CN104237527A - Kit and method for detecting specific allergen antibody IgE in high-sensitivity manner - Google Patents
Kit and method for detecting specific allergen antibody IgE in high-sensitivity manner Download PDFInfo
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- G—PHYSICS
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- 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
Abstract
The invention provides a kit and a method for detecting specific allergen antibody IgE in a high-sensitivity manner. The kit mainly comprises a fiber material membrane provided with allergen protein to be detected, double biotin-streptavidin optimized liquid, a biotin coupled anti-human IgE antibody, biotin or streptavidin marked polymerase, and a substrate color developing agent corresponding to polymerase in a curing manner; the kit can qualitatively or semi-quantitatively detect the concentration of the specific allergen antibody IgE in human serum or plasma rapidly in the high-sensitivity manner, can screen dozens of allergens, is rapid and accurate, and is suitable for high-throughput testing, and a relatively small quantity of samples are used.
Description
(1) technical field
The present invention relates to a kind of qualitative or semi-quantitatively sieve Check or detect the detection kit of the multiple allergenic specific IgE antibody levels in human serum or blood plasma with sensitivity, and detection method.
(2) background technology
2011 ~ 2012 years world's allergic reaction tissue (WAO) point out about the white paper of allergic reaction (also claiming allergic reaction, Allergy): " the popular of anaphylactia rises just dramatically in developed country and developing country's worldwide.Anaphylactia comprises allergic asthma, allergic rhinitis, severe allergic reaction, and medicine, food and insect allergy react, eczema, nettle rash (hay fever) and angioedema.The increase that especially children suffer from anaphylactia number of past Two decades years has the trend broken out." (Pawankar R et al, WAO White Book on Allergy2011-2012:Executive Summary.) prevalence rate of anaphylactia that only food causes just reaches 3% ~ 6%.(Sicherer S H, Epidemiology of food allergy.J Allergy Clin Immunol.2011; 127:594-602.) anaphylactia is that patient sucks or ingests and (is called anaphylactogen or allergen into the material containing allergen, Allergen) B cell triggering body afterwards produces excessive immunoglobulin E (Immunoglobulin E, IgE), just be attached on the high affinity receptor Fc ε R1 on mast cell and basophilic granulocyte surface with Allergens cross-link when contact allergy is former again in vivo for IgE antibody, cause the gathering of Fc ε R1 acceptor, make mast cell and Basohil activation.Mast cell retting conditions in reactivation process also discharges the inflammatory mediator be stored in cytoplasmic granules: histamine, with cell factor and chemotactic factor (CF)s such as the leukotriene synthesized by arachidonic acid pathway, immunoreactivity prostaglandin and IL4, IL5, cause anaphylactoid disease symptoms.The generation of anaphylactia, IgE antibody plays a crucial role, and is called the allergic reaction (i.e. Gell-CoombsI type hypersensitivity, or the speed property the sent out hypersensitivity of title IgE mediation) that IgE mediates.The feature of anaphylactia of IgE mediation is high under allergenic specific IgE (sIgE) the antibody concentration compared with normal situation in patient body Inner eycle blood, and illness is more serious, and sIgE antibody concentration is higher.（Bloebaum?R?M?et?al，2004，Mechanisms?of?IgE-mediated?allergic?reactions，In：Lockey?RF?et?al?ed，Allergens?and?allergen?immunotherapy，Marcel?Dekker，Inc，New?York，USA，PP65-84.）
