CN104237527B - The test kit of highly sensitive detection allergen specificity antibody IgE and method - Google Patents
The test kit of highly sensitive detection allergen specificity antibody IgE and method Download PDFInfo
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Classifications
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- 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
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- Urology & Nephrology (AREA)
- Hematology (AREA)
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- Biotechnology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides test kit and the detection method of a kind of highly sensitive detection allergen specificity antibody IgE, described test kit specifically includes that the fibrous material film being solidified with allergen protein to be measured, double biotinstreptatins optimize liquid, the antihuman IgE antibody of biotin coupling, biotin or the polymerase of marked by streptavidin, and the substrate developer corresponding with described polymerase；Test kit of the present invention can the most qualitative or the most quick allergen specificity antibody IgE concentration in detection human serum or blood plasma, and can tens of kinds of anaphylactogens of an examination, quickly, accurately, amount of samples few, suitably carry out high throughput testing.
Description
(1) technical field
The present invention relates to one the most qualitative or semi-quantitatively sieve or detect human serum or blood plasma
In the detection kit of multiple allergenic specific IgE antibody levels, and detection method.
(2) background technology
2011～2012 world's allergy tissue (WAO) about allergy (also referred to as allergy
Reaction, Allergy) white paper point out: " anaphylactic disease popular developed country and development in
Rise the most dramatically in country's worldwide.Anaphylactic disease includes allergic asthma, anaphylaxis
Rhinitis, severe allergic reaction, medicine, food and insect hypensensitiveness reaction, eczema, urticaria (hay
Heat) and angioedema.The increase that past 20 years, especially child suffered from anaphylactic disease number has cruelly
The trend sent out." (Pawankar R et al, WAO White Book on Allergy2011-2012:
Executive Summary.) prevalence rate of anaphylactic disease that only food causes just reaches 3%～6%.
(Sicherer S H, Epidemiology of food allergy.J Allergy Clin Immunol.
2011；127:594-602.) anaphylactic disease is that patient sucks or ingests into containing allergen
Material (referred to as anaphylactogen or allergen, Allergen) triggers the B cell of body afterwards and produces excess
IgE (Immunoglobulin E, IgE), when IgE antibody contacted the most again
Quick former time be just attached on mastocyte and basophil cellular surface with Allergens cross-link height affine
On receptor Fc ε R1, cause the gathering of Fc ε R1 receptor, make mastocyte and basophilic granulocyte live
Change.The mastocyte inflammatory that retting conditions release are stored in cytoplasmic granules in activation process is situated between
Matter: histamine, with the leukotriene synthesized by arachidonic acid pathway, immunoreactivity prostaglandin and
The cytokines such as IL4, IL5 and chemotactic factor, cause anaphylactoid disease symptoms.Anaphylaxis disease
Sick generation, IgE antibody plays a crucial role, anaphylaxis (the i.e. Gell-Coombs of referred to as IgE mediation
I type allergy, or claim the IgE speed property the sent out allergy of mediation).The anaphylactic disease of IgE mediation
Feature be allergenic specific IgE (sIgE) the antibody concentration calibration in blood circulation in the patient
Often high under situation, and disease is the most serious, sIgE antibody concentration is the highest.(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 anaphylactic disease is pointed out, according to disease in U.S. clinical doctor's practical guide
History combines pricking method or blood testing allergenic specific IgE (sIgE) antibody concentration, and examination was caused a disease
Quick former (Siles R I, Hsieh F H, Allergy blood testing:A practical guide for
clinicians.Am Clin J Medicine.2011；78:585-592.).At present, in detection blood
