EP1019730A1

EP1019730A1 - STRIP TEST FOR DIAGNOSING ALLERGIES $i(IN-VITRO)

Info

Publication number: EP1019730A1
Application number: EP98928264A
Authority: EP
Inventors: Rüdiger WAHL; Oliver Cromwell; Helmut Fiebig
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 1997-05-21
Filing date: 1998-05-14
Publication date: 2000-07-19
Also published as: WO1998053321A1; HUP0003843A2; PL192349B1; PL336978A1; JP2002514306A; DE19721151A1; DE19721151C2; TR199902855T2

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

Strip test for in vitro allergy diagnostics

The invention relates to a strip test for in vitro allergy diagnostics

Assessment of specific immunoglobulin E (IgE) in sera and whole blood from allergy sufferers.

Various methods of in-vivo and in-vitro allergy diagnostics are known. In-vitro allergy diagnostics are becoming increasingly important in addition to in-vivo allergy diagnostics, which include, for example, skin, prick, intracutaneous and provocation tests, since the allergen spectrum is constantly increasing and in-vivo testing in many cases due to side effects is not feasible for the patient.

In addition, there are often no commercial test solutions available for numerous new allergens, since the effort involved in producing the extract and thus also in producing the test solution is often not in proportion to the number of patients when it comes to rare allergies. These include: the professional allergens, e.g. in woods,

Green coffee, hygiene articles such as Latex gloves, etc., which often only affect small groups. In the case of high-grade allergy sufferers, in vivo tests with new, not yet adequately characterized allergen solutions can lead to unacceptable side effects that significantly impair well-being. In vitro diagnostics, on the other hand, has the advantage that it is completely harmless to the patient, since the test is carried out with the patient's serum outside the organism.

The specific IgE plays the decisive role in the in vitro determination of specific IgE and total IgE in patient sera. IgE (immunoglobulin E) is a protein and, from a serological point of view, is primarily responsible for the clinical picture of an allergy. So far, the in vitro determination of allergies is known by means of the allergy test, which was developed by Johansson, Wide and Bennick and is described in Lancet 2, 1105-1107 (1967). This method is the most commonly used test today. It is also the first test system to measure specific IgE in sera from allergy sufferers. Similar test systems are described, for example, in EP 203 238.

All of these known tests can only be performed with sera made from whole blood and are relatively insensitive, i.e. 1-2 ml of serum are required for a meaningful diagnosis per test.

It is also important for many patients that in vitro testing involves a variety of allergens, including rare allergens such as e.g. Belong to latex, so as to allow an accurate and reliable diagnosis. Another disadvantage of the tests known to date, in addition to an inadequate allergen spectrum which these tests cover, is an elaborate and lengthy procedure which cannot be carried out by everyone in a simple manner and, in particular, cannot be evaluated. In addition, such a test should be able to be used for diagnosis both in the human-medical field and in veterinary medicine and should be carried out equally with serum and whole blood without the result being falsified.

It is therefore an object of the present invention to develop a strip test for in-vitro allergy diagnostics which can be handled in a simple manner and which can be carried out both with serum and with whole blood and includes a sufficient number of allergens.

The invention relates to a strip test for in-vitro diagnosis for the assessment of specific IgE, characterized in that the test can be carried out both with whole blood and with serum. The invention further relates to a strip test for in-vitro diagnosis for the assessment of specific IgE in sera and / or whole blood, characterized in that each stick is coated with a positive, a negative control and at least nine different allergens.

The strip test is used to assess specific IgE in whole blood or sera from allergy sufferers and can also be used in the veterinary area in addition to the human area. The evaluation is preferably carried out visually by comparison with a color card. The coloring of the allergen pads on the respective test strips is compared with a given color scale. The color intensity is the indicator for the findings of the allergy diagnosis.

A typical strip test preferably includes 9 allergens, a negative and a positive control. A complete test preferably contains different allergy test strips from different areas. These include, for example, grasses / cereals / herbs (G / G / K), animals / feathers / mites (T / F / M), trees (B), mushrooms (P), food (NM), screening (S) [Phadiatop ].

A typical allergen spectrum is listed in the table below. In addition to these standard allergens there are also allergens of a special kind, such as those found in roasted coffee, certain woods, latex etc.