The clinical diagnosis of anaphylactia is pointed out in U.S. clinical doctor practical guide, according to patient's medical history binding site thorn or blood testing allergenic specific IgE (sIgE) antibody concentration, examination is caused a disease anaphylactogen (Siles R I, Hsieh F H, Allergy blood testing:A practical guide for clinicians.Am Clin J Medicine.2011; 78:585-592.).At present, detect allergenic specific IgE (sIgE) antibody concentration in blood, the cause a disease method of anaphylactogen of examination has enzyme immunoassay (EIA), Western blot (Immunoblotting Assay), collaurum Sidestream chromatography method (LFA), protein chip (Proteins microarray) etc., development trend also meets market demands: robotization, fast, accurately, amount of samples is few, an examination tens kinds of anaphylactogens.On market, product is many, wherein, Pharmacia Inc., Sweden's product I mmunoCAP250 system is the representative of enzyme immunoassay, and ImmunoCAP Rapid is the representative of collaurum Sidestream chromatography method, ImmunoCAP ISAC is the representative of protein chip, has started allergen molecule diagnosis; The AllergyScreen of German Mediwiss-analytic company be then Western blot representative (
immunoblot for analysing specific IgE in human serum).Because of IgE antibody mean concentration ~ 0.005ug/ml in human blood, 0.002% of total immunoglobulin (Ig) mean concentration, sIgE antibody concentration is lower, for meeting robotization, fast, accurate, amount of samples is few, an examination tens kinds of anaphylactogens, the requirement of allergenic specific IgE (sIgE) antibody concentration in sxemiquantitative or quantitatively detection sample, must configure the detector that photosignal amplifies, or the signal amplifying system of biochemical method.As, Pharmacia Inc., Sweden product I mmunoCAP Rapid once can only examination about ten kinds of anaphylactogens, be unworthy of reading apparatus, the sensitivity of naked eyes interpretation sIgE antibody concentration only has 1.0IU/ml(1IU IgE=2.44ngIgE), distinguish negative and positive findings with 1.49IU/ml.
(3) summary of the invention
The object of the invention is to provide a kind of detection kit and the detection method that use the signal amplifying system of biochemical method (the second signal amplification system of two biotin-Streptavidin and polymerase) to detect allergen specificity antibody IgE with sensitivity, in order to allergen specificity antibody IgE concentration that is qualitative or that semi-quantitatively detect fast in human serum or blood plasma, and can an examination tens of kinds of anaphylactogens.
The technical solution used in the present invention is:
A kind of kit of highly sensitive detection allergen specificity antibody IgE, mainly comprise: the fibrous material film being solidified with allergen protein to be measured, two biotin-Streptavidin optimizes liquid, antihuman IgE antibody's (commercially available acquisition of biotin coupling, during use, the PBS containing 0.05%Tween20 of pH7.4 is mixed with the working fluid of concentration 1:100 ~ 10000), polymerase (the commercially available acquisition of biotin or marked by streptavidin, the working fluid of concentration 1:5000 ~ 50000 is mixed with enzyme buffer liquid) during use, and substrate developer (the commercially available acquisition corresponding with described polymerase, also can prepare voluntarily), it is two biotins that described pair of biotin-Streptavidin optimizes liquid: the mixture solution of Streptavidin molar concentration rate 24 ~ 39:18, described people's IgE antibody is sheep antihuman IgE antibody or rabbit antihuman IgE antibody, and described polymerase is poly alkaline phosphatase or poly horseradish peroxidase.Above-mentioned is the principal ingredient of reagent, does not comprise and detects conventional reagent used as dilution, cleansing solution etc.
Described pair of biotin-Streptavidin is optimized liquid and can be prepared as follows:
(1) be dissolved in ethanol/dimethyl sulfoxide (DMSO) (85/15, V/V) solution by two biotin, obtained concentration is 1.1mmol/L, obtains A liquid;
(2) prepare the solution of streptavidin of 1.0mg/mL with the PBS of 0.05mol/L, obtain B liquid;
(3) under violent vortex, according to its mol ratio, slowly A liquid is added in B liquid, obtain described pair of biotin-Streptavidin and optimize liquid.
The ratio optimizing twin biotin and Streptavidin molecular mixing in liquid can be 12:9,7:6,19:9,13:6 etc., sees shown in Fig. 1 and Fig. 2.