Allergenic specific IgE (sIgE) antibody concentration, the cause a disease method of anaphylactogen of examination has enzyme immunoassay
(EIA), immunoblotting (Immunoblotting Assay), gold colloidal Sidestream chromatography method (LFA),
Protein chip (Proteins microarray) etc., development trend also meets market demands: from
Dynamicization, quickly, accurately, amount of samples few, tens kinds of anaphylactogens of an examination.On market, product is not
Few, wherein, Pharmacia Inc., Sweden's product I mmunoCAP250 system is enzyme immunoassay
Representing, ImmunoCAP Rapid is the representative of gold colloidal Sidestream chromatography method, ImmunoCAP ISAC
It is the representative of protein chip, has started allergen molecule diagnosis；And Germany Mediwiss-analytic
The AllergyScreen of company be then immunoblotting representative (Immunoblot
For analysing specific IgE in human serum).Because of IgE antibody mean concentration in human blood
～0.005ug/ml, it is the 0.002% of total immunoglobulin mean concentration, sIgE antibody concentration is lower,
For meeting automatization, quick, accurate, amount of samples is few, tens kinds of anaphylactogens of an examination, semidefinite
The requirement of allergenic specific IgE (sIgE) antibody concentration in amount or detection by quantitative sample, it is necessary to
The detector that configuration photosignal amplifies, or the signal amplifying system of biochemical method.As, Sweden
Pharmacia Products ImmunoCAP Rapid once can only about ten kinds of anaphylactogens of examination, no
Joining reading apparatus, the sensitivity of naked eyes interpretation sIgE antibody concentration only has 1.0IU/ml(1IU IgE=2.44ng
IgE), negative and positive findings are distinguished with 1.49IU/ml.
(3) summary of the invention
It is an object of the present invention to provide a kind of signal amplifying system (double biotin using biochemical method
The second signal amplification system of-Streptavidin and polymerase) to detect anaphylactogen with sensitivity special
The detection kit of property antibody IgE and detection method, in order to qualitative or the most quickly detect human blood
Allergen specificity antibody IgE concentration clearly or in blood plasma, and can tens of kinds of anaphylactogens of an examination.
The technical solution used in the present invention is:
The test kit of a kind of highly sensitive detection allergen specificity antibody IgE, specifically includes that solidification
The fibrous material film of allergen protein to be measured, double biotin-Streptavidin is had to optimize liquid, biotin
The antihuman IgE antibody of coupling (commercially available acquisition, pH7.4's containing 0.05%Tween20 during use
PBS be configured to the working solution of concentration 1:100～10000), biotin or marked by streptavidin
Polymerase (commercially available acquisition is configured to concentration 1:5000～50000 with enzyme buffer liquid during use
Working solution), and the substrate developer corresponding with described polymerase (commercially available acquisition, it is possible to from
Row preparation)；It is double biotin that described pair of biotin-Streptavidin optimizes liquid: Streptavidin mole
The concentration mixture solution than 24～39:18, described people's IgE antibody is sheep antihuman IgE antibody or rabbit
Antihuman IgE antibody, described polymerase is poly alkali phosphatase or poly horseradish peroxidase.On
State the main component for reagent, do not comprise the conventional reagent used by detection such as diluent, cleaning mixture etc..
Described pair of biotin-Streptavidin optimizes liquid and can prepare as follows:
(1) double biotin are dissolved in ethanol/dimethyl sulfoxide (85/15, V/V) solution, system
Obtaining concentration is 1.1mmol/L, obtains A liquid；
(2) by the solution of streptavidin of the PBS preparation 1.0mg/mL of 0.05mol/L, B is obtained
Liquid；
(3) under violent vortex, according to its mol ratio, slowly A liquid is added in B liquid, to obtain final product
Described pair of biotin-Streptavidin optimizes liquid.
Optimize the ratio of twin biotin and Streptavidin molecular mixing in liquid can be 12:9,7:
6,19:9,13:6 etc., as shown in Fig. 1 and Fig. 2.
