DE202009006503U1 - Assay devices for the determination of immunoglobulins E - Google Patents

Abstract

Assay devices for the determination of immunoglobulins E, consisting of an allergy lateral flow assay (ALFA), which consists of allergen solutions (screens) and of an ALFA base set as a base unit with a ready-to-use test unit made of cassettes.

Description

The This invention relates to assay devices for the determination of immunoglobulins E - IgE or sIgE (specific IgE, allergen - specific IgE) - in rapid tests for the qualitative determination of Total IgE in human serum or plasma or sIgE in whole blood, Serum or plasma. Designed as the Allergy Lateral Flow Assay (ALFA) Devices are designed for a screening test to the user Being able to give an allergy test very quickly and be able to perform clearly. By the Selecting suitable allergen solutions becomes a symptom-based Allergy diagnostics possible.

The incidence of allergies has increased dramatically worldwide in recent decades. Allergies are pathological immune reactions of type I (immediate-type reaction) whose symptoms generally occur within 30 to 60 minutes. As a disease symptoms include: hay fever (rhinitis), conjunctivitis (conjunctivitis), hives (urticaria), allergic asthma and the most dangerous manifestation of anaphylaxis - the anaphylactic shock ( Jörg Rinne, Jens Becker: The 1 × 1 allergy. Synergia Verlag, Darmstadt 2007 ).

Known methods of determining analytes include the lateral flow assays (LFA) with their test systems. They are based on the same principle as other immunological assays (eg ELISA - enzyme-linked immunosorbent assay, enzyme immunoassay) because they are based on the effect of the antibody-antigen reaction. In addition, LFA have chromatographic properties because the antibodies are bound to a membrane and the sample to be analyzed is pulled over the test strip by the capillary forces and thereby a rapidly visible result is achieved ( Konrad Faulstich et al.: Point-of-Care Technologies; Devices and applications for lateral-flow immunodiagnostics archive, Jul 07 ).

In the EP 291194 (Analytical test device) z. For example, the use of lateral flow assay devices for detecting analytes in fluid samples is proposed (Holders: Inverness Medical Switzerland GmbH, CH and Umilever NV, NL - with a family of 61 patents). According to this invention, a sample on a test strip initially flows through a reaction zone containing a labeled specific binding reagent which is free to move within the wet test strip. Thereafter, the sample enters a test zone downstream of the reaction zone which contains an immobilized specific binding reagent which binds either the labeled binding reagent or the analyte. The test strip may also be provided with a liquid effluent which allows unbound reagent or unbound analyte to be washed out of the test zone, and downstream of the test zone also a control zone may be provided DE 21 2005 000 034 U1 ).

The Utility Model DE 21 2005 000 034 U1 (Improvements in or relating to lateral flow assay devices; owner: Inverness Medical Switzerland GmbH, CH) itself has an assay device for content identified as a "lateral flow immunoassay device". This involves the determination of analytes in a sample, the device comprising a test strip with a reading zone in which the result of the test can be viewed and also a mobile part is provided, which can be moved from a first (test) position to a read zone second (reading) position is movable.

Other descriptions based on lateral flow assays include methods and apparatus therefor ( WO 2006/013329 A1 ), the test strips of the assay ( WO 2008/018073 A1 ), Test equipment for performing specific binding tests EP 560410 B1 ) or devices for reading test strips EP 653625 B1 ).

Of the Invention is based on the object, an improved allergy test (Search test) to provide. The task was According to the invention by rapid tests for qualitative Determination of immunoglobulins (Ig) E dissolved, both a measurement of total IgE in human serum or plasma as well of sIgE (specific IgE, allergen-specific IgE) in whole blood, Serum or plasma allows.

To is a device designed as an Allergy lateral flow assay (ALFA) developed from allergen solutions (screens) and from an ALFA base set as a base unit with a ready to use one Test unit composed of cassettes.

The qualitative determination of total IgE is based on the following findings:
Allergic reactions of the immediate type (type I allergy) are mediated by allergen-specific immunoglobulins of class E (IgE). The normal serum IgE concentration is age-dependent and reaches its highest value at the age of 6 to 15 years. In many cases, the occurrence of allergen-specific IgE is accompanied by a significant increase in the total IgE concentration in the blood of the patients.

The test principle consists of a combination of a test unit - the ALFA Total IgE Basic Set - with an anti-IgE solution (more details in Example 1) the embodiments).