Preferably, described fibrous material film can be fixedly arranged in the liquid filling hole of reactive tank, using this reactive tank directly as Kit components.Described reactive tank can be as shown in Figure 4.
Described fibrous material film is nitrocellulose filter, mixed cellulose ester membrane or porous PVF film etc. (commercially available acquisition), has the counter-bending and antifungal gene physical strength of sufficient Enough, is fixedly arranged in reactive tank after solidifying allergen protein to be measured.Fibrous material film is different with the allergen protein that need solidify, the method activated fiber material membranes such as Bian bromination nitrile, glutaraldehyde, carboxyl sex change, amino sex change, chloromethyl sex change can be distinguished, make it the corresponding allergen protein of covalent bond enough (0.2 ~ 2.0ug), fully in conjunction with the sIgE antibody in sample liquid, the accuracy of testing result can be guaranteed.
Described allergen protein can extract according to a conventional method from following natural material, comprising: plant pollen class (tree pollen class, showy flowers of herbaceous plants powder class and weeds pollen class etc.) anaphylactogen; Der Pilz anaphylactogen; Animal scurf class anaphylactogen; Insects anaphylactogen; Vegetation foodstuff class (comprising fruits, greengrocery, nut fruits, edible fungi and cereals) anaphylactogen and animal food class (comprising meat, Poultry and Eggs, Fish and shell-fish and milk) anaphylactogen etc.
Detection reagent of the present invention uses antihuman IgE antibody's trapping agent solution of biotin coupling simultaneously, two biotin-Streptavidin optimizes liquid and solution, both the biomagnification system of the two biotin-Streptavidin of tool, have again polymerase-colour developing amplification, very big raising detection sensitivity, anticipates as shown in Figure 3.
When described polymerase is poly alkaline phosphatase, corresponding developer is the potpourri (NBT/BCIP of nitro tetrazole orchid and the chloro-3-indoylphosphate of the bromo-4-of 5-, Nitroblue Tetrazolium/5-Bromo-4-chloro-3-Indolyl Phosphate), or paranitroanilinum (p-nitrophenyl phosphate).
When described polymerase is poly horseradish peroxidase, corresponding developer is 3,3 ', 5,5 '-tetramethyl benzidine (TMB, Tetramethylbenzidine), or o-phenylenediamine (o-Phenylenediamine), or benzidine (DAB, 3,3'-diaminobenzidine tetrahydrochloride) and substrate carbamide peroxide.
The invention still further relates to the method utilizing described kit high flux to detect allergen specificity antibody IgE, described method comprises following sequential steps:
(1) reactive tank being installed with the fibrous material film being solidified with allergen protein to be measured is got in liquid filling hole, horizontal positioned under room temperature, for subsequent use;
(2) 200 ~ 400 μ L serum or blood plasma are added in 1 ~ 2mL sample diluting liquid, add after mixing in the liquid filling hole of reactive tank, reactive tank is placed on vortex mixer, incubated at room 30 ~ 60 minutes;
(3) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(4) antihuman IgE antibody's working fluid of 200 ~ 400 μ L biotin couplings is added in 1 ~ 2mL sample diluting liquid, add after mixing in reactive tank, to be placed on vortex mixer incubated at room 30 ~ 60 minutes;
(5) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(6) in reactive tank, add 1 ~ 3ml two biotin-Streptavidin optimize liquid, to be placed on vortex mixer incubated at room 10 ~ 20 minutes;
(7) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(8) add in reactive tank 1 ~ 3ml biotin or marked by streptavidin poly alkaline phosphatase working fluid, to be placed on vortex mixer incubated at room 10 ~ 30 minutes;
(9) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(10) in reactive tank, add the substrate chromogenic reagent solution of 1 ~ 2ml dilution, to be placed on vortex mixer incubated at room 10 ~ 20 minutes;
(11) running water stops enzyme reaction;
(12) by reactive tank and colorimetric card comparison interpretation, or interpretation is carried out with reading apparatus.
