathon CG, paraben mixture, Span 83, Span 80, acetylsalicylic acid, aromatic mixture II and rosin.

[0079] In another preferred embodiment, the allergen combination further includes one or more selected from Group A3: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortisonol pivalate, dodecyl glycoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile.

[0080] In another preferred embodiment, the allergen combination further includes one or more selected from Group A4: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin.

[0081] In another preferred embodiment, the allergen combination further comprises one or more selected from group A5: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, p-tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, thiol mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, iodoquinol and budesonide.

[0082] In another preferred embodiment, the allergen combination is selected from the following group:

[0083] (i) 12 allergens in Group A1;

[0084] (ii) 12 allergens of group A1 + 8 allergens of group A2;

[0085] (iii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3;

[0086] (iv) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4;

[0087] (v) 12 allergens of Group A1 + 8 allergens of Group A2 + 10 allergens of Group A3 + 10 allergens of Group A4 + one or more allergens of Group A5; and

[0088] (vi) 12 allergens of Group A1 + 8 allergens of Group A2 + 10 allergens of Group A3 + 10 allergens of Group A4 + 20 allergens of Group A5.

[0089] Typically, when used, the allergen reagent combination of the present invention can be divided into a basic antigen series (a total of 20 A1+A2), a screening antigen series (a total of 40 A1+A2+A3+A4), and a standard antigen series (a total of 60 A1+A2+A3+A4+A5).

[0090] In another preferred embodiment, the subject is Chinese.

[0091] In another preferred embodiment, the subject is a patient with chronic eczema, contact dermatitis, or other skin diseases associated with delayed-type allergic reactions (or the offspring of a person with a history of skin allergies).

[0092] Kit for diagnosing potential allergens for skin allergy in subjects

[0093] The kit of the present invention comprises the above-mentioned contact allergen reagent combination.

[0094] In another preferred embodiment, the kit comprises one or more prefabricated spot testers loaded with the allergen.

[0095] The kit of the present invention has no special requirements on the structure of the spot tester, and the spot tester commonly used in the art can be used.

[0096] In another preferred embodiment, the spot tester comprises:

[0097] A substrate, and n independent patch chambers (i.e., test chambers) arranged on the substrate, wherein n is a positive integer of 5-100, wherein the patch chambers are independently loaded with the same or different allergens.

[0098] In another preferred embodiment, preferably n is ≥8, preferably ≥10, and more preferably ≥20.

[0099] In another preferred embodiment, n is 10-80, preferably 20-70, more preferably 30-60.

[0100] In another preferred embodiment, each of the patch chambers is independently loaded with different allergens.

[0101] In another preferred embodiment, the distance between any two independent patch chambers is ≥ 0.5 cm, preferably ≥ 0.8 cm.

[0102] In another preferred embodiment, the substrate and the patch chamber are made of non-allergenic materials.

[0103] In another preferred embodiment, the patch chamber contains or includes a material that can adsorb the allergen.

[0104] In another preferred embodiment, an absorbent lining is fixed in the patch chamber for absorbing (or adhering to or fixing) the allergen (or the test agent containing the allergen).

[0105] In another preferred embodiment, the patch chamber is internally provided with filter paper for absorbing liquid allergens.

[0106] In another preferred embodiment, the test agent is selected from the following group: solution, emulsion, cream, gel, powder or a combination thereof.

[0107] In another preferred embodiment, the test agent is prepared by mixing water or vaseline with the allergen.

[0108] In another preferred embodiment, the kit contains a blank spot tester not loaded with the allergen, and an allergen (or a test agent containing the allergen) for preparing or preparing the spot tester loaded with the allergen.

[0109] Generally, the allergens of the present invention can be loaded on the same spot tester, or loaded on different spot testers and attached separately.

[0110] application

[0111] Apply the allergen reagent to the patient's back (and / or the extended side of the upper arm), gently press the patch tester to ensure close contact with the skin, and fix it with materials such as tape. After a certain period of time (such as 24 or 48 hours), remove the patch tester, gently wipe off the allergens remaining on the skin, and mark it with a marker. Sit quietly for about 20-60 minutes (such as 30 minutes) and then score the patch test reaction. Score again at a predetermined time (such as 24 hours and 48 hours) after removing the patch tester. In special cases, the number of scoring times can be increased, such as scoring again 4 days, 6 days, or even 1 week after removing the patch tester.

[0112] In another preferred embodiment, the allergen reagent contacts the back and the extended side of the upper arm of the subject.

[0113] In another preferred embodiment, the antigen combination is in contact with the subject's skin for 24-96 hours, preferably 24-48 hours, and more preferably 48 hours.

[0114] The judgment criteria are: (-) is a negative reaction; (±) is a questionable reaction, with only slight erythema; (+) is a positive reaction, with erythema, infiltration, and possibly small papules; (2+) is a strong positive reaction, with erythema, infiltration, papules, and blisters; (3+) is an extremely positive reaction, with obvious erythema, severe infiltration (edema), and confluent blisters; the reaction exceeds the test area; IR is an irritation reaction.

[0115] The main advantages of the present invention include:

[0116] 1. The allergen reagent combination of the present invention has a wide coverage and a high detection rate, and is particularly suitable for diagnosing (or identifying) contact allergens of patients.

[0117] 2. The allergens included in the allergen reagent combination of the present invention have been verified by a large number of literature statistics and clinically verified by the inventors, and are the most suitable combination for identifying skin allergens in China at this stage.

[0118] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0119] Example 1

[0120] Identification of allergens

[0121] New chemical products have exploded in the past few decades, and the types and quantities of skin allergens that Chinese people may be exposed to have also changed dramatically. Skin allergies have become very common, and some allergens show obvious changes in different regions and time spans.

[0122] Based on the analysis and research of the inventors, and in combination with a large amount of clinical practice, skin allergens suitable for China's national conditions have been determined, as shown in Table 1.

[0123] Table 1 List of allergens

[0124]

[0125]

[0126] When the excipient is vaseline, it is solid (or quasi-solid), and the concentration is expressed as a percentage (%) based on the total weight; when the excipient is water, it is liquid, and the concentration is expressed as a volume percentage based on the total volume.

[0127] Example 2.

[0128] Epidemiological validation based on literature data

[0129] In order to verify whether the allergens screened by the present invention (60 allergens in Table 1) are common contact allergens in China, epidemiological verification was performed based on literature data, and a summary of all patch test studies reported in China was conducted to verify and analyze the proportion of positive reactions to allergens detected in China, thereby determining or verifying the allergens with high positive reactions included in the patch tester of the present invention.

[0130] Three Chinese databases were searched: Chinese National Knowledge Infrastructure (CNKI), Chinese Technical Journal VIP Database and Wanfang Data, as well as Chinese and English literature published in PubMed. The content includes the results of Chinese patch tests from 1987 to June 2018. The search keywords include: patch test, and allergic contact dermatitis, facial dermatitis, eczema, atopic dermatitis, hand dermatitis or eczema, dermatitis. A total of 3977 documents were obtained, including 3656 Chinese documents (1784 in CNKI, 1332 in Wanfang Data, and 540 in VIP database), 320 in PubMed English, and 1 unpublished research data from Shanghai Dermatology Hospital.

[0131] After screening, 1872 duplicate articles included in different databases and 1838 articles without clear data lists or with obvious data errors were deleted during the final data input and analysis. The final statistical database included 253 articles, including 224 Chinese articles and 29 English articles. The literature screening results are as follows: Figure 1 shown.

[0132] Record information included in each article: name of the first author, year of publication, study area, allergen, allergen series, number of subjects, number of positive reactions. Some additional record information: percentage of males and females, age range, diagnosis, other skin diseases, patch reading time.

[0133] The positivity rate for each allergen was then calculated as the total number of patients with positive reactions divided by the total number of patients tested with that allergen. The positive reactions for each allergen tested (included in at least two studies) were calculated and ranked to analyze the trend of allergen sensitization.

[0134] For the 60 allergen types listed in Table 1, the statistical results are shown in Table 2.

[0135] Table 2 Statistical results of allergen positive rate

[0136]

[0137]

[0138]

[0139] result

[0140] On the one hand, the 60 allergens listed in Table 1 (i.e., G1-G60) almost cover the vast majority of allergen cases reported in the literature, indicating that the coverage of G1 to G60 is very wide. On the other hand, among G1 to G60, the highest positive rate is 25.58 (G1), the positive rates of the first 12 allergens (G1-G12) are all about 10 or higher; the positive rates of the first 20 allergens (G1-G20) are all about 6 or higher; the positive rates of the 21st to 30th allergens (G21-G30) are all between about 5.66 and 4.50; the positive rates of the 31st to 40th allergens (G31-G40) are all between about 4.47 and 3.58; the positive rates of the 41st to 60th allergens (G21-G30) are relatively low, between about 3.51 and 1, that is, common allergens (positive rates > 1% can be regarded as common allergens).