The Qualitative determination of allergen-specific according to the invention IgE (sIgE, specific IgE) is in human whole blood, serum or plasma. The method of determination is an Allergy lateral flow assay (ALFA) designed a device for a screening test, which gives the user the option of an allergy test to carry out quickly and clearly. By the selection of suitable allergen solutions becomes symptom-based Allergy diagnostics possible.

The triggering allergens of the type I reaction with a few exceptions to the group of proteins and are mostly derived from the natural environment, such. From plant pollen, Animal hair, food, mites or insecticides. A characteristic Characteristic of a type I allergy is the involvement of allergen-specific Immunoglobulins (antibodies) of class E (sIgE). Of the Evidence of sIgE is therefore an integral part of the current Allergy diagnosis. The test principle according to the invention consists of a combination of a test unit - the ALFA basic set as single and eight-fold cassete - with variable usable allergen solutions (closer in the example 2 of the embodiments).

The evaluation of the test results is based on a test scheme ( 1 ), which consists of a sample application (S) and an evaluation window (TC) with a test line (T) and a control line (C).

For the testing will transfer the sample material to the sample application site S of the base set pipetted and then immediately the desired allergen solution added. While During the incubation period, the fluid becomes due to capillary forces pulled through the test unit. The sIgE binds the sample specifically to the antigens contained in the allergen solution. The Antigens are tagged and become via a catcher molecule (Anti-IgE labeled with a ligand) retained on the test line T. simultaneously the sIgE is bound by an antibody that binds to colored Particle is coupled (conjugate). Unbound conjugate will retained at the control line C and forms within the incubation period, a dark colored line.

The inventive method for the determination of Immunoglobulins E is an allergy lateral flow assay (ALFA) is made up of allergen solutions (screens) and from an ALFA base set as a base unit with a ready to use one Test unit composed of cassettes. With the immunoglobulins it is IgE or sIgE and allergen solutions to those from serum, plasma or whole blood.

By the choice of allergen solutions will be surprising possible that the test method is symptom- and patient-specific can be carried out.

Among other things, the following single allergens and allergen mixtures can be determined:
Mites (D. pterus, d1, D. farinae, d2), birch (t3), meadowgrass (g6), cat (e1), dog (e5), Alternaria alternata (m6).

Of the Seasonal Screening affects canine grass (g2), lolk (g5), common grass (g6), meadow bluegrass (g8), rye (g12), Alternaria alternata (m6), birch (t3), mugwort (w6).

Of the perennial screen refers to mites (D. pter., d1; D. farinae, d2), cat (e1), dog (e5), Aspergillus fumigatus (m3), Aspergillus niger (m33).

• • eine kurze Testdauer (Resultat nach 20 min)
• • die Verwendbarkeit von Serum, Plasma und von Vollblut
• • eine hervorragende Übereinstimmung mit dem Skin Prick-Test und anderen in-vitro sIgE-Testmethoden.

The test according to the invention is characterized by

• • a short test duration (result after 20 min)
• • the usability of serum, plasma and whole blood
• • Excellent agreement with the Skin Prick test and other in-vitro sIgE test methods.

The Features of the invention will become apparent from the elements of the claims and from the description, showing both individual characteristics as well as several in the form of combinations advantageous embodiments for which protection is sought with this document becomes. The essence of the invention is a combination of known (Lateral flow assay, determination of IgE) and new elements (Allergy Lateral flow assay, use also of whole blood, determination of sIgE), which influence each other and in their new overall effect a use advantage and the desired success result in it lies that the assay devices of the invention a very fast and clear allergy test and a symptom-based Allow allergy diagnostics.

The sensitivity and specificity of Allergy lateral flow assay (ALFA) was determined against various in vitro diagnostic testing methods (Immuno CAP ^® and ALLERG-O-LIQ).

The Invention is intended to be based on exemplary embodiments be explained without being limited to these examples to be.

embodiments

Example 1: Qualitative determination of Total IgE in human serum or plasma

allergic Immediate type reactions (type I allergy) are caused by allergen-specific Immunoglobulins of class E (IgE) mediated. The normal serum concentration IgE is age-dependent and reaches the highest Value at the age of 6 to 15 years. In many cases it works the occurrence of allergen-specific IgE with a clear Increase in total IgE concentration in the blood of the patients. In these cases, the IgE level in the blood can be 1000 times increase. The IgE concentration is given in IU / mL, where 1 IU corresponds to about 2.4 ng IgE.