Described colorimetric card makes according to 0 ~ 6 grade of stage division of specificity antibody IgE concentration in international serum, and form can be strip, square, triangle and circle etc.Color is different with developer, can be yellow, Lan Se and black etc., but the intensity of color must require consistent with specificity antibody IgE concentration in international serum 0 ~ 6 grade.
According to the inventive method, adopt colorimetric card maybe need not with the reading apparatus of data processing function, according to operation steps operation, just can with the naked eye qualitative or multiple allergenic specific IgE antibody levels semi-quantitatively in interpretation detection human serum or blood plasma easily; Use the reading apparatus of band data processing function, the multiple allergenic specific IgE antibody concentration in human serum or blood plasma can be detected quantitatively.
Beneficial effect of the present invention is mainly reflected in: kit of the present invention can the qualitative or allergen specificity antibody IgE concentration that semi-quantitatively detects fast in human serum or blood plasma with sensitivity, and can an examination tens of kinds of anaphylactogens, fast, accurately, amount of samples is few, is suitable for carrying out high flux detection.
(4) accompanying drawing explanation
Fig. 1 is the schematic diagram of twin biotin and Streptavidin molecule combination in the two biotins-Streptavidin optimization liquid for biotin labeled polymerase bonding; In figure
represent Streptavidin;
represent two biotin;
Fig. 2 is the schematic diagram of twin biotin and Streptavidin molecule combination in two biotins-Streptavidin optimization liquid of the polymerase bonding supplying marked by streptavidin; In figure
represent Streptavidin;
represent two biotin;
Fig. 3 is the second signal amplification system schematic diagram (A) of two biotin-Streptavidin and polymerase-developer and does not use the signal amplifying system schematic diagram (B) of biochemical method;
In figure
represent the allergen protein being incorporated into cellulose membrane;
represent anaphylactogen sIgE antibody;
represent biotin labeled antibody;
represent Streptavidin;
represent two biotin;
represent biotin labeled poly alkaline phosphatase;
represent the alkaline phosphatase of marked by streptavidin;
represent color signal; A and B diagram compares, and A comparatively B signal amplifies 35 times.
Fig. 4 is reagent place's reactive tank structural representation.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: detection reagent preparation of the present invention
1. nitrocellulose filter solidification (covalent bond) allergen protein (with reference to Hermanson GT et al, Immunobilized affinity ligand techniques.Academic Press(New York), 1992, pp53-56.)
(1) nitrocellulose filter bar activation
A. after wearing safety protection equipment, in vent cabinet, compound concentration is the Sigmaaldrich company of the CNBr(U.S. of 1mg/ml, C91492) acetonitrile solution;
B. the nitrocellulose filter (Bio-Rad company of the U.S., 162-0112) of well cutting is suspended in the Na of 1mol/L, pH11
2cO
3in solution, be transferred in the glass beaker of band stirrer, then lie in vent cabinet on magnetic stirrer plate;
C. limit is stirred, while add CNBr solution; Stir 10 minutes, survey the pH value of solution therebetween every the accurate pH test paper of more than the pH8 of 2 minutes; If necessary, the pH of activating solution is regulated to be 11 by NaOH solution;
D. the nitrocellulose filter bar in activating solution is transferred to the glass funnel of band core, vacuum filtration removing CNBr solution (in 2%FeSO4 solution);
E. 100ml ddH is used
2after O washing, then wash with the acetone soln of 20ml90%; Finally repeat 5 cleaning final vacuums with the sodium phosphate buffer of 100ml pH7.5 dry, stand-by.