Preferably, described fibrous material film can be fixedly arranged in the liquid filling hole of reactive tank, by straight for this reactive tank
Connect 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
Deng (commercially available acquisition), there is sufficient counter-bending and antifungal gene mechanical strength, solidify anaphylactogen to be measured
It is fixedly arranged in reactive tank after albumen.Fibrous material film is different with the allergen protein that need to solidify, and can distinguish
With method activated fibers such as bromination nitrile, glutaraldehyde, carboxyl degeneration, amino degeneration, chloromethyl degeneration
Material membrane, makes it the corresponding allergen protein of covalent bond enough (0.2～2.0ug), in order to
Can fully combine the sIgE antibody in sample liquid, it is ensured that the accuracy of testing result.
Described allergen protein can extract according to a conventional method from following natural material, including: 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 (include fruits, greengrocery,
Nuts, edible fungi and cereals) anaphylactogen and animal food class (include meat, Poultry and Eggs,
With shell-fish and milk) anaphylactogen etc..
The detectable of the present invention uses antihuman IgE antibody's trapping agent solution of biotin coupling simultaneously,
Double biotin-Streptavidin optimizes liquid and solution, and the biology both having had double biotin-Streptavidin is put
Big system, has again polymerase-colour developing amplification, detection sensitivity is greatly improved, as shown in Figure 3
Meaning.
When described polymerase is poly alkali phosphatase, corresponding developer be nitro tetrazole blue and
Mixture (NBT/BCIP, the Nitroblue Tetrazolium of the bromo-4-of 5-chloro-3-indoylphosphate
/ 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
And substrate carbamide peroxide tetrahydrochloride).
The invention still further relates to utilize described test kit high throughput testing allergen specificity antibody IgE
Method, described method includes order below step:
(1) reaction of the fibrous material film being solidified with allergen protein to be measured it is installed with in taking liquid filling hole
Groove, horizontal positioned under room temperature, standby；
(2) 200～400 μ L serum or blood plasma are added in 1～2mL sample diluting liquid, add after mixing
Enter in the liquid filling hole of reactive tank, reactive tank is placed on vortex mixer, incubated at room 30～60
Minute；
(3) with cleaning mixture flushing liquor reactive tank, repeated washing 3～5 times, each 10～30 seconds；
(4) antihuman IgE antibody's working solution of 200～400 μ L biotin couplings is added 1～2mL sample
In product diluent, add in reactive tank after mixing, be placed in incubated at room on vortex mixer
30～60 minutes；
(5) with cleaning mixture flushing liquor reactive tank, repeated washing 3～5 times, each 10～30 seconds；
(6) in reactive tank, add 1～3ml double biotin-Streptavidins optimize liquid, be placed in mixing
Incubated at room 10～20 minutes on device；
(7) with cleaning mixture flushing liquor reactive tank, repeated washing 3～5 times, each 10～30 seconds；
(8) add in reactive tank 1～3ml biotin or marked by streptavidin poly alkalescence
Phosphatase working solution, is placed on vortex mixer incubated at room 10～30 minutes；
(9) with cleaning mixture flushing liquor 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, be placed on vortex mixer
Incubated at room 10～20 minutes；
(11) flowing water rinses and terminates enzyme reaction；
(12) by reactive tank and colorimetric card comparison interpretation, or interpretation is carried out with reading apparatus.
Described colorimetric card is according to specificity antibody IgE concentration in international serum 0～6 grades of classification sides
Method makes, and form can be strip, square, triangle and circle etc..Color is with developer not
With, can be yellow, blue and black etc., but the intensity of color must with in international serum
Specificity antibody IgE concentration 0～6 grades of requirements are consistent.
According to the inventive method, use colorimetric card maybe without the reading apparatus of band data processing function, according to
Operating procedure operates, so that it may the most qualitative or semi-quantitatively interpretation detection human serum or blood plasma
In multiple allergenic specific IgE antibody levels；Use the reading apparatus of band data processing function, can
Detect the multiple allergenic specific IgE antibody concentration in human serum or blood plasma quantitatively.
The beneficial effects are mainly as follows: test kit of the present invention can the most qualitative or semidefinite
Allergen specificity antibody IgE concentration in amount ground quickly detection human serum or blood plasma, and can once sieve
Looking into tens of kinds of anaphylactogens, quickly, accurate, amount of samples is few, suitably carries out high throughput testing.