Tree pollen (B) grasses / cereals / herbs (G / G / K)

Birch mugwort

Oak barley

Alder glass herb

Hazel 6 grasses

Poplar oats

Plane ragweed

European beech rye

Elm plantain

Willow wheat

Neg. Control neg. control

Pos. Control Pos. Control

A commercial test of the present invention includes, for example

Inhalation allergen test strips Food allergen test strips Cross-reactivity allergen test strips and Mediterranean allergen test strips.

The test strips according to the invention are referred to as Allergodip ™. The above-mentioned exemplary composition of such an Allergodip test system in relation to the allergens to be tested can look as follows:

The code numbers were taken from the allergy disc catalog as of October 1993:

^* here officinales

** M-A must still specify whether short, giant ragweed etc.

Other typical components of the test system are also patient color cards, test glass tubes with markings, test stands, conjugate, substrate, disposable pipettes and a regulation for performing and evaluating the test. 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 carried out in the form of a 1-day and a 2-day test. The main difference between the two tests is the duration of incubation with conjugate and substrate. The test can also be performed with whole blood or with serum made from whole blood. The individual test strips are preferably produced by bromine cyan activation, as is also known from allergen pane testing.

BrCN-activated sheets of paper are used to make the Allergodip. The BrCN activation of the paper sheets was based on the allergen disk activation, whereby quantities and volumes were significantly reduced.

Cellulose paper is activated with BrCN. The allergens are covalently coupled to the activated papers. One allergen is coupled for each BrCN activated paper sheet (A4). 4 mm wide strips are cut and glued onto a plastic strip (A4). The plastic strips are e.g. 9 different allergens, a positive and a negative control glued. The A4 plastic strip is cut into 4 mm wide plastic strips (Allergodips) that are approx. 7 cm long.

The allergen test strips are preferably produced in such a way that strips 4 mm wide are cut from the allergen-linked sheets. These strips are glued to each other on a polystyrene plate, approx. 30 cm long and 8 cm wide, using an adhesive tape on both sides. The different allergen strips are glued on top of each other and pressed on firmly. The polystyrene plates so glued with allergen strips are now added overnight using a specially developed press (using a torque wrench)

Room temperature pressed. After pressing, 4 mm wide Allergodip (strips) are cut from the allergen-coated polystyrene plate using a cutting machine. The strips are stored in tubes with 10-piece drying plugs, labeled and stored at -20 ° C. They are stable for at least 2 years under these conditions.

The polystyrene plate is also color-coded at the top to indicate which stripes are involved, e.g. trees. Every Allergodip has a different color. Another object of the invention is a method for the detection of specific IgE in whole blood or sera, which is characterized in that at least the following steps are carried out in succession:

1. Pipetting the serum or whole blood into a test tube;

2. Incubation with the test strip (Allergodip ™);

3. washing under running water;

4. Short drying;

5. Allergodip ™ incubation with the conjugate;

6. washing;

7. Short drying;

8. Allergodip ™ incubation with the substrate;

9. Brief drying with blotting paper;

10. removal of the developed test strip;

11. Comparison against the color card or comparison and documentation using the combined patient / color card.

The test can be carried out in the form of a 1-day or 2-day test:

1 day test

The Allergodip is incubated in a 6.5 mm x 7 cm test tube with the serum for 30 minutes at room temperature. The strip is then washed in an ultrasonic bath for 2 minutes.

The strip is incubated with AP-Anti-IgE for 2 hours at room temperature.

The strip is then washed in an ultrasonic bath for 2 minutes.

The strip is incubated with substrate for 1.5 hours at room temperature.

The strip is dried with filter paper and the color reaction is assessed against a color card (possibly a densitometer).

-Day test

The Allergodip is placed in a test tube containing the serum for 3 hours

Incubated at room temperature.

Washing analogous to the 1-day test.

The Allergodip is incubated with AP-Anti-IgE overnight at room temperature.

Washing analogous to the 1-day test.

The strip is incubated with substrate for 1.5 hours at room temperature.

About the blue color of the allergen pads the height of the spec. IgE level determined in the patient's serum. The 1-day test is divided into 3 allergen classes and the overnight test is divided into 4 classes.