(2) allergen protein solidification
A. by the allergen protein 0.1mol/L through purifying of dialysing, the PBS of pH7.4 is mixed with applicable concentration (as: plant pollen class allergen protein: 0.1 ~ 7.0mg/ml; Der Pilz allergen protein: 2.0 ~ 5.0mg/ml; Animal scurf class allergen protein: 0.05 ~ 3.0mg/ml; Vegetation foodstuff class allergen protein: 1.0 ~ 8.0mg/ml; Animal food class allergen protein: 1.5 ~ 10.0mg/ml; Insects allergen protein: 0.1 ~ 3.0mg/ml etc.) for subsequent use;
B. the nitrocellulose filter bar shifting CNBr activation enters in the allergen protein solution of 50ml, and at 4 DEG C, on shaking table, hybrid reaction spends the night (more than 16 hours) lentamente;
C. centrifugal 10 minutes of 1000rpm at above-mentioned reaction mixture 4 DEG C; Measure the allergen protein concentration of supernatant, make the allergen protein of solidification reach more than 85%;
D. add 40ml PBS, make cellulose membrane bar resuspension, then centrifugal 10 minutes of 1000rpm at 4 DEG C; Repeat-secondary;
E. use 0.1mol/L, the ethanolamine solutions 40ml of pH7.5 makes cellulose membrane bar resuspension, cleans 4 hours lentamente under room temperature on shaking table;
F. repeat to wash three times with PBS again, centrifugal; Add 1% antiseptic BND(5-Bromo-5-Nitro-1,3-Dioxane again) solution mixing reaction final vacuum drying in 1 hour, stand-by.
2. be solidified with the fibrous material film reaction cartridge groove of allergen protein
After identifying the mark of allergen protein and nitrocellulose filter bar top and bottom, upward, the nitrocellulose filter bar being solidified with allergen protein is loaded reactive tank mark to be had in the aperture of corresponding allergen protein film upper surface, and epiphragma.
3. antihuman IgE antibody's solution preparation of biotin coupling
The mouse antihuman IgE antibody (Life Technologies company of the U.S., 05-4740) of biotin coupling, during use, the PBS containing 0.05%Tween20 of pH7.4 is mixed with the working fluid of concentration 1:100 ~ 10000.
4. pair biotin-Streptavidin optimizes liquid preparation
A. be dissolved in ethanol/dimethyl sulfoxide (DMSO) (85/15, V/V) solution by two biotin (Pierce Chemical company of the U.S., IL40), obtained concentration is 1.1mmol/L;
B. Streptavidin (Sigmaaldrich company of the U.S., the 85878) solution of 1.0mg/ml is prepared with the PBS of 0.05mol/L; Then be 24 ~ 39:18 according to two biotin and Streptavidin volumetric molar concentration ratio under violent vortex, slowly add two biotin solution and enter in solution of streptavidin; Take out two biotins that minority measures this optimization liquid: Streptavidin site ratio is 0.8 ~ 0.85.
5. biotin labeled poly alkaline phosphatase solution preparation
With enzyme buffer liquid (5mmol/L Tris-HCl, pH9.3,1mmol/L MgCl
2, 0.1mmol/LZnCl
2, 1mmol/L arginine and antiseptic 1%BND) and biotin labeled poly alkaline phosphatase (BRL company of the U.S., BPA20, the degree of polymerization 20) is mixed with the working fluid of concentration 1:5000 ~ 50000.
Note: use this poly alkaline phosphatase enzyme solutions more general alkaline phosphatase enzyme solutions can amplify nearly 100 times.
6. the substrate chromogenic reagent solution preparation of alkaline phosphatase
Use 10ml ddH
2o lucifuge dissolves substrate developer (BCIP/NBT) (the Sigmaaldrich company of the U.S. of alkaline phosphatase, B5655-5tab), this solution is containing the chloro-3-indyl-phosphate of the bromo-4-of 0.15mg/ml BCIP(5-), 0.3mg/ml NBT(NBT), 0.1mol/L pH ~ 9.5Tris buffer and 5mmol/L MgCl
2.
The solution of above-mentioned preparation, be equipped with the reactive tank of fibrous material film bar of solidification allergen protein and conventional reagent as dilution, cleansing solution etc. constitute detection reagent of the present invention.
