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Allergen-microarray assay

A technology of allergens and microarray chips, which is applied in the field of immunoglobulins combined with allergens, can solve the problems of ineffectiveness, time-consuming and heavy workload, and inability to perform allergies

Inactive Publication Date: 2007-07-11
PHADIA MULTIPLEXING DIAGNOSTICS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0034] However, these traditional immunoassay methods are not very effective for detecting allergies in patients, because the number of various allergies has exceeded hundreds and is still increasing steadily
For a laboratory that tests hundreds of patient samples per day, the time and effort required to analyze patient samples to identify all possible and rare allergies is enormous, so unable to proceed

Method used

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  • Allergen-microarray assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Allergen spotting

[0105] Allergens were arrayed using a Genetic Microsystems GMS 417 spotter. Proteins were spotted onto derivatized glass slides. Dot each allergen once on a dot, 3 dots in total. The dots (features) are approximately 200 microns in diameter.

[0106] Allergens are functionally combined in the following way:

[0107] (A) outdoors (I. trees [1=rBetv1, 2=rBetv2, 3=rBetv4, 4=rJunO2, 5=Cass1], II. grass [6=rPhlp2, 7=rPhlp4, 8=rPhlp5a, 9=rPhlp1, 10=rPhlp6, 11=rPhlp7, 12=rPhlp11, 13=rPhlp12], III. Seed [14=rPhlp2, 15=Artv1a, 16=Mugwort profilin]),

[0108] (C) Food allergens (X. Vegetables [17=Apig1, 18=Apig1.0201, 19=Dauc1.2, 20=rArah2, 21=rArah5], XI. Fruits [22=Mald1, 23=Mald2], XII .shrimp[24=rPena1], XIII.fish[25=recCarp]),

[0109](B) indoor (IV.Mites [26=rDerp1, 27=rDerp2.0101, 28=rDerp2, 29=rDerp2b, 30=rDerp5, 31=rDerp5a, 32=rDerp7, 33=rDerp8, 34=rDerp10, 35=rTyrp2 ], V. Animals [36=rLepd2.01, 37=rLepd13, 38=rEurm2.0101, 39=rFeld1, 40=rFeldla...

Embodiment 2

[0115] SOPHIA (Solid Phase Immunosorbent Assay)

[0116] After incubation overnight from CEL A associates (aldehyde slides) or self-made slides, they were treated with 1×TBST (10 mM Tris pH 8.0 / 150 mM NaCl / 0.5 mM Tris pH 8.0 / 150 mM NaCl / 0.5 %Tween 20) shaking and washing. Slides were then transferred to 1X TBST solution containing 0.01% BSA and blocked for 2 hours at ambient temperature. Slides were next washed in 1×TBST for 15 minutes and rinsed briefly with distilled water.

[0117] Allergen microarrays were incubated with diluted sera (diluted 1 :5 in 1 x TBST) at 37°C for (at least) 60 minutes with shaking. Various dilutions of serum have been tested, ranging from 1:1 to 1:15. Usually a 1:5 dilution gives the best results. Thirty microliters of diluted sera were applied to slides in PressSeal Chambers from SIGMA Technologies. The use of the pressurized sealing box refers to the scheme provided by the manufacturer. Incubation times with serum ranged from 60 minutes to...

Embodiment 3

[0122] Reproducible Assay Analysis of Serum from Patient C

[0123] Patient C's serum was analyzed three times using separately prepared allergen microarrays. The experimental steps are basically the same as above.

[0124] After analysis, slides are scanned using the same hardware facility. Data analysis was performed using the GenePix software package. The data obtained from 3 repeated experiments were calculated and the mean value of the signal intensity of 3 experiments for each allergen was compared.

[0125] Only the corresponding values ​​of the signals which were at least 1.5 times higher than the mean value of the signals of the buffer spots were used for the final analysis. Table 1 shows the mean, standard deviation and percentage of marked difference of the corresponding signals.

[0126] Table 1

[0127] allergen

[0128] The average standard deviation calculated from the values ​​obtained from the analysis of 3 independent experiments was 12.36%. ...

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Abstract

A method is provided for the detection of an immunoglobulin which binds to an allergen in a sample, whereby one or more allergens are immobilized on a microarray chip after which the sample is incubated with the immobilized allergens so that immunoglobulins which are specific for the allergens bind to the specific allergen after which the immunoglobulins which are bound to the specific immobilized allergens are detected, as well as a method for in vitro diagnosis of allergies in a patient.

Description

technical field [0001] The present invention relates to a method for detecting immunoglobulins bound to allergens in a sample and a method for in vitro diagnosis of allergy patients. Background technique [0002] An allergy is a reaction of the body's immune system to a substance that is normally considered harmless. It is this reaction that leads to a class of symptoms known as anaphylaxis. Therefore, allergies are not due to the failure of the immune system, but the overactivation of the immune system. Allergy sufferers can have a wide range of symptoms in response to an allergen. For example, symptoms in some people can include asthma, eczema, rashes, itchy eyes, sinusitis, stuffy or runny nose, and hay fever, but more serious symptoms can also occur. Allergies to things like snake venom, nuts, and shellfish can lead to a critical condition called "anaphylaxis." This is when the body reacts so strongly that it causes swelling in the throat, a drop in blood pressure an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6854
Inventor 赖因哈德·希勒克里斯蒂安·哈瓦内格曼弗雷德·W·穆勒
Owner PHADIA MULTIPLEXING DIAGNOSTICS GMBH
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