(4) accompanying drawing explanation
Fig. 1 is that the double biotin for biotin labeled polymerase bonding-Streptavidin optimizes in liquid twin
Biotin and the schematic diagram of Streptavidin molecule combination；In figureRepresent Streptavidin；Represent double biotin；
Fig. 2 is that the double biotin-Streptavidin of the polymerase bonding for marked by streptavidin optimizes in liquid
Twin biotin and the schematic diagram of Streptavidin molecule combination；In figureRepresent strepto-parent
And element；Represent double biotin；
Fig. 3 is double biotin-Streptavidin and the second signal amplification system schematic diagram of polymerase-developer
And do not use the signal amplifying system schematic diagram (B) of biochemical method (A)；
In figureRepresent the allergen protein being incorporated into cellulose membrane；Represent anaphylactogen sIgE antibody；
Represent biotin labeled antibody；Represent Streptavidin；Represent double biotin；Table
Show biotin labeled poly alkali phosphatase；Represent the alkali phosphatase of marked by streptavidin；Represent color signal；A and B diagram compares, and A relatively B signal amplifies 35 times.
Fig. 4 is reagent place's reactive tank structural representation.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention
It is not limited to that:
Embodiment 1: prepared by the detectable of the present invention
1. nitrocellulose filter solidification (covalent bond) allergen protein is (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 ventilated chamber, compound concentration is CNBr(U.S. of 1mg/ml
Sigmaaldrich company of state, 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, pH112CO3In solution, it is transferred in the glass beaker of band stirrer, then
Lie in ventilated chamber on magnetic stirrer plate；
C. limit stirring, limit adds CNBr solution；Stir 10 minutes, the most at intervals of two minutes
The accurate pH test paper of more than pH8 surveys the pH value of solution；If necessary, live with NaOH solution regulation
The pH changing liquid is 11；
D. the nitrocellulose filter bar in activating solution is transferred to the glass funnel of band core, vacuum filtration
Remove CNBr solution (in 2%FeSO4 solution)；
E. 100ml ddH is used2After O washing, then wash with the acetone soln of 20ml90%；Finally use
The sodium phosphate buffer of 100ml pH7.5 is repeated 5 times cleaning final vacuum and is dried, stand-by.
(2) allergen protein solidification
A. it is configured to be suitable for by the PBS of allergen protein 0.1mol/L, pH7.4 through dialysis purification
Concentration (such as: plant pollen class allergen protein: 0.1～7.0mg/ml；Der Pilz anaphylactogen egg
White: 2.0～5.0mg/ml；Animal scurf class allergen protein: 0.05～3.0mg/ml；Plant eats
Species allergen protein: 1.0～8.0mg/ml；Animal food class allergen protein: 1.5～10.0
mg/ml；Insects allergen protein: 0.1～3.0mg/ml etc.) standby；
B. in the nitrocellulose filter bar of transfer CNBr activation enters the allergen protein solution of 50ml, 4 DEG C
Under on shaking table hybrid reaction overnight (more than 16 hours) lentamente；
At the most above-mentioned reaction mixture 4 DEG C, 1000rpm is centrifuged 10 minutes；Measure the allergy of supernatant
Former protein concentration, makes the allergen protein of solidification reach more than 85%；
D. adding 40ml PBS, make cellulose membrane bar resuspension, then 1000rpm is centrifuged at 4 DEG C
10 minutes；Repeat-secondary；
E. cellulose membrane bar resuspension is made with the ethanolamine solutions 40ml of 0.1mol/L, pH7.5,
Clean lentamente on shaking table 4 hours under room temperature；
Repeat to wash three times with PBS the most again, centrifugal；Add 1% preservative BND
(5-Bromo-5-Nitro-1,3-Dioxane) solution mixing 1 hour final vacuum of reaction is dried, stand-by.