[0141] The above analysis results indicate that the allergens in Table 1 (i.e., G1-G60) are particularly suitable for diagnosing skin allergens in Chinese people.

[0142] For some representative allergens among G1-G60, further description is given below.

[0143] Thimerosal

[0144] Thimerosal is another common mercury allergen. As an organic preservative, thimerosal has long been widely used in over-the-counter ophthalmic preparations, contact lens solutions, vaccines, antitoxins, and desensitization preparations for skin tests.

[0145] The research analysis showed that the thimerosal patch-positive rate was 14.65% (Table 2).

[0146] Data show that the contact allergy positivity rate in my country is significantly higher than that in Turkey, but similar to that in the United States and India. The reason for the difference may be related to the frequency of allergen exposure and the different population densities in these regions.

[0147] CL+ME-Isothiazole (Methylisothiazolinone and Methylchloroisothiazolinone (MCI / MI)) / Methylisothiazolinone (Methylisothiazolinone (MI))

[0148] CL+ME-isothiazole and methylisothiazolinone belong to the isothiazolinone class and are commonly used preservatives. Since isothiazolinone was added to cosmetics, household and industrial products, the growth rate of epidemiological allergy rates of this ingredient has reached a new high. In China, the sensitization rate of CL+ME-isothiazole was first reported in 2013.

[0149] The research analysis showed that the positive rate of methylisothiazolinone was 16.37% (Table 2).

[0150] CL+ME-isothiazole (also known as Kasong CG, Euxyl K100, etc.) has been sold as a preservative since 1980, and its usage has increased rapidly, but the increase in usage has also caused controversy over the safety of this chemical. In the early 1980s, CL+ME-isothiazole / methylisothiazolinone saw explosive growth after it was put into use. In order to reduce its risk of sensitization, relevant regulations have more strictly limited its maximum concentration to 15p.pm, but their sensitization positive rate in patients is very high, and they are allergens that are very easy to cause sensitization.

[0151] Methylisothiazolinone has been used in cosmetics since 2005 and was first mentioned in the literature on cosmetic contact allergic dermatitis in 2010. The efficacy of this ingredient is lower than that of CL+ME-isothiazole, so it is believed that its sensitization ability is also lower. The Chinese literature involving the patch test results of CL+ME-isothiazole (positive rate 2.36%) was published in 2008, but the positive rate gradually increased and remained at a high level of about 8%. The trend of methylisothiazolinone is similar. In 2013, the first literature reported its patch test positive rate, which was only 0.5%, and then the positive rate increased year by year.

[0152] The high sensitization rates of methylisothiazolinone and CL+ME-isothiazole are related to their widespread use in the market.

[0153] According to China's regulations before December 2016, the upper limits of methylisothiazolinone and CL+ME-isothiazole in leave-on or rinse-off cosmetics were 100 p.pm and 15 p.pm, respectively, while the addition of CL+ME-isothiazole was banned in leave-on cosmetics afterwards.

[0154] Methylisothiazolinone and CL+ME-isothiazole have a cross-reactivity due to the same components, and several surveys have shown a cross-reactivity rate of 40-70%. Therefore, when patch testing is performed on patients suspected of being allergic to isothiazoles, both methylisothiazolinone and CL+ME-isothiazole should be tested, and if there is an allergy to either of them, the other substance should also be included in the reference.

[0155] Formaldehyde and formaldehyde releasers

[0156] Formaldehyde is a commonly used preservative and a strong allergen. As the simplest organic substance, it has a small molecular structure that can penetrate the stratum corneum, which is one of the reasons why it is an effective hapten.

[0157] Research analysis showed that the positive reaction rate of formaldehyde was 10.54% (Table 2).

[0158] Formaldehyde is a chemical substance that is widely present in various items, such as skin care products, vinyl and rubber gloves, clothing, paper, polishes, disinfectants, medicines, smoke, etc. Formaldehyde sensitization often occurs in special occupational exposure groups. The most likely potential exposure objects include some hair and body care products and cosmetics, as well as non-ironing fabrics; some extremely sensitive individuals may be allergic to formaldehyde in paper or smoke. As a volatile organic compound, formaldehyde can also cause contact allergic dermatitis in exposed areas through the air.