The highest IgE concentrations (up to 50,000 IU / mL) found in patients with atopic dermatitis. About that In addition, increased total IgE levels also occur in parasitoses on. Deviating from the standard titers are u. a. in parasitic infections and in certain autoimmune diseases.

1.1 .: Test principle

ALFA Total IgE consists of one test unit - the ALFA Total IgE Basic Set - in combination with an anti-IgE solution.

To perform the test, the patient sample (serum or plasma) is placed in the sample application site of the base set. Immediately thereafter, the anti-IgE solution is also dropped into the sample application site. During the incubation period of 25 to 30 min, the liquid is drawn by capillary force through the test unit. The anti-IgE binds specifically to the IgE of the sample. Anti-IgE is labeled with a ligand and is isolated by capture molecules (recognition elements or binding partners immobilized) on the three test lines (TC in 1 ) withheld.

simultaneously is the IgE bound to anti-IgE by an anti-IgE conjugate (anti-IgE gold), which has a red color bound. The intensity the color reaction of the three test lines is proportional to the amount the bound immune complexes consisting of ligand-labeled anti-IgE, IgE, and anti-IgE gold conjugate. For interpretation of the result is the number of visible test lines and their intensity used.

Unbound conjugate is added to the control line (C in 1 ) withheld. After incubation for 25 to 30 minutes, a dark red line develops there.

1.2 .: Test procedure

• a. 30 μl of serum or plasma transferred to the sample order window of the base set.
• b. Immediately add 2 drops of the anti-IgE solution into the Sample order window of the basic set given.
• c. After 25 to 30 minutes, the test result in the evaluation window of the basic set.

Of the Test must be evaluated within 25 to 30 minutes after the start of the test become. A later or earlier assessment may cause a wrong interpretation of the result have as a consequence.

1.3 .: Evaluation of the test results

The Test result is only valid if the control line (C) the test unit is clearly visible. Based on the number of visible Test lines (T) and their intensity will be the result of ALFA Total IgE interpreted with the help of an evaluation card.

Example 2: Qualitative determination of Allergen-specific IgE in human serum, plasma or whole blood

allergy Lateral Flow Assay (ALFA) is a rapid test for qualitative determination of sIgE in human serum, plasma or whole blood. ALFA is as a search test designed and intended to give the user the opportunity an allergy test very fast and unmistakable to carry out. By selecting suitable Allergen solutions will be a symptom-based allergy diagnosis allows.

2.1 .: Test principle

The ALFA Basic Set is a single and eight-fold cassette with variable usable allergen solutions have been developed.

For testing, the sample material is placed in the sample application site (S in 1 ) of the base set and then immediately added the desired allergen solution. During the incubation period of 20 min, the liquid is drawn through the test unit due to capillary forces. The sIgE of the sample specifically binds to the antigens contained in the allergen solution. The antigens are labeled and are retained by a capture molecule on the test line (T).

At the same time, the sIgE is bound by an antibody that is coupled to colored particles (conjugate). The color intensity at the test line is proportional to the amount of immune complexes Ligand-labeled antigens, sIgE and IgE-specific conjugate. The signal intensity ranges from light pink (very little sIgE in the sample) to dark red (a lot of sIgE in the sample).

Unbound Conjugate is retained at the control line (C) and forms a dark red line within the incubation period.

2.2 .: Test procedure

• a. 30 μl serum or plasma or 30 μl Whole blood is transferred to the sample application window (S) of the Basic Set.
• b. Immediately add 2 drops of the desired allergen solution into the sample order window of the base set.
• c. After 25 to 30 minutes, the test result in the evaluation window (TC) of the basic set.

Of the Test must be evaluated within 25 to 30 minutes after the start of the test become. A later or earlier assessment may cause a wrong interpretation of the result have as a consequence.

2.3 .: Evaluation of the test results

The Test result is only valid if the control line (C) the test unit is clearly visible.

• • Schwache Resultate sind erst nach Ablauf von 20 min sichtbar.
• • Die Kontrolllinie ist in der Regel stärker gefärbt ist als die Testlinie.
• • Stark-positive Resultate sind eventuell schon vor Ablauf der Inkubationszeit sichtbar.
• • Testeinheit ins Licht halten.

The evaluation is carried out on the basis of the evaluation of the test line (T). The color intensity of the test line correlates with the amount of sIgE contained in the sample. Please note:

• • Weak results are only visible after 20 min.
• • The control line is usually more colored than the test line.
• • Strong positive results may be visible before the incubation period.
• • Hold the test unit in the light.