2. it is 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, film upper surface upward,
The nitrocellulose filter bar being solidified with allergen protein is loaded reactive tank and is identified with corresponding anaphylactogen egg
In white aperture, and epiphragma.
3. antihuman IgE antibody's solution preparation of biotin coupling
The Mus 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 configured to concentration 1:100～10000
Working solution.
4. prepared by pair biotin-Streptavidin optimization liquid
A. double biotin (Pierce Chemical company of the U.S., IL40) are dissolved in ethanol/diformazan
In base sulfoxide (85/15, V/V) solution, prepared concentration is 1.1mmol/L；
B. with the Streptavidin (U.S. Sigmaaldrich of the PBS preparation 1.0mg/ml of 0.05mol/L
Company, 85878) solution；Then according to double biotin and Streptavidin mole under violent vortex
Concentration ratio is 24～39:18, slowly adds double biotin solution and enters in solution of streptavidin；Take out
Minority measures double biotin of this optimization liquid: Streptavidin site ratio is 0.8～0.85.
The most biotin labeled poly alkaline phosphatase enzymatic solution is prepared
With enzyme buffer liquid (5mmol/L Tris-HCl, pH9.3,1mmol/L MgCl2, 0.1mmol/L
ZnCl2, 1mmol/L arginine and preservative 1%BND) and by biotin labeled poly alkaline phosphatase
Enzyme (BRL company of the U.S., BPA20, the degree of polymerization 20) is configured to concentration 1:5000～50000
Working solution.
Note: use this poly alkaline phosphatase enzymatic solution more general alkaline phosphatase enzymatic solution can amplify nearly 100
Times.
6. the substrate chromogenic reagent solution preparation of alkali phosphatase
Use 10ml ddH2The substrate developer (BCIP/NBT) that O lucifuge dissolves alkali phosphatase is (beautiful
Sigmaaldrich company of state, B5655-5tab), the bromo-4-of this solution BCIP(5-Han 0.15mg/ml
Chloro-3-indyl-phosphate), 0.3mg/ml NBT(NBT), 0.1mol/L pH～9.5Tris
Buffer and 5mmol/L MgCl2。
The solution of above-mentioned preparation, equipped with the reactive tank and often of the fibrous material film bar of solidification allergen protein
Rule reagent such as diluent, cleaning mixture etc. constitute the detectable of the present invention.
Embodiment 2(does not uses the signal amplifying system of biochemical method):
Detectable preparation sees embodiment 1(and does not include that unparalleled biotin-Streptavidin optimizes liquid
And biotin labeled poly alkaline phosphatase enzymatic solution).
Detection method is as follows:
(1) pretreatment: (structure sees Fig. 3, has been loaded with in aperture to take out experiment required detection reactive tank
Be solidified with the fibrous material film of allergen protein to be measured), horizontal positioned under room temperature, numbering or
Labelling patient's name；
(2) primary incubation: by 300 HL serum add 1.7ml sample diluting liquids (pH7.4's
PBS+10% new-born calf serum) interior mixing, pour in the reactive tank removing epiphragma, will reaction
Groove is placed on vortex mixer, and room temperature (20～25 DEG C) hatches 45 minutes.
(3) clean: with cleaning mixture (the PBS+0.05% tween 20 of pH7.4) flushing liquor reactive tank, weight
Cleaning 5 times again, each 10 seconds；During flushing, cleaning mixture is made to sufficiently flow through reactive tank.
Liquid runs down reactive tank is allowed to flow down when noting toppling over cleaning mixture, it is to avoid cross-contamination.
(4) second time is hatched: add antihuman IgE antibody's work of 6 (about 300 microlitre) biotin couplings
Make liquid (solvent is the PBS of pH7.4, concentration 1:10000) and enter 1.7ml antibody diluent
Middle mixing, pours in reactive tank, and reactive tank is placed in room temperature on vortex mixer (20～25 DEG C)
Hatch 45 minutes.
(5) clean: course synchronization is rapid (3).
(6) third time is hatched: add the alkali phosphatase of 2.0ml marked by streptavidin in reactive tank
Working solution (solvent is the TBS of pH9.2,1:500), is placed in room temperature on vortex mixer (20～25 DEG C)
Hatch 20 minutes.
(7) clean: course synchronization is rapid (3).
Hatch for (8) the 4th times: in reactive tank, add the NBT/BCIP substrate nitrite ion of 2.0ml, put
On vortex mixer, room temperature (20～25 DEG C) hatches 15 minutes.
(9) reaction is terminated: flowing water rinses and terminates enzyme reaction.
(10) reading: by reactive tank and colorimetric card comparison interpretation.The semi-quantitatively people of interpretation record detection
Multiple allergenic specific IgE antibody levels in blood serum sample, result example is shown in Table 1.
Double biotin-the Streptavidin of embodiment 3(application and the second signal amplification system of polymerase):
Detectable preparation sees embodiment 1.
Detection method is as follows:
(1) pretreatment: (structure sees Fig. 3, in aperture to take out experiment required detection reactive tank
Fibrous material film equipped with being solidified with allergen protein to be measured), horizontal positioned under room temperature, number or mark
Note patient's name；
(2) primary incubation: by 300 HL serum add 1.7ml sample diluting liquids (pH7.4's
PBS+10% new-born calf serum) interior mixing, pour in the reactive tank removing epiphragma, reactive tank is put
On vortex mixer, room temperature (20～25 DEG C) hatches 45 minutes.
(3) clean: use cleaning mixture (the PBS+0.05% tween 20 of pH7.4) flushing liquor reactive tank,
Repeated washing 5 times, each 10 seconds；During flushing, cleaning mixture is made to sufficiently flow through reactive tank.Note
Liquid runs down reactive tank is allowed to flow down when toppling over cleaning mixture, it is to avoid cross-contamination.
(4) second time is hatched: add the antihuman IgE antibody of 6 (about 300 microlitre) biotin couplings
Working solution (solvent is the PBS of pH7.4, concentration 1:10000) enters in 1.7ml antibody diluent
Mixing, pours in reactive tank, reactive tank is placed in room temperature on vortex mixer (20～25 DEG C) and hatches 45
Minute.
(5) clean: course synchronization is rapid (3).
(6) third time is hatched: add the double biotin-Streptavidin optimization of 2.0ml in reactive tank
Liquid (double biotin and Streptavidin molar concentration ratio are 30:18), is placed in room temperature on vortex mixer
(20～25 DEG C) hatch 15 minutes.
(7) clean: course synchronization is rapid (3).
Hatch for (8) the 4th times: in reactive tank, add 2.0ml biotin labeled poly alkaline phosphatase
Enzyme working solution (solvent is the TBS of pH9.5,1:5000), is placed in room temperature on vortex mixer (20～25 DEG C)
Hatch 20 minutes.
(9) clean: course synchronization is rapid (3).
Hatch for (10) the 5th times: in reactive tank, add the NBT/BCIP substrate nitrite ion of 2.0ml,
It is placed in room temperature on vortex mixer (20～25 DEG C) and hatches 15 minutes.
(11) reaction is terminated: flowing water rinses and terminates enzyme reaction.
(12) reading: by reactive tank and colorimetric card comparison interpretation.The semi-quantitatively people of interpretation record detection
Multiple allergenic specific IgE antibody levels in blood serum sample, result example is shown in Table 1.
Double biotin-the Streptavidin of application simultaneously and the second signal amplification system (embodiment of polymerase
3) and do not use signal amplifying system (embodiment 2) the experiment detection of biochemical method with a
Allergenic specific IgE antibody level in blood sample of patient basis, testing result is listed in table 1, in order to
Relatively.
Table 1: the comparison of the two kinds of system detection results of allergenic specific IgE antibody level in blood sample
In comparison, the testing result of table is visible, owing to embodiment 1 does not uses the signal of biochemical method
Amplification system, detection sensitivity is relatively low so that 2 grades (including some 2 grades) sIgE below resists
Bulk concentration does not detects.
Claims (2)
1. the test kit of a highly sensitive detection allergen specificity antibody IgE, including: it is solidified with the fibrous material film of allergen protein to be measured, double biotin-Streptavidin optimizes liquid, the antihuman IgE antibody of biotin coupling, biotin or the polymerase of marked by streptavidin, and the substrate developer corresponding with described polymerase；It is double biotin 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 alkali phosphatase or poly horseradish peroxidase；
The method of described test kit highly sensitive detection allergen specificity antibody IgE includes order below step:
(1) reactive tank of the fibrous material film being solidified with allergen protein to be measured it is installed with in taking liquid filling hole, horizontal positioned under room temperature, standby；
(2) 200～400 μ L serum or blood plasma are added in 1～2mL sample diluting liquid, add in the liquid filling hole of reactive tank after mixing, reactive tank is placed on vortex mixer, incubated at room 30～60 minutes；
(3) reactive tank is rinsed with cleaning mixture, repeated washing 3～5 times, each 10～30 seconds；
(4) antihuman IgE antibody's working solution of 200～400 μ L biotin couplings is added in 1～2mL sample diluting liquid, add in reactive tank after mixing, be placed on vortex mixer incubated at room 30～60 minutes；
(5) reactive tank is rinsed with cleaning mixture, repeated washing 3～5 times, each 10～30 seconds；
(6) in reactive tank, add 1～3ml double biotin-Streptavidins and optimize liquid, be placed on vortex mixer incubated at room 10～20 minutes；
(7) reactive tank is rinsed with cleaning mixture, repeated washing 3～5 times, each 10～30 seconds；
(8) in reactive tank, add 1～3ml biotin or the poly alkali phosphatase of marked by streptavidin or poly horseradish peroxidase working solution, be placed on vortex mixer incubated at room 10～30 minutes；
(9) reactive tank is rinsed with cleaning mixture, repeated washing 3～5 times, each 10～30 seconds；
(10) in reactive tank, add the substrate chromogenic reagent solution of 1～2ml dilution, be placed on vortex mixer incubated at room 10～20 minutes；
(11) flowing water rinses and terminates enzyme reaction；
(12) by reactive tank and colorimetric card comparison interpretation, or interpretation is carried out with reading apparatus.
2. test kit as claimed in claim 1, it is characterised in that described fibrous material film is fixedly arranged in the liquid filling hole of reactive tank.
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| CN113125739B (en) * | 2021-04-16 | 2022-07-26 | 杭州浙大迪迅生物基因工程有限公司 | Fluorescent chip quantitative detection kit |
| CN113125753B (en) * | 2021-04-16 | 2022-07-26 | 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 |
| CN114636820B (en) * | 2022-05-07 | 2022-10-21 | 珠海圣美生物诊断技术有限公司 | Kit and method for detecting circulating PD-L1 positive cells |
| CN114814204A (en) * | 2022-05-31 | 2022-07-29 | 南京岚煜生物科技有限公司 | Detection kit for semi-quantitative allergen screening and preparation method thereof |
| CN115980369B (en) * | 2023-02-22 | 2024-10-25 | 广州医科大学附属第二医院 | An allergen screening kit based on inflammatory response typing and its application |
| CN119574891A (en) * | 2025-02-06 | 2025-03-07 | 杭州浙大迪迅生物基因工程有限公司 | A fluorescent percolation kit for detecting allergen-specific IgE antibodies |
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| 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 |
| US7604956B2 (en) * | 2004-03-01 | 2009-10-20 | Biotraces, Inc. | 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 |
| CN101178402B (en) * | 2007-12-06 | 2011-08-24 | 武汉赛欣生物技术有限公司 | Preparation method of polyase marker antibody |
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| 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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