[0159] The analysis found that the formaldehyde positive rate has increased linearly over time since the 1980s. The possible reason is related to the increase in exposure frequency, and some studies believe it is related to the development of China's construction industry.

[0160] Common preservatives that can release formaldehyde include bronopol, diazolidinyl urea, DMDM ​​hydantoin, imidazolidinyl urea and quart 15. The free formaldehyde they decompose plays an antimicrobial role. Therefore, patients who are allergic to formaldehyde, especially those with strong positive patch tests, should be careful to avoid contact with these formaldehyde releasers.

[0161] Iodopropynyl Butylcarbamate

[0162] Iodopropynyl aldehyde is widely used as a preservative in polymer emulsions and pigments, such as water-based paints, adhesives, cement, inks, and as a wood preservative in the metal processing industry and daily products. Today, it can also be used in various cosmetics such as shampoos, lotions, creams, powders and baby products.

[0163] Research analysis showed that the sensitization rate of iodopropynyl aldehyde was 10.28% (Table 2).

[0164] The occurrence of allergic contact dermatitis may be related to cosmetics, sanitary wipes, metal workers' working environment, etc.

[0165] Octyl Gallate

[0166] Gallic acid esters, including propyl, octyl, and dodecyl esters, are the most common antioxidants. They are easily soluble in fat and are often used in cosmetics and foods (especially baked and fried foods). When mixed with heated chicken fat, they can cause airborne contact dermatitis and occasionally cheilitis or oral inflammation.

[0167] The research analysis showed that the positive reaction rate (sensitization rate) of octyl gallate allergen was 8.44% (Table 2).

[0168] As it is more likely to cause irritation reactions in patch tests, it is recommended to conduct more testing trials in China to collect more sample data for further research.

[0169] Disperse Blue Mix

[0170] Disperse dyes are commonly found in dye synthetic materials (especially polyesters and acetates), triacetate polyamide, polyacrylonitrile, printed products, and plastic products. Disperse dyes are reported to be more likely to cause allergic contact dermatitis than other types of textile dyes. The results showed that disperse blue 106 (DB 124) is the most common dye allergen. Both of them can be used alone or as a mixture as allergens for patch testing screening. Patients who are strongly suspected of being allergic to textiles in clinical practice should pay attention to other disperse dyes, such as some acidic, alkaline, reactive, and direct dyes.

[0171] The research analysis showed that the positive reaction rate of Disperse Blue 106 was 18.77% (Table 2).

[0172] Shellac

[0173] Shellac is a resinous excretion of insects that protects certain host trees. It is mainly used as a paint for wood products and is also used in cosmetics.

[0174] Shellac is widely used as a colorant in the cosmetic industry, particularly in eyeliner and mascara, and may be included in additional patch testing for patients with suspected cosmetic allergic contact dermatitis and in routine patch testing for suspected allergic contact dermatitis.

[0175] The research analysis showed that the positive reaction rate of shellac was 4.47% (Table 2).

[0176] Sorbitan Sesquioleate

[0177] Emulsifiers help water and fat mix together and are common ingredients in skin care products, cosmetics and topical medications. Sorbitan sesquioleate is a common emulsifier in skin care products.

[0178] Patients who are allergic to emulsifiers are often more likely to be allergic to other common ingredients in topical preparations (such as preservatives or active ingredients). It is recommended that patients with contact dermatitis that may be caused by topical preparations be tested for emulsifiers to avoid missing them.

[0179] Research analysis showed that in China, the positive reaction rate of sorbitan sesquioleate was 6.84% (Table 2).

[0180] Cocamidopropyl Betaine (CAPB)

[0181] Cocamidopropyl betaine is a surfactant used in a variety of skin care products, especially soaps, shampoos and conditioners, but also in other products such as skin lotions, makeup removers, and contact lens cleaners. Since the 1970s, CAPB has gradually replaced sodium lauryl sulfate (SLS) and other anionic surfactants due to its lower irritation.

[0182] According to the research analysis, the positive reaction rate of cocamidopropyl betaine is 3.51% (Table 2), and it should be included in the antigen series.