Embodiment 2(does not use the signal amplifying system of biochemical method):
Detect preparation of reagents and do not comprise unparalleled biotin-Streptavidin optimization liquid and biotin labeled poly alkaline phosphatase enzyme solutions see embodiment 1().
Detection method is as follows:
(1) pre-service: take out the required detection reaction groove (structure, see Fig. 3, has been equipped with the fibrous material film being solidified with allergen protein to be measured in aperture) of experiment, horizontal positioned under room temperature, numbering or mark patient's name;
(2) primary incubation: 300 HL serum are added mixing in 1.7ml sample diluting liquid (the PBS+10% NBCS of pH7.4), pour into and remove in the reactive tank of epiphragma, be placed in by reactive tank on vortex mixer, room temperature (20 ~ 25 DEG C) hatches 45 minutes.
(3) clean: with cleansing solution (the PBS+0.05% Tween-20 of pH7.4) washing fluid reactive tank, repeated washing 5 times, each 10 seconds; During flushing, cleansing solution is made to sufficiently flow through reactive tank.Note allowing liquid runs down reactive tank flow down when toppling over cleansing solution, avoid cross pollution.
(4) second time is hatched: (solvent is the PBS of pH7.4 to add antihuman IgE antibody's working fluid of 6 (about 300 microlitre) biotin couplings, concentration 1:10000) enter in 1.7ml antibody diluent to mix, pour in reactive tank, reactive tank is placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatches 45 minutes.
(5) clean: course synchronization rapid (3).
(6) third time hatches: the alkaline phosphatase working fluid (solvent is TBS, the 1:500 of pH9.2) adding 2.0ml marked by streptavidin in reactive tank, is placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatches 20 minutes.
(7) clean: course synchronization rapid (3).
Hatch for (8) the 4th times: the NBT/BCIP substrate nitrite ion adding 2.0ml in reactive tank, be placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatch 15 minutes.
(9) cessation reaction: running water stops enzyme reaction.
(10) reading: by reactive tank and colorimetric card comparison interpretation.Semi-quantitatively interpretation record detect human serum sample in multiple allergenic specific IgE antibody levels, result example is in table 1.
The second signal amplification system of the two biotin-Streptavidin of embodiment 3(application and polymerase):
Detect preparation of reagents see embodiment 1.
Detection method is as follows:
(1) pre-service: take out the required detection reaction groove (structure, see Fig. 3, has been equipped with the fibrous material film being solidified with allergen protein to be measured in aperture) of experiment, horizontal positioned under room temperature, numbering or mark patient's name;
(2) primary incubation: 300 HL serum are added mixing in 1.7ml sample diluting liquid (the PBS+10% NBCS of pH7.4), pour into and remove in the reactive tank of epiphragma, be placed in by reactive tank on vortex mixer, room temperature (20 ~ 25 DEG C) hatches 45 minutes.
(3) clean: with cleansing solution (the PBS+0.05% Tween-20 of pH7.4) washing fluid reactive tank, repeated washing 5 times, each 10 seconds; During flushing, cleansing solution is made to sufficiently flow through reactive tank.Note allowing liquid runs down reactive tank flow down when toppling over cleansing solution, avoid cross pollution.
(4) second time is hatched: (solvent is the PBS of pH7.4 to add antihuman IgE antibody's working fluid of 6 (about 300 microlitre) biotin couplings, concentration 1:10000) enter in 1.7ml antibody diluent to mix, pour in reactive tank, reactive tank is placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatches 45 minutes.
(5) clean: course synchronization rapid (3).
(6) third time hatches: in reactive tank, add the two biotin-Streptavidin of 2.0ml optimize liquid (two biotin and Streptavidin volumetric molar concentration ratio are 30:18), be placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatch 15 minutes.
(7) clean: course synchronization rapid (3).