Possible Test results are:

• Variant 1: Test line (T) and control line (C) are colored → that Test result is positive, the patient sample contains sIgE against the allergen / allergen mixture used
• Variant 2: Test line (T) is not colored, but the Control line (C) is colored → the test result is negative, the patient sample contains no measurable Concentration of sIgE against the allergen used / the allergen mixture
• Variant 3: Test line (T) and control line (C) are not colored → that Test result is invalid, the test must be with a new one Basic set to be repeated
• Variant 4: Test line (T) is colored, but control line (C) is not colored → the test result is invalid, the test must be repeated with a new base set become.

limits:

Height Titers of allergen-specific IgG can mask sIgE. Negative test results may be a weak sensitization of the patient against the allergen investigated. The sensitivity may be at the test execution with whole blood with some allergens, compared with the sensitivity of serum or plasma, lower. The test procedure is recommended with serum or plasma.

Example 3: Seasonal Screen

trigger an allergy in spring or summer can Allergens of birch, mugwort or grass pollen, Molds or other allergens - like dog tooth grass, Lolch, periwinkle, meadow bluegrass, rye or Alternaria alternata - be, which fly through the air at this time and against the body has formed allergen-specific antibodies (sensitization). These allergens are taken via the respiratory tract. pollen are the second most common cause of house dust mites an allergy. With the help of the test according to the invention May cause sensitization to these allergens within 20 be detected up to 30 min.

3.1: test principle

To Purpose of the blood and the allergen solution bind allergen-specific Antibody (sIgE) to the allergens in the solution. The allergens are labeled and bind via this label to the test line. A conjugate (red) contained in the test cassette binds the sIgE. After 20 to 30 minutes, the test result can be read off become. A slightly pink to dark red test line is a positive one Result and confirms the suspicion of sensitization against the allergens mentioned.

Example 4: Perennial Screen

Allergy allergens can be caused by dust mite allergens, cat and dog epithelia, mold or other allergens - such as Aspergillus fumigatus or Aspergillus niger - that fly through the air all year round and have been sensitized by the body to allergen-specific antibodies (sensitization). , These allergens are taken via the respiratory tract. Dust mites are the most common cause of allergy to pollen. With the aid of the test according to the invention, a sensitization to these allergens in be detected within 20 to 30 min.

4.1 .: Test principle

To Task of the blood and the allergen solution bind alergen-specific Antibody (sIgE) to the mentioned allergens in the solution. The allergens are labeled and bind via this label to the test line. A conjugate (red) contained in the test cassette binds the sIgE. After 20 to 30 minutes, the test result can be read off become. A slightly pink to dark red test line is a positive one Result and confirms the suspicion of sensitization against the allergens mentioned.

1 : Test scheme ALFA

• S

  Sample application window

  TC

  evaluation window

  T

  test line

  C

  line of control

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - EP 291194 [0004]
• - DE 212005000034 U1 [0004, 0005]
• WO 2006/013329 A1 [0006]
• WO 2008/018073 A1 [0006]
• - EP 560410 B1 [0006]
• - EP 653625 B1 [0006]

Cited non-patent literature

• - Jörg Rinne, Jens Becker: The 1 × 1 allergy. Synergia Verlag, Darmstadt 2007 [0002]
• Konrad Faulstich et al .: Point-of-Care Technologies; Devices and applications for lateral-flow immunodiagnostics archive, Jul 07 [0003]

Claims (5)

Assay devices for the determination of immunoglobulins E, consisting of an allergy lateral flow assay (ALFA), which is from allergen solutions (screens) and from an ALFA basic set as a base unit with a ready-to-use test unit made of cassettes composed. Assay devices according to claim 1, characterized in that that the immunoglobulins are IgE and / or sIgE (specific IgE, Allergen-specific IgE) and that the allergen solutions consist of serum, plasma or whole blood. Assay devices according to claim 1 or 2, characterized indicated that they use a seasonal screen as an allergy test show proof of sensitization to allergens from birch, mugwort or grass pollen, from mold fungi or from dog tooth grass, lolk, elmgrass, meadow bluegrass, rye or Alternaria alternata. Assay devices according to claim 1 or 2, characterized characterized in that it is a perennial screen as an allergy test show proof of sensitization to allergens from house dust mites, cat and dog epithelia, molds, Aspergillus fumigatus or Aspergillus niger. Assay devices according to claim 1 or 2, characterized characterized in that the ALFA base set as a base unit a ready to use Test unit consisting of single cassettes or eight-cassettes - with variably applicable allergen solutions - exists.