PL192349B1 - Strip-type test for performing an allergy diagnosis in vitro - Google Patents

Abstract

The invention relates to a strip test for diagnosing allergies in-vitro, and a method for detecting specific IgE in whole blood or in sera. The invention also relates to the use of said strip test for detecting inhaled allergens, food allergens, cross-reactivity allergens and Mediterranean allergens.

Description

Description of the invention

The invention relates to an in vitro allergy diagnosis test strip for the detection of specific immunoglobulin E (IgE) in whole blood or serum.

Many methods are known for the diagnosis of allergy in vivo and in vitro. In addition to in vivo allergy diagnostics, which includes, for example, skin, prick, intradermal and provocation tests, in vitro allergy diagnostics is gaining more and more importance because the range of allergens is constantly growing, and in vivo tests in many cases cannot be performed on patients due to side effects.

For many new allergens, we often do not have any commercially available tests, because, in rare cases of allergies, the effort required to produce the extract, and thus obtaining the test, often does not correspond to the number of patients. These include occupational allergies, such as wood allergies, raw coffee, hygiene products such as latex gloves, etc., which often only affect small professional groups. In the case of people with acute allergies, in vivo tests with new, insufficiently tested allergen solutions may lead to unexpected side effects that seriously threaten the health of the patient. An important advantage of in vitro diagnostics lies in the complete safety of the patient, because the test performed on the patient's serum is performed outside the patient's body.

The determination of specific and total IgE in the patient's serum is of key importance in the in vitro test. IgE (immunoglobulin E) is a protein that, from a serological point of view, is responsible for the disease picture of allergies.

So far, in vitro allergy tests in the form of plate tests are known, which were developed by Johansson, Widy and Bennick (Lancet 2, 1105-1107 (1967)). This procedure is the most commonly used test today. It is also the first test system to measure specific IgE in the sera of allergic persons. A similar test system is described e.g. in EP 203 238.

All these known tests can only be performed with serum prepared from whole blood and are relatively insensitive, ie approx. 1-2 ml of serum are needed for a definitive diagnosis.

Furthermore, it is important for many patients that in vitro tests cover multiple allergens, including rare allergens such as latex, for example, allowing an accurate and reliable diagnosis. A further disadvantage of the tests known to date, in addition to the insufficient spectrum of allergens they cover, is the large scale and length of the procedures used, which means that they cannot be carried out simply by every person, and this applies in particular to the analysis of their results. Similar tests should enable diagnosis not only in the medical field, but also in the veterinary field, and the results should not give a false picture, whether serum or whole blood is used for the diagnosis.

The object of the present invention is to develop easy-to-use in vitro allergy diagnosis test strips. Such tests would cover a sufficient spectrum of allergens and could be applied to both serum and whole blood.

The essence of the invention is an in vitro allergy diagnosis strip test, where specific immunoglobulin E (IgE) is detected in total blood or serum, which is a complete test for the detection of inhalation allergens, food allergens, cross-reactive allergens and Mediterranean allergens, which is characterized by this that a positive control sample, a negative control sample and at least nine allergens are applied to each of the test strips, and these allergens are covalently bound.

The test strip is used to detect specific IgE in total blood or serum of allergic patients and, apart from medicine, can also be used in veterinary medicine. The result is preferably analyzed visually by comparison with a color chart. The colors taken on by the allergen impregnations on each test strip are compared with the enclosed color scale. The intensity of the colors is an indicator of the result of an allergy diagnosis.

A typical test strip contains 9 allergens, a positive and negative control. The complete test preferably comprises different allergen test strips of different types. These include, for example, the following types: grasses / grains / herbs (G / G / K), animals / feathers / mites (T / F / M), trees (B), fungi (P), foodstuffs (NM), screening (s).

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A typical allergen spectrum is summarized in the following table. In addition to the standard allergens, there are specific allergens, such as roasted coffee, certain trees, latex etc.

Tree pollen (B) Grasses / Cereals / Herbs (G / G / K) birch mugwort oakwood barley alder glasses hazelnut 6-grass poplar oat plane tree old man red beech rye elm grandmother willow wheat negative control negative control positive control positive control

Animals / Feathers / Mites (T / F / M) Mushrooms (P) dermat. pter. Alternaris tennis dermat. far. Aspergillus fum. a mixture of feathers (goose, hen, duck) Botrytis cinerea hamster Cladosp. herbarum dog Curvularia lunata rabbit Fusarium monoliforme cat Helminthosporium halodes Guinea pig Penicillium praktum horse Rhizopus nigricans negative control negative control positive control positive control

Foodstuffs (NM) peanuts hazelnuts poultry chicken cod crabs cow's milk rye flour celery tuber soybean negative control positive control

PL 192 349 B1

A test according to the invention suitable for commercial use includes, for example:

• inhalation allergen test strips, • food allergen test strips, • cross-reactive allergen test strips, as well as • Mediterranean allergen test strips.