Hatch for (8) the 4th times: in reactive tank, add 2.0ml biotin labeled poly alkaline phosphatase working fluid (solvent is TBS, the 1:5000 of pH9.5), be placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatch 20 minutes.
(9) clean: course synchronization rapid (3).
Hatch for (10) the 5th times: the NBT/BCIP substrate nitrite ion adding 2.0ml in reactive tank, be placed in room temperature on vortex mixer (20 ~ 25 DEG C) and hatch 15 minutes.
(11) cessation reaction: running water stops enzyme reaction.
(12) reading: by reactive tank and colorimetric card comparison interpretation.Semi-quantitatively interpretation record detect human serum sample in multiple allergenic specific IgE antibody levels, result example is in table 1.
Apply the second signal amplification system (embodiment 3) of two biotin-Streptavidin and polymerase simultaneously and do not use the signal amplifying system of biochemical method (embodiment 2) to test detection with the allergenic specific IgE antibody level in a trouble person blood Cheongju sample, testing result lists in table 1, to compare.
Table 1: the comparison of the allergenic specific IgE antibody level two kinds of system detection results in blood Cheongju sample
The testing result of relatively upper table is visible, and because embodiment 1 does not use the signal amplifying system of biochemical method, detection sensitivity is lower, and 2 grades of (comprising some 2 grades) sIgE antibody concentration are not below detected.
Claims (3)
1. the kit of a highly sensitive detection allergen specificity antibody IgE, mainly comprise: the fibrous material film being solidified with allergen protein to be measured, two biotin-Streptavidin optimizes liquid, the antihuman IgE antibody of biotin coupling, the polymerase of biotin or marked by streptavidin, and the substrate developer corresponding with described polymerase; It is two biotins that described pair of biotin-Streptavidin optimizes liquid: the mixture solution of Streptavidin molar concentration rate 24 ~ 39:18, described people's IgE antibody is sheep antihuman IgE antibody or rabbit antihuman IgE antibody, and described polymerase is poly alkaline phosphatase or poly horseradish peroxidase.
2. kit as claimed in claim 1, is characterized in that described fibrous material film is fixedly arranged in the liquid filling hole of reactive tank.
3. utilize the method for the highly sensitive detection allergen specificity antibody IgE of kit described in claim 1 or 2, described method comprises following sequential steps:
(1) reactive tank being installed with the fibrous material film being solidified with allergen protein to be measured is got in liquid filling hole, horizontal positioned under room temperature, for subsequent use;
(2) 200 ~ 400 μ L serum or blood plasma are added in 1 ~ 2mL sample diluting liquid, add after mixing in the liquid filling hole of reactive tank, reactive tank is placed on vortex mixer, incubated at room 30 ~ 60 minutes;
(3) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(4) antihuman IgE antibody's working fluid of 200 ~ 400 μ L biotin couplings is added in 1 ~ 2mL sample diluting liquid, add after mixing in reactive tank, to be placed on vortex mixer incubated at room 30 ~ 60 minutes;
(5) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(6) in reactive tank, add 1 ~ 3ml two biotin-Streptavidin optimize liquid, to be placed on vortex mixer incubated at room 10 ~ 20 minutes;
(7) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(8) add in reactive tank 1 ~ 3ml biotin or marked by streptavidin poly alkaline phosphatase working fluid, to be placed on vortex mixer incubated at room 10 ~ 30 minutes;
(9) with cleansing solution washing fluid reactive tank, repeated washing 3 ~ 5 times, each 10 ~ 30 seconds;
(10) in reactive tank, add the substrate chromogenic reagent solution of 1 ~ 2ml dilution, to be placed on vortex mixer incubated at room 10 ~ 20 minutes;
(11) running water stops enzyme reaction;
(12) by reactive tank and colorimetric card comparison interpretation, or interpretation is carried out with reading apparatus.