[0183] Example 3

[0184] Test kits for identifying potential allergens

[0185] The kit of this embodiment includes 60 allergen reagents as described in Table 1 (i.e., G1-G60 in Table 2), excipients (including glycerol and water), and a patch tester, which includes: a substrate, which is a polyurethane film with a low-sensitivity adhesive attached; and a circular patch chamber (diameter 8 mm) of polyethylene terephthalate (PET) fixed on the substrate, the patch chamber has a filter paper lining and can be used to load the allergen reagent; each substrate has four rows of five patch chambers spaced apart from each other. The patch chambers are numbered on the back of the substrate.

[0186] When in use, the allergen reagents are mixed with the corresponding excipients at corresponding concentrations as shown in Table 1, loaded on the filter paper lining of the patch chamber, and then attached to the skin of the subject.

[0187] The 60 allergens can be loaded in sequence according to A1-A5. A set is divided into 3 spot testers. The first spot tester loads the basic antigen series (all 20 of A1+A2, i.e. G1-G20), the second spot tester loads all 20 of A3+A4 (i.e. G21-40), and the third spot tester loads all 20 of A5 (i.e. G41-60).

[0188] You can test the first spot tester first, and if no result is detected, then test the second spot tester, and so on, which is flexible to use.

[0189] Of course, all (most of) the 60 allergens, such as the top 40, the top 50, or the top 55, can also be loaded on a spot tester to conduct an allergen test.

[0190] Example 4

[0191] Verification of high frequency allergens

[0192] The test kit described in Example 3 was used to test patients visiting Shanghai Dermatology Hospital. These patients (subjects) were Chinese who were clinically diagnosed with contact dermatitis, eczema, and facial dermatitis.

[0193] For 1003 subjects, the test was performed using a patch tester containing the basic antigen series (the first patch tester, containing the first 20 allergens, ie, G1-G20).

[0194] The statistical results of the test are as follows:

[0195] The positive rates of the 20 allergens G1-G20 detected in these test objects were in the range of 29.8%-3.1% (from high to low). In addition, the results show that the ranking of the 20 allergens from high to low according to the positive rate in this embodiment is very consistent with the ranking from G1 to G20, especially the ranking of the top 12 is consistent with G1 to G12. The highest positive rate is the G1 allergen, that is, nickel sulfate (29.8%), while the lowest positive rate (3.1%) falls into the A2 allergen group.

[0196] The results showed that G1 to G20 (especially G1 to G12) are allergens with a high positive rate in the Chinese population and are particularly suitable for use as differential skin allergens in the Chinese population.

[0197] Example 5

[0198] Verification of common allergens

[0199] The test kit described in Example 3 was used to test patients visiting Shanghai Dermatology Hospital. These patients (subjects) were Chinese who were clinically diagnosed with contact dermatitis, eczema, and facial dermatitis.

[0200] For 768 subjects, the second patch tester (containing 20 allergens, ie, G21-G40) was tested.

[0201] The statistical results of the test are as follows:

[0202] The positive rates of the 20 allergens G21-G40 detected in these test objects were in the range of 5.75%-1.44% (from high to low). In addition, the results show that the ranking of the 20 allergens from high to low according to the positive rate in this embodiment is basically consistent with the ranking from G21 to G40.

[0203] The results showed that G21 to G40 are common allergens in the Chinese population and are suitable for use as differential skin allergens in the Chinese population.

[0204] Example 6

[0205] Verification of common allergens

[0206] The test kit described in Example 3 was used to test patients visiting Shanghai Dermatology Hospital. These patients (subjects) were Chinese who were clinically diagnosed with contact dermatitis, eczema, and facial dermatitis.

[0207] For 341 subjects, the third patch tester (containing the last 20 allergens, ie, G41-G60) was tested.

[0208] The statistical results of the test are as follows:

[0209] The positive rates of the 20 allergens G41-G60 detected in these test objects were in the range of 3.22%-1.15% (from high to low). In addition, the results show that the ranking of the 20 allergens from high to low according to the positive rate in this embodiment is basically consistent with the ranking from G41 to G60.

[0210] The results showed that G41 to G60 are also common allergens in the Chinese population and are suitable for use as differential skin allergens in the Chinese population.

[0211] In addition, the comprehensive results of Examples 4-6 show that the allergens G1-G60 of the present invention can detect the corresponding allergens of the vast majority of patients with skin allergies (including patients with contact dermatitis, eczema, and facial dermatitis).

[0212] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a contact allergen reagent combination for preparing a kit for diagnosing contact allergens that cause skin allergies in a subject, wherein the reagent combination comprises: Allergens of Group A1: nickel sulfate, potassium dichromate, disperse blue 106, cobalt chloride, aroma mixture I, methylisothiazolinone, thimerosal, sodium lauryl sulfate, carbazide mixture, formaldehyde, iodopropynyl aldehyde, and p-phenylenediamine; and Allergens in group A2: octyl gallate, kason CG, paraben mixture, Span 83, Span 80, acetylsalicylic acid, aroma mixture II and rosin; Wherein, the subject is Chinese; The combination of the allergens of group A1 and the allergens of group A2 constitutes the basic antigen series.

2. The use according to claim 1, characterized in that In the kit, the concentration of nickel sulfate is 5%, and the excipient is vaseline; the concentration of potassium dichromate is 0.5%, and the excipient is vaseline; the concentration of disperse blue 106 is 1%, and the excipient is vaseline; the concentration of cobalt chloride is 1%, and the excipient is vaseline; the concentration of aromatic mixture I is 8%, and the excipient is vaseline; the concentration of methylisothiazolinone is 0.2%, and the excipient is water; the concentration of thimerosal is 0.05%, and the excipient is vaseline; the concentration of sodium lauryl sulfate is 0.25%, and the excipient is water; the concentration of carba mixture is 3%, and the excipient is vaseline; the concentration of formaldehyde is 1%, and the excipient is water; the concentration of iodopropynyl aldehyde is 0.2%, and the excipient is vaseline; the concentration of p-phenylenediamine is 1%, and the excipient is vaseline.

3. The use according to claim 1, characterized in that The allergen combination further comprises one or more selected from Group A3: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortison pivalate, dodecyl glucoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile.

4. The use according to claim 1, characterized in that The allergen combination further comprises one or more selected from Group A4: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin.

5. The use according to claim 1, characterized in that The allergen combination also includes one or more selected from Group A5: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, p-tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, thiol mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, iodoquinol and budesonide.

6. The use according to claim 1, characterized in that The allergen combination is selected from the following group: (i) 12 allergens in group A1 + 8 allergens in group A2; (ii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3; (iii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4; (iv) 12 allergens from Group A1 + 8 allergens from Group A2 + 10 allergens from Group A3 + 10 allergens from Group A4 + one or more allergens from Group A5; and (v) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4 + 20 allergens of group A5; Among them, the 10 allergens in group A3 are: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortison pivalate, dodecyl glucoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile; The 10 allergens in group A4 are: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin; The 20 allergens in group A5 are: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, mercapto mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, clioquinol and budesonide.

7. The use according to claim 1, characterized in that The subject is a patient with chronic eczema, contact dermatitis, or a patient with a skin disease associated with delayed-type allergy.

8. A kit for diagnosing potential allergens that cause skin allergies in a subject, characterized in that: The kit comprises an allergen reagent combination, wherein the reagent combination comprises: Allergens of Group A1: nickel sulfate, potassium dichromate, disperse blue 106, cobalt chloride, aroma mixture I, methylisothiazolinone, thimerosal, sodium lauryl sulfate, carbazide mixture, formaldehyde, iodopropynyl aldehyde, and p-phenylenediamine; and Allergens in group A2: octyl gallate, kason CG, paraben mixture, Span 83, Span 80, acetylsalicylic acid, aroma mixture II and rosin; The combination of the allergens of group A1 and the allergens of group A2 constitutes the basic antigen series.

9. The kit according to claim 8, characterized in that In the kit, the concentration of nickel sulfate is 5%, and the excipient is vaseline; the concentration of potassium dichromate is 0.5%, and the excipient is vaseline; the concentration of disperse blue 106 is 1%, and the excipient is vaseline; the concentration of cobalt chloride is 1%, and the excipient is vaseline; the concentration of aromatic mixture I is 8%, and the excipient is vaseline; the concentration of methylisothiazolinone is 0.2%, and the excipient is water; the concentration of thimerosal is 0.05%, and the excipient is vaseline; the concentration of sodium lauryl sulfate is 0.25%, and the excipient is water; the concentration of carba mixture is 3%, and the excipient is vaseline; the concentration of formaldehyde is 1%, and the excipient is water; the concentration of iodopropynyl aldehyde is 0.2%, and the excipient is vaseline; the concentration of p-phenylenediamine is 1%, and the excipient is vaseline.

10. The kit according to claim 8, characterized in that The allergen combination further comprises one or more selected from Group A3: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortison pivalate, dodecyl glucoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile.

11. The kit according to claim 8, characterized in that The allergen combination further comprises one or more selected from Group A4: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin.

12. The kit according to claim 8, characterized in that The allergen combination also includes one or more selected from Group A5: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, p-tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, thiol mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, iodoquinol and budesonide.

13. The kit according to claim 8, characterized in that The allergen combination is selected from the following group: (i) 12 allergens in group A1 + 8 allergens in group A2; (ii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3; (ii) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4; (iv) 12 allergens from Group A1 + 8 allergens from Group A2 + 10 allergens from Group A3 + 10 allergens from Group A4 + one or more allergens from Group A5; and (v) 12 allergens of group A1 + 8 allergens of group A2 + 10 allergens of group A3 + 10 allergens of group A4 + 20 allergens of group A5; Among them, the 10 allergens in group A3 are: Peru balsam, ethylenediamine, N-cyclohexylthiophthalide, and tixocortison pivalate, dodecyl glucoside, cinnamyl alcohol, quart 15, tert-butylhydroquinone, lyral and methyl dibromoglutaronitrile; The 10 allergens in group A4 are: black rubber mixture, shellac, sesquiterpene lactone mixture, bronopol, 6-hydro-1,3,5-tris(2-hydroxyethyl)triazine, thiuram mixture, benzocaine, lanolin alcohol, Tween 80 and epoxy resin; The 20 allergens in group A5 are: cocamidopropyl betaine, neomycin sulfate, diazolidinyl urea, tert-butylphenol formaldehyde resin, benzyl salicylate, DMDM ​​hydantoin, chlorhexidine gluconate, mercapto mixture, triethanolamine, stearyl alcohol, imidazolidinyl urea, dodecanoate, benzophenone-3, isopropyl myristate, N-isopropyl-N-phenyl-p-phenylenediamine, peppermint oil, mercaptobenzothiazole, drometrizole, clioquinol and budesonide.

14. The kit according to claim 8, characterized in that The kit includes one or more pre-made spot testers loaded with the allergen.

15. The kit according to claim 14, characterized in that The spot tester comprises: A substrate, and n independent patch chambers, i.e., test chambers, arranged on the substrate, wherein n is a positive integer of 5-100, wherein the patch chambers are independently loaded with the same or different allergens.

16. The kit according to claim 15, characterized in that The contact area, i.e., the cross-sectional area, of the n independent patch chambers is 0.1-1.5 cm 2 .

17. The kit according to claim 15, characterized in that The contact area, i.e., the cross-sectional area, of the n independent patch chambers is 0.3-1.0 cm 2 .

18. The kit according to claim 15, characterized in that The contact area, i.e., the cross-sectional area, of the n independent patch chambers is 0.5-0.8 cm 2 .

19. The kit according to claim 15, characterized in that The cross section of the patch chamber is circular, rectangular, elliptical, regular hexagonal or oval.

20. The kit according to claim 15, characterized in that n is 10-80.

21. The kit according to claim 15, characterized in that n is 20-70.

22. The kit according to claim 15, characterized in that n is 30-60.

23. The kit according to claim 15, characterized in that The patch chambers are each independently loaded with different allergens.

24. The kit according to claim 15, wherein The distance between any two independent patch chambers is ≥0.5cm.

25. The kit according to claim 15, characterized in that The distance between any two independent patch chambers is ≥0.8cm.

26. The kit according to claim 8, characterized in that The kit contains a blank spot tester not loaded with the allergen, and an allergen or a test agent containing the allergen for preparing or making the spot tester loaded with the allergen.

27. The kit according to claim 8, characterized in that The kit also includes a label or instructions.

28. The kit according to claim 27, characterized in that The label or instructions record the judgment criteria, which are: (-) is a negative reaction; (±) is a suspicious reaction, with only weak erythema; (+) is a positive reaction, with erythema, infiltration, and possibly small papules; (2+) is a strong positive reaction, with erythema, infiltration, papules and blisters; (3+) is an extremely positive reaction, with obvious erythema, severe infiltration, and confluent herpes; the reaction exceeds the test area; IR is an irritation reaction.

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