The test strips of the invention are named Allergodip ™. In view of the allergens tested, the examples of compositions of such Allergodip test systems mentioned so far may be as follows. Code numbers are listed in the Platelet Allergen Catalog, October 1993.

Allergodip test lineups

Inhalative No code Food No code Crosswords No code Mediterranean skies No code grass dx901 chicken poultry f 74 birch f 03 grass dx901 rye pollen g 12 cow milk f 02 alder f 02 Parietaria at 19 * birch f 03 rye flour f 05 hazel f 04 birch f 03 mugwort in 06 peanuts f13 apple tree f 49 olive f 09 grandmother in 09 hazelnuts f 17 hazelnuts f 17 grandmother in 09 dog e 02 celery s 901 carrot f 31 old man ** cat e 01 soybeans f 14 peach f 95 cat e 01 Alternaria m 06 crabs f 23 mugwort in 06 Alternaria m 06 D. pteron d 01 cod f 03 celery s 901 D. pteron d 01 negative control k 905 negative control k 905 negative control k 905 negative control k 905 positive control AP positive control AP positive control AP positive control AP

* healing here ** still has to be precisely defined whether it is a short old man, a giant, etc.

Further typical components of the test systems are furthermore color cards and patient cards, glass test tubes, test stands, conjugate, substrate, disposable pipettes and a description of the procedure for performing and evaluating the test result. The conjugate is preferably AP-Anti-IgE, the substrate is preferably AP-purple IgE or BM-purple IgE.

The test is preferably carried out at room temperature.

The test can also be performed in the form of a one-day and two-day test. The main differences between the two assays are the incubation times with the conjugate and substrate. In addition, the test can be performed on whole blood or serum.

As with known plate tests, individual test strips are preferably obtained by activation with cyanogen bromide.

BrCN activated sheets of paper are used to prepare the Alergodip tests. The activation of the BrCN paper sheets is analogous to the activation of allergenic plaques, the amounts and volumes being clearly reduced.

The cellulose paper is activated with BrCN. Allergens are covalently bound to the activated paper. The allergen is bound to the sheets of paper (DIN A4) of activated BrCN. Strips 4 mm wide are cut and glued to plastic strips (DIN A4). Plastic strips are stuck one above the other, e.g. 9 different allergens, a negative and a positive control. DIN A4 plastic strips are cut into plastic strips 4 mm wide and about 7 cm long (Allergodipes).

The preparation of the allergen strips is preferably carried out in such a way that 4 mm strips are cut from the sheets of the bound allergens. These strips are glued one above the other with double-sided adhesive tape on polystyrene plates approx. 30 cm long and 8 cm wide. The various allergy strips are stuck on top of each other and pressed firmly. The polystyrene plates obtained in this way with adhered allergens are now processed (dynamometrically) over a period of one night in a specially developed press at room temperature. After pressing, 4 mm wide Allergodip strips are cut from the polystyrene plates with allergens glued on them with a cutting machine. The strips are placed in vials of 10 with desiccant, labeled and stored at -20 ° C. In such conditions, they are durable for at least 2 years.

To indicate which stripes are meant, e.g. trees, polystyrene plates are also marked with different colors. Each Allergodip has a different color.

The invention also relates to a method for determining specific IgE in total blood or serum, characterized in that the following steps are carried out one after another:

1.pipetting of serum or total blood into a test tube,

2.incubation with a test strip (Allergodip ™),

3.washing with running water,

4.short drying,

5. incubation of Allergodip ™ with the conjugate,

6.washing,

7.short drying,

8. Incubation of Allergodip ™ with the substrate,

9.short drying with tissue paper,

10.taking the developed test strips,

11. comparison with the color card or comparison and documentation using the color card and the patient's card.

The test can be conducted as a one-day or two-day test.

One-day test • The Allergodip test is incubated with serum for 30 min. in a 6.5 mm x 7 cm test tube.

• The strips are then washed in an ultrasonic cleaner for 2 min.

• The strips are incubated with AP-Anti-IgE for 2 hours at room temperature. • The strips are then washed in an ultrasonic bath for 2 min.

• The strips are incubated with the substrate for 1.5 hours at room temperature. • The strips are blotted dry and the color reaction is assessed against the color chart (optionally by densitometry)

Two-Day Test • The Allergodip test is incubated with the serum in the test tube for 3 hours.

• Washing in analogy to the one-day test • The strips are incubated with AP-Anti-IgE overnight at room temperature. • Washing in the same way as for the one-day test. • The strips are incubated with the substrate for 1.5 hours at room temperature. reaction to the color card (possibly densitometrically)

The level of specific IgE in the serum of the patients is determined by comparing the blue color of the allergenic impregnation with the color chart. In the case of a one-day test, it is divided into three classes of allergens, in the case of an overnight test, it is divided into four classes.

The following auxiliary reagents are used in addition to the test strips:

Conjugate

The conjugate used is an AP-Anti-IgE conjugate

Substrate

AP-purple is used as the substrate. Alternatively, for example, BM-purple can be used.

Washing solution

The washing solution used is preferably 0.9% NaCl solution + 1% TWEEN 20 (Merck Index 12 ed. 7742) + 0.05% NaN3. The washing solution is preferably used in a 1:15 dilution.

Studies have shown that tap water can be used for rinsing in an ultrasonic cleaner. Washing can also be done under the tap (1 minute).

Various ancillary equipment is required to perform the Allergodip test, e.g. ultrasonic cleaner, glass reaction tubes, Allergodip holder, reaction tube rack, disposable pipettes and a color chart. Ultrasonic rinsing may be replaced by tap rinsing.

PL 192 349 B1

It is preferable to use calibrated glass reaction tubes (1.5 ml), which allows the amount of serum + reagents to be pipetted to be determined.

Test tubes should be fixed in plastic or plexiglass racks. The washing of Allergodip can take place in an ultrasonic bath, which is filled with a washing solution. However, studies have shown that it is also possible to rinse under the tap.

In order to hang the allergy test strips in an ultrasonic cleaner filled with a rinse solution, a special attachment is required, consisting of a plexiglass plate in which the grooves for the attachment clips are milled. Similarly, when rinsing under the tap, the straps should be placed in the appropriate holder.

After the test is completed, the allergy test strips must be dried briefly with tissue paper, and then the results can be read.

The evaluation of the Allergodip results is carried out on the basis of a color chart that corresponds to the appropriate color, corresponding to a one- or two-day test.

The color chart allows grading from 0 to 4. Allergodip is offered as a one or two day test.

Preferably, a patient card is used to document the results. The card reproduces, among other things, the composition of allergens and is marked with a different color for each individual test.

The invention further relates to the use of test strips for the diagnosis of allergies in vitro in human and veterinary medicine. In particular, the test strips can be used to detect inhalation allergens, food allergens, cross-reactive allergens or Mediterranean allergens and to diagnose allergies associated therewith.

All water-absorbing matrices used for similar tests can be used as indicator papers. Filter paper is most commonly used for this purpose, but other water-absorbent cellulose or plastic products can also be used. Water-absorbent carriers, preferably filter paper, are impregnated in a known manner with an impregnating solution which contains the reagents necessary for chlorine detection. The impregnated and dried papers can be converted into square or rectangular strips, which can be stuck or unsealed on a foil, e.g. plastic foil, paper foil, or metal strips in a known manner. The bonding area is limited to a fraction of the indicator paper, e.g. 10 to 20%. The indicator papers may also be applied to the adhesive tape in the form of strips prior to impregnation, which after impregnation may be cut vertically to the direction of the stripes into commercial strips.

The following examples are intended to illustrate the invention; however, they are by no means intended to limit the invention.

Example 1

Binding of allergens to filter papers for the production of Allergodip test strips

All data is for the binding of allergens to size BrCN Filter Paper sheets

16x26 cm = 416 cm ² and the amount of protein to be injected 10 μg / ml of the allergen dilution solution.

Only BrCN Filter Paper sheets may be incubated in a metal dish.

It has been found in work that in one allergen binding batch it is advantageous to produce six allergen sheets.

In the case of solutions containing proteins, the formation of foam leads to the uncontrolled denaturation of the protein. Hence, the formation of foam by vigorous shaking or stirring should be avoided.

By incubating the allergen solution with sheets of cyanogen bromide-activated filter paper (for preparation see PA 6.02.03), the allergens are immobilized by forming covalent bonds.

In order to bind a given allergen, 115 ml of the allergen binding solution are introduced into a metal dish. The steamer is moved so that the sheets are completely covered with the allergenic binder solution.

Incubation: IKA shaker, speed: 45 rpm, 4 ° C, for 1 night.

The following washes are then carried out:

5-10 ml of the allergenic solution is withdrawn for protein determination. The allergen solution is decanted or drained as completely as possible.

Washing solution No. 1:

1x120 ml, shake for 10 min. on an IKA shaker at 4 ° C.

1x120 ml, shake for 3 h on an IKA shaker at 4 ° C.

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Washing solution number 2:

x 120 ml, shake each time for 10 minutes. on an IKA shaker at 4 ° C.

Washing solution no.3:

x 120 ml, shake each time for 10 minutes. on an IKA shaker at 4 ° C.

Secure sheets of paper in incubation buffer until further use.

No foam may form during this.

For protection, the test sheets or strips are protected by freeze-drying.

If lyophilization is not possible immediately after binding of the allergen, individual sheets of allergens are sealed hermetically without buffer in a foil tube and stored at -20 ° C.

The sheets or strips are individually lyophilized without buffer in evaporators. Up to 8 allergen sheets can be lyophilized in one cycle. One night freeze drying is sufficient.

Individual sheets of allergens must be sealed and labeled immediately after freeze-drying. Store at -20 ° C.

Example of a d 2

The course of a one-day test performed on the patient's serum

Before using the reagents and Allergodip in the test, they must be brought to room temperature.

Before use, the substrate is mixed by gently tilting it several times. Before starting work, turn on the incubator in order to achieve the substrate incubation temperature of 37 ° C.

The patient's serum is pipetted until it reaches the height marked on the glass tube. The Allergodip test strip is placed in the serum. All allergen impregnations must be completely immersed in the liquid.

Incubation is carried out for 30 minutes at room temperature. Allergodip is removed from the serum and placed in a special holder in an ultrasonic bath filled with washing buffer.

• All impregnations must be covered with the washing buffer.

• Washing is carried out for 5 minutes. in an ultrasonic cleaner.

• The strips are removed and briefly dried with filter paper.

The second test tube is filled to the marked height with conjugate. Allergodip is placed in conjugation and incubated for 2 hours at room temperature.

Washing is carried out for 5 minutes in an ultrasonic bath with the same buffer as before. The Allergodip is then removed and briefly dried with filter paper. The substrate is pipetted to the marked height into the third test tube and the Allergodip is placed in it. Incubation is carried out in an incubator for 3 hours at 37 ° C. The Allergodip test strips are then removed from the tube, dried with filter paper and the color reaction is assessed.

The results are assessed by comparing them with a color chart (one-day test - classes 0-4).

Allergodip test strips can be pasted into the documentation. The colored complex on the strip is stable at room temperature for about one year.

Pr z c a d 3

The course of the two-day test performed on the patient's serum

Before using the reagents and Allergodip, they must be brought to room temperature. Before use, the substrate is mixed by gently tilting it several times.

The patient's serum is pipetted in this amount. that it reaches the height marked on the glass tube. The Allergodip test strip is placed in the serum. All allergen impregnations must be completely immersed in the liquid. Incubation is carried out for 3 hours at room temperature. Allergodip is removed from the serum and placed in a special holder in an ultrasonic cleaner filled with washing buffer. All impregnations must be covered with the washing buffer. Washing is carried out for 2 minutes. in an ultrasonic cleaner. Finally, the strips are pulled out and briefly dried with filter paper.

The second test tube is filled to the marked height with AP-A-IgE conjugate. Allergodip is placed in the conjugate and incubated for 18 hours at room temperature.

The strips are then removed and washed in an ultrasonic bath for 2 minutes with washing buffer (0.9% NaCl, 1% Tween, 0.5% NaN3, water).

PL 192 349 B1

After this operation, the test strips are removed and dried with filter paper. Substrate AP-purple IgE is pipetted into the next test tube and the test strips are incubated for 90 min. in room temperature.

The Allergodip test strips are then removed from the tube, dried with filter paper and the color reaction is analyzed (qualitatively and quantitatively). The results are evaluated by comparing them with the Allergodip color cards. Then Allergodip is pasted into the documentation in the patient's card.

Example 4

Total blood test

The tests are performed analogously to the procedure described for the one-day test. The time intervals specified are adhered to. No differences from the serum test were observed. The color reaction is distinct and gives bright results that correspond to the results obtained with the serum test.

Claims (11)

1. In vitro allergy diagnosis strip test, where specific immunoglobulin E (IgE) is detected in total blood or serum, is a complete test for the detection of inhalation allergens, food allergens, cross-reactive allergens and Mediterranean allergens, characterized by each test strip is loaded with a positive control sample, a negative control sample and at least nine allergens, and these allergens are covalently bound. 2. An in vitro allergy diagnosis test strip according to claim 1; The method of claim 1, in the form of a test kit containing test strips, an AP-Anti-IgE conjugate, an AP-purple or BM-purple substrate, patient cards with color codes for visual evaluation, and instructions for describing the procedure. 3. A method for detecting specific immunoglobulin E (IgE) in whole blood or serum according to claim 1; A process as claimed in claim 1 or 2, characterized in that the following steps are carried out consecutively: 1) pipetting of serum or total blood into a test tube, 2) incubation with a test strip, 3) washing with running water, 4) short drying, 5) incubation of the test strip with the conjugate, 6) washing, 7) short drying, 8) incubation of the test strip with the substrate, 9) short drying with tissue paper, 10) collection of the developed test strips, 11) comparison with the color card or comparison and documentation with the use of the color card and the patient's card. 4. Use of a test strip as defined in claim 1. 1 or 2 for the diagnosis of allergies in human and veterinary medicine.