Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins

Definitions

• the invention relates to a strip test for in vitro allergy diagnostics
• IgE immunoglobulin E
• 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.
• IgE immunoglobulin E
• IgE immunoglobulin E
• 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 ].
• allergen spectrum is listed in the table below.
• allergens of a special kind such as those found in roasted coffee, certain woods, latex etc.
• Tree pollen grasses / cereals / herbs (G / G / K)
• 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.
• Allergodip TM The test strips according to the invention are referred to as Allergodip TM.
• the above-mentioned exemplary composition of such an Allergodip test system in relation to the allergens to be tested can look as follows:
• test system 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)
• Allergodip strips
• 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:
• test can be carried out in the form of a 1-day or 2-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).
• the Allergodip is placed in a test tube containing the serum for 3 hours
• the Allergodip is incubated with AP-Anti-IgE overnight at room temperature.
• 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).
• the 1-day test is divided into 3 allergen classes and the overnight test is divided into 4 classes.
• Conjugate AP-Anti-IgE conjugate is used as conjugate.
• AP-purple is used as the substrate.
• 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.
• 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.
• studies have shown that washing under the tap is also possible.
• a special holder 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.
• 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.
• 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.
• the strip test can be used to detect inhalation allergens, food allergens, cross-reactivity allergens or Mediterranean allergens and to diagnose the associated allergies.
• 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
• Foam formation in protein-containing solutions leads to uncontrollable denaturation of proteins. Foaming by vigorous shaking or stirring should therefore be avoided.
• allergens are immobilized by forming a covalent bond.
• allergen coupling solution 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.
• test sheets or strips are freeze-dried for storage.
• 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.
• 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.
• 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
• 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.
• 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.
• test strip is removed and with a washing buffer
• test strip (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.
• 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.
• 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.

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• Immunology (AREA)
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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 1997
  • 1997-05-21 DE DE19721151A patent/DE19721151C2/de not_active Expired - Fee Related
• 1998
  • 1998-05-14 EP EP98928264A patent/EP1019730A1/de not_active Ceased
  • 1998-05-14 HU HU0003843A patent/HUP0003843A2/hu unknown
  • 1998-05-14 TR TR1999/02855T patent/TR199902855T2/xx unknown
  • 1998-05-14 PL PL336978A patent/PL192349B1/pl not_active IP Right Cessation
  • 1998-05-14 WO PCT/EP1998/002850 patent/WO1998053321A1/de not_active Application Discontinuation
  • 1998-05-14 JP JP54991598A patent/JP2002514306A/ja not_active Ceased

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