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| CN113125739A (en) * | 2021-04-16 | 2021-07-16 | 杭州浙大迪迅生物基因工程有限公司 | Fluorescent chip quantitative detection kit |
| CN113125753A (en) * | 2021-04-16 | 2021-07-16 | 杭州浙大迪迅生物基因工程有限公司 | Kit for detecting specific antibody of dust mite component |
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010055766A1 (en) * | 1999-04-02 | 2001-12-27 | Alexander Aristarkhov | Immunosorbant assay using branched bis-biotin/avidin/multiple label complex as a detection reagent |
| US20050186642A1 (en) * | 2004-02-24 | 2005-08-25 | Biocare Medical, Inc. | Immunoassay reagents and methods of use thereof |
| WO2005084283A2 (en) * | 2004-03-01 | 2005-09-15 | Drukier Andrzej K | Supersensitive immunoassays |
| CN1869699A (en) * | 2006-06-09 | 2006-11-29 | 深圳市博卡生物技术有限公司 | Method of anaphylactogen screening |
| CN101178402A (en) * | 2007-12-06 | 2008-05-14 | 武汉赛欣生物技术有限公司 | Preparation method of polyase marker antibody |
| CN201654038U (en) * | 2010-03-16 | 2010-11-24 | 湖北楚冠生物药业有限公司 | Allergen specific antibody IgE immuno-blotting detection reagent kit |
| CN102317780A (en) * | 2009-02-23 | 2012-01-11 | 普利玛食品株式会社 | Allergen detection method using immunochromatography |
| CN203324273U (en) * | 2013-06-14 | 2013-12-04 | 杭州浙大迪迅生物基因工程有限公司 | Reactive tank with high throughput for screening allergen specificity IgE (immunoglobulin E) antibodies |
| CN103529200A (en) * | 2013-10-25 | 2014-01-22 | 中南大学 | Colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on competitive hybridization reaction |
-
2014
- 2014-03-18 CN CN201410099290.XA patent/CN104237527B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010055766A1 (en) * | 1999-04-02 | 2001-12-27 | Alexander Aristarkhov | Immunosorbant assay using branched bis-biotin/avidin/multiple label complex as a detection reagent |
| US20050186642A1 (en) * | 2004-02-24 | 2005-08-25 | Biocare Medical, Inc. | Immunoassay reagents and methods of use thereof |
| WO2005084283A2 (en) * | 2004-03-01 | 2005-09-15 | Drukier Andrzej K | Supersensitive immunoassays |
| CN1869699A (en) * | 2006-06-09 | 2006-11-29 | 深圳市博卡生物技术有限公司 | Method of anaphylactogen screening |
| CN101178402A (en) * | 2007-12-06 | 2008-05-14 | 武汉赛欣生物技术有限公司 | Preparation method of polyase marker antibody |
| CN102317780A (en) * | 2009-02-23 | 2012-01-11 | 普利玛食品株式会社 | Allergen detection method using immunochromatography |
| CN201654038U (en) * | 2010-03-16 | 2010-11-24 | 湖北楚冠生物药业有限公司 | Allergen specific antibody IgE immuno-blotting detection reagent kit |
| CN203324273U (en) * | 2013-06-14 | 2013-12-04 | 杭州浙大迪迅生物基因工程有限公司 | Reactive tank with high throughput for screening allergen specificity IgE (immunoglobulin E) antibodies |
| CN103529200A (en) * | 2013-10-25 | 2014-01-22 | 中南大学 | Colorimetric method for simultaneously detecting multiple miRNA (micro-ribonucleic acid) sequences based on competitive hybridization reaction |
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| CN105044326A (en) * | 2015-09-11 | 2015-11-11 | 苏州浩欧博生物医药有限公司 | Kit and method for achieving highly-sensitive multiterm joint inspection of allergen-specific IgE antibodies |
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| WO2022217731A1 (en) * | 2021-04-16 | 2022-10-20 | 杭州浙大迪迅生物基因工程有限公司 | Kit for quantitative measurement using fluorescent chip |
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