In addition to the test strips, the following additional reagents are required:

Conjugate AP-Anti-IgE conjugate is used as conjugate.

Substrate

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

A 0.9% NaCl solution is preferably used as the washing solution + 1% TWEEN 20 + 0.05% NaN ₃ . The washing solution is preferably diluted accordingly as a 1:15 concentrate.

Investigations have shown that tap water in the ultrasonic bath can be used for washing, and washing under the tap (1 minute) can also be carried out.

Various devices are required to carry out the Allergodip, e.g. an ultrasonic bath, calibrated test tube, holder for Allergodip, stand for the test tube, disposable pipettes and a color card. Ultrasonic bath washing can be replaced by washing under the tap.

Calibrated test tube tubes (1.5 ml) are preferably used to specify how much serum + reagents have to be pipetted.

The tubes should be placed in plastic or plexiglass stands.

The Allergodip can be washed in an ultrasonic bath, which is filled with the washing solution. However, studies have shown that washing under the tap is also possible.

In order to hang the allergy test strips in the ultrasonic bath filled with washing solution, a special holder is required, consisting of a plexiglass pane into which grooves for the holder clamps are milled. A holder for the strips should also be used for washing under the tap.

After the end of the test, the allergy test strips must be dabbed briefly with blotting paper in order to be able to read them. The Allergodip is evaluated using a color card that is adapted to the colors that correspond to the 1 or 2-day test. The color card enables a classification from class 0 - 4.

The Allergodip is offered as a 1 and 2 day test.

A patient card is preferably used to document the results. Among other things, the card gives the allergen composition again and is color coded individually for each test strip.

The invention furthermore relates to the use of the strip test for in vitro allergy diagnosis in the human and veterinary field. In particular, the strip test can be used to detect inhalation allergens, food allergens, cross-reactivity allergens or Mediterranean allergens and to diagnose the associated allergies.

All absorbent matrices that are usually used for such tests can be used as reagent papers. The use of filter paper is the most common, but other absorbent cellulose or plastic products can also be used. The absorbent carriers, preferably filter paper, are impregnated in a manner known per se with impregnation solutions which contain the reagents necessary for the determination of chlorine. The soaked and dried papers can be processed into square or rectangular zones, which in turn are applied in a known manner to the carrier film, e.g. Plastic films, paper or metal strips can be glued or sealed. The adhesive area is limited to a fraction of the reagent paper area, e.g. to 10 to 20%. The reagent papers can also be coated in strips before being impregnated

Plastic tape applied, and after impregnation cut into handy sticks perpendicular to the direction of the strip.

The following examples serve to explain the invention in more detail, but in no way to limit it. example 1

Allergy coupling to BrCN-activated filter papers for the production of the test strips (Allergodip)

All information relates to an allergen coupling to a BrCN filter paper sheet of the size 16 x 26 cm = 416 cm ² and a protein specification of 10 μg / ml allergen dilution solution.

Only one sheet of BrCN filter paper can be incubated in a metal tray.

For allergen coupling, the production of 6 allergen leaves in one work step has proven to be practical.

Foam formation in protein-containing solutions leads to uncontrollable denaturation of proteins. Foaming by vigorous shaking or stirring should therefore be avoided.

By incubating an allergen solution with bromine-activated filter paper sheets (for production see PA 6.02.03), allergens are immobilized by forming a covalent bond.

To couple the respective allergen, 115 ml of allergen coupling solution are placed in an incubation dish. The bowl is moved so that the sheet is completely covered with allergen coupling solution.

Incubation: IKA shaker, speed: 45 red / min, 4 ° C, overnight.

The following washes are carried out:

Take 5 - 10 ml of allergen solution for protein determination. Decant or aspirate the allergen solution as completely as possible. Washing solution No. 1:

Move 1 x 120 ml, 10 min. At 4 ° C on the IKA shaker. 1 x 120 ml, agitate for 3 h at 4 ° C on the IKA shaker.

Washing solution no.2:

Move 3 x 120 ml, each 10 min. At 4 ° C on the IKA shaker.

Washing solution No. 3:

2 x 120 ml, each on the IKA shaker at 4 ° C for 10 min. Store sheets of paper under incubation buffer until further processing.

There should be no foam.

The test sheets or strips are freeze-dried for storage.

If freeze-drying is not possible immediately after coupling the allergen, the allergen sheets are sealed individually, airtight and without buffer in a film tube and stored temporarily at -20 ° C.

The sheets or strips are freeze-dried without a buffer, individually in GT trays. 8 sheets of allergen can be dried in one pass. Freeze drying overnight is sufficient.

After drying in the freezer, the allergen sheets must be individually sealed and labeled immediately. Storage at -20 ° C. Example 2

Carrying out the 1-day test with patient serum.

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

Mix the substrate by gently tipping it several times before use. Switch on the incubator before working to reach the working temperature of 37 ° C for the substrate incubation.

Patient serum is pipetted into the test glass tube up to the ring marker. Allergodip, the test strip, is placed in the serum. All allergen pads must be under liquid.

Incubation takes place for 30 minutes at room temperature. The Allergodip is removed from the serum and hung on the holder in an ultrasonic bath filled with washing buffer.

- All allergen pads must be covered with the wash buffer. Wash for 5 minutes in an ultrasonic bath. Remove the sticks and dab them briefly with filter paper.

The conjugate is pipetted into a second test tube up to the ring marker. The Allergodip is placed in the conjugate and added for 2 hours

Incubated at room temperature.

The wash is carried out with the same wash buffer as before in an ultrasonic bath for 5 minutes. The Allergodip is removed and dabbed briefly with filter paper, then the substrate is pipetted up to the ring marker in a third test tube and the Allergodip is placed in the substrate. Incubation takes place for 3 hours at 37 ° C in the incubator. The Allergodip test strip is then removed from the tube, blotted with filter paper and the color reaction is read. The results are assessed by comparison with a color chart (1-day test - class 0 - 4).

Allergodip can be stuck on for documentation. The color complex of the test strip is stable for at least one year at room temperature.

Example 3

Carrying out the 2-day test with patient serum

Before the reagents and Allergodip are used in the test, they must be brought to room temperature. Mix the substrate by gently tipping it several times before use.

The patient serum is pipetted into the test glass tube up to the ring maker. Allergodip is placed in the serum. All allergen pads must be under liquid. The incubation is carried out for 3 hours at room temperature. The Allergodip test strip is removed from the serum and hung in an ultrasonic bath filled with washing buffer using the fastening clip or a holder. All allergen pads must be covered with the wash buffer and washed in an ultrasonic bath for 2 minutes. Then the sticks are removed and dabbed briefly with filter paper.

The conjugate AP-A-IgE to ring marker is then pipetted into a second test tube and the test strip is placed in the conjugate. The incubation takes place over a period of 18 hours at room temperature.

Then the test strip is removed and with a washing buffer

(0.9% NaCl, 1% Tween 20, 0.5% NaN ₃ , water) washed in an ultrasonic bath for 2 minutes. The test strip is then removed and blotted with filter paper. The AP-purple IgE substrate is then pipetted into another test tube and the test strip is incubated for 90 minutes at room temperature.

Finally, the Allergodip test strip is removed, dried with filter paper and the color reaction is read and evaluated. The results are assessed by comparison with the Allergodip color chart. The Allergodip is stuck in the patient card for documentation.

Example 4

Testing with whole blood

The test is carried out in the same way as the 1-day test. All time intervals are retained. There were no differences in the serum test. The color reaction was clear and distinct and corresponded to that of the serum test.

Claims

- 17 patent claims

1. Strip test for the in vitro allergy diagnosis of specific IgE, characterized in that the test can be carried out both with whole blood and with serum.

2. Strip test according to claim 1, characterized in that each stick is coated with a positive, a negative control and at least 9 allergens.

3. Strip test according to claim 1, characterized in that it represents a complete test for the detection of inhalation allergens, food allergens, cross-reactivity allergens and Mediterranean allergens.

4. Strip test according to one or more of the preceding claims, characterized in that it is in the form of a test system (kit).

5. Strip test system according to claim 4 containing 10 allergen

Dipsticks, conjugate, substrate, patient color cards with color code for visual evaluation and an implementation regulation.

6. A method for the detection of specific IgE in whole blood or sera, characterized in that at least the following

Steps to be performed in sequence: