CN108646027A - ELISA kit based on IgG4 antibody test food allergens - Google Patents
ELISA kit based on IgG4 antibody test food allergens Download PDFInfo
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- 238000008157 ELISA kit Methods 0.000 title claims abstract description 26
- 238000012360 testing method Methods 0.000 title claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 70
- 239000000243 solution Substances 0.000 claims abstract description 59
- 239000007788 liquid Substances 0.000 claims abstract description 35
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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
- G01N33/6854—Immunoglobulins
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
ELISA kit provided by the invention based on IgG4 antibody test food allergens, belongs to food allergen detection technique field.The kit includes following reagent, is coated with the detection plate of food allergen；Washing buffer；Sample diluting liquid；Standard items or positive quality control product；4 antibody of anti-human igg of biotin labeling；The avidin solution of enzyme label；Substrate developing solution；Terminate liquid.The characteristics of present invention selects IgG4 to detect food allergen for the first time, and is the ELISA kit prepared based on indirect method, has detection sensitivity strong, and accuracy is high, favorable reproducibility, and being capable of the large batch of sample of single treatment.Embodiment proves that kit detection sensitivity provided by the invention reaches 1ng/ml.
Description
Technical field
The present invention relates to food allergen detection technique fields, and in particular to based on IgG4 antibody test food allergens
Kit, i.e. food anaphylactogen specificity IgG 4 detection kit.
Background technology
Anaphylactia incidence accounts for 30% of total world population or more, and the four big of 21 century is classified as by the World Health Organization
One of noninfectious disease.The incidence of anaphylactia significantly rises in recent years, becomes the public health problem of global concern,
Guo Jia et al. is to anaphylactia epidemiological survey the results show that there is 22% people to suffer from anaphylaxis disease in 1,200,000,000 total populations
Disease, such as allergic rhinitis, asthma, conjunctivitis, eczema, food hypersenstivity, drug allergy and serious allergic reaction etc., serious shadow
Ring people’s lives quality and life security.
Food allergen such as egg product, peanut, milk, soya bean, wheat, tree nuts, fish and crust group food, are main
Food allergen.Wherein in crust group food, shrimp allergen is concerned, it was reported that 0.6%~2.8% anaphylactia is suffered from
Person's prawn allergy；People detect at least 13 kinds of IgE binding proteins in shrimp, but tropomyosin is accredited as only master
Anaphylactogen is wanted, relative molecular mass is between 34000~39000；It is reported that tropomyosin is shrimp, crab, oyster, cuttlefish
The important antigen of equal invertebrates, has highly conserved amino acid sequence.In egg allergy, albumen is more easy to cause than yolk
Allergy, ovomucoid are main allergen；Its positive rate is up to 35% in children's food hypersenstivity, and allergy of being grown up also is up to
12%.Due to the seriousness and frequently-occurring of peanut allergy reaction, which causes the extensive concern of medical institutions, it was reported that food
In the allergic reaction of object induction, peanut allergy accounts for 10%~47%.Such disease is increasing in recent years, becomes common disease, more
Morbidity is that China's health and economic development field need the significant problem solved.
Currently, patent CN1620504A is disclosed with based on the information design obtained by a part of gene of predetermined substance
Primer carries out PCR, for measuring the method in food whether there is or not predetermined substance, micro constitutent and identification contained in detection food
It is excellent in terms of harmful predetermined substance anaphylactogen, but this method relies on the primer of known detectable substance, but PCR method is multiple
Miscellaneous, detection time is long, of high cost；Patent CN101285840A discloses a kind of detection method of anaphylactogen in food, although should
Method does not disclose relevant detection antibody type hypotype in capable of quickly and easily obtaining testing result but the patent, does not have simultaneously
There are the detection sensitivity and accuracy of open this method detection food allergen.In addition, with the molecular weight peaks and figure of flight mass spectrum
Spectral shape carrys out the protein site of identification mark for basic parameter.By the feature of diagnosis allergy original albumen to anaphylactogen in food into
Row Testing and appraisal, but need special large-scale precision instrument, testing cost is high, analysis difficulty need special testing staff and
Analysis personnel
In conclusion although there are many method for the related food allergen detection studied at present, all there is detection speed
Slowly, of high cost, the problem of being not suitable for batch detection, and also it is machine-readable to need specific analytical instrument to carry out, and constrains mistake in food
The development of quick former detection.
Invention content
In view of this, it is an object of the invention to detect the kit of food allergen based on IgG4, the kit is made to have
There are stronger specificity and higher sensitivity.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides the ELISA kits based on IgG4 antibody test food allergens, including following components：
It is coated with the detection plate of food allergen；
Washing buffer；
Sample diluting liquid；
Standard items or positive quality control product；
4 antibody-solutions of anti-human igg of biotin labeling；
The avidin solution of enzyme label；
Substrate developing solution；
Terminate liquid.
Preferably, the peridium concentration of the food allergen is 0.1~2000 μ g/ml.
Preferably, the type for being coated with food allergen in the detection plate of food allergen includes aquatic products category, poultry
Birds, egg-milk, greengrocery, fruits, cereals or nut fruits.
Preferably, the washing buffer is PBS buffer solution.
Preferably, the mass concentration of 4 antibody-solutions of anti-human igg of the biotin labeling is 0.01~8000 μ g/ml.
Preferably, the substrate developing solution is tetramethyl benzidine substrates solution or 2,2- hydrazines-bis- -3- ethyl benzo thiophenes
Oxazoline -6- sulfonic acid substrate solutions.
Preferably, the sample diluting liquid is PBS buffer solution.
Preferably, the PBS buffer solution includes the component of following parts by weight：8.0 parts of NaCl, 0.20 part of KCl, 1.16 parts
Na2HPO4, 0.20 part of KH2PO4With 1000 parts of water；The pH value of the PBS buffer solution is 7.0~7.6.
Preferably, the hydrogen-oxygen that the sulfuric acid solution or mass concentration that the terminate liquid is 0.5~2mol/L are 30%~40%
Change sodium solution.
Preferably, 1~5000 times of working solution when a concentration of use for the avidin solution that the enzyme marks.
ELISA kit provided by the invention based on IgG4 antibody test food allergens, including following reagent, coating
There is the detection plate of food allergen；Washing buffer；Sample diluting liquid；Standard items or positive quality control product；Biotin labeling resists
4 antibody of human IgG；The avidin solution of enzyme label；Substrate developing solution；Terminate liquid.The present invention selects IgG4 antibody to detect food
Anaphylactogen, and the ELISA kit prepared based on indirect method, have the characteristics that detection sensitivity is strong, and being capable of single treatment
Large batch of sample.Embodiment proves that kit detection sensitivity provided by the invention reaches 1ng/ml.
ELISA kit provided by the invention based on IgG4 antibody test food allergens, standard items or positive quality control
Product are 4 albumen of human IgG, and food allergen albumen coated elisa plate can detect the oral feeding in the liquid such as blood plasma, serum
The allergen specificity IgG4 that any food-induced body generates has the type of detection food allergen more, has a wide range of application
The characteristics of.The sensitivity of testing result can be effectively improved using biotin-avidin system.
Description of the drawings
Fig. 1 is that kit of the present invention is prepared and process for using figure；
Fig. 2 is the standard curve of the ELISA kit of IgG4 antibody test food allergens in embodiment 1.
Specific implementation mode
The present invention provides the ELISA kits based on IgG4 antibody test food allergens, including following reagent：
It is coated with the detection plate of food allergen；
Washing buffer；
Sample diluting liquid；
Standard items or positive quality control product；
4 antibody-solutions of anti-human igg of biotin labeling；
The avidin solution of enzyme label；
Substrate developing solution；
Terminate liquid.
ELISA kit provided by the invention includes being coated with the detection plate of food allergen.The food allergen
Peridium concentration is preferably 0.1~2000 μ g/ml, more preferably 0.1~100 μ g/ml.It is described to be coated with food mistake in the present invention
The type of food allergen includes aquatic products category, fowl poultry kind, egg-milk, greengrocery, fruits, cereals in the detection plate of quick original
Or nut fruits.The aquatic products category includes fish, shrimp, crab, shellfish and clam etc..The fowl poultry kind includes pork, chicken, beef and sheep
Meat.Egg-milk includes egg and milk etc..The vegetables include radish, mushroom, celery, capsicum and green pepper.The fruits include
Citrus, orange, banana, apple, Kiwi berry, mango, grape, pineapple and tomato.The cereals include wheat, oat, rice,
Corn, soya bean and pea.The nut fruits include pine nut, peanut, fibert, walnut and American pistachios.
In the present invention, the type of the detection plate is preferably transparent polystyrene board and opaque white polyphenyl second
Alkene plate.The transparent polystyrene board is for quantitatively detecting；The opaque white polystyrene plate is used for qualitative detection.
ELISA kit provided by the invention includes washing buffer.In the present invention, the washing buffer is preferably
PBS buffer solution.The solute of the PBS buffer solution preferably includes component 8.0 parts of NaCl, the 0.20 part of KCl of following parts by weight,
1.16 part Na2HPO4With 0.20 part of KH2PO4With 1000 parts of water；The pH value of the PBS solution is 7.0~7.6.
In the present invention, the ELISA kit includes sample diluting liquid.The sample diluting liquid is that pH value is 7.0~7.6
PBS buffer solution.The solute of the PBS buffer solution preferably includes component 8.0 parts of NaCl, the 0.20 part of KCl of following parts by weight,
1.16 part Na2HPO4, 0.20 part of KH2PO4With 1000 parts of water；The pH value of the PBS solution is 7.0~7.6.
ELISA kit provided by the invention includes 4 antibody of anti-human igg of biotin labeling.The biotin labeling
The mass concentration of 4 antibody of anti-human igg is preferably 0.01~8000 μ g/ml.4 antibody of anti-human igg of the biotin labeling adds
It is preferably the holes 0.02~0.2ml/ to add volume.In the present invention, 4 antibody of the anti-human igg is preferably that the monoclonal of anti-human igg 4 is anti-
Body.
ELISA kit provided by the invention includes standard items or positive quality control product.The standard items or positive quality control product
For people IgG4.The mass concentration of the positive quality control product is preferably 0.01~1000 μ g/ml；The additive amount of the positive quality control product
The holes preferably 0.02~0.2ml/.
ELISA kit provided by the invention includes enzyme label avidin solution.In the present invention, the enzyme marks Avidin
Solution is 1~5000 times of working solution when using.The present invention marks the source of Avidin to be not particularly limited the enzyme, uses
Enzyme well-known to those skilled in the art marks avidin solution.
ELISA kit provided by the invention includes substrate developing solution.The substrate developing solution is tetramethyl benzidine bottom
Object solution or 2,2- hydrazines-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid substrate solutions.The quality of the tetramethyl benzidine is dense
Degree preferably 0.02~0.05%；The mass concentration of the 2,2- hydrazines-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid is preferably
0.05~0.1mg/ml.
ELISA kit provided by the invention includes terminate liquid.The terminate liquid is the sulfuric acid solution of 0.5~2.0mol/L
Or the sodium hydroxide solution that mass concentration is 30%~40%, the more preferably sulfuric acid solution of 1.0mol/L or mass concentration are
35% sodium hydroxide solution.
In the present invention, the preparation method of the ELISA kit based on IgG4 antibody test food allergens, preferably
Preparation method including the detection plate for being coated with food allergen.
In the present invention, the preparation method of the detection plate for being coated with food allergen preferably includes following steps：
1) food allergen is diluted with coating buffer solution, obtains food allergen coating buffer；
2) the food allergen coating buffer that the step 1) obtains is added in the reacting hole of detection plate, 4 DEG C overnight；
3) next day adds confining liquid, is washed again after incubation in step 2) described in once washing after overnight detection plate hole
It washs, is coated with the detection plate of food allergen.
The present invention coating buffer solution dilution food allergen and 4 albumen of human IgG, obtain coating buffer.It is described in the present invention
Diluted method is not particularly limited, using diluted technical solution well-known to those skilled in the art.In the present invention,
The coating buffer solution is preferably carbonate buffer solution.The concentration of the carbonate buffer solution is preferably 0.01~0.2mol/L.
The pH value of the carbonate buffer solution is preferably 8.5~9.5.
After obtaining coating buffer, the coating buffer is added in the reacting hole of detection plate by the present invention, and 4 DEG C overnight.The present invention
In, the volume of coating buffer is preferably the holes 0.02~0.2ml/ in each reacting hole.
In the present invention, described 4 DEG C preferably carry out under conditions of standing overnight, are coated on conducive to what food allergen consolidated
In detection plate.
Next day after overnight detection plate hole described in once washing, adds confining liquid, is washed again after incubation.The present invention couple
There is no limit using the technical solution of washing well-known to those skilled in the art for the method for the once washing.This hair
In bright, the additive amount of the washing buffer is preferably at least the volume of coating buffer；The number of the once washing is preferably 2~
6 times；The incubation time of the single wash is preferably every time 0~5min (similarly hereinafter).
After the once washing, confining liquid is added in the detection plate after once washing by the present invention, is washed again after incubation.This
In invention, the confining liquid is preferably the balf serum albumin PBS buffer solution that mass concentration is 1%~5%.The confining liquid
Additive amount be preferably at least the volume of coating buffer.In the present invention, the time of the incubation is preferably 10~200min.It is described to incubate
The temperature educated is preferably 10~40 DEG C, more preferably 15~35 DEG C.
After the incubation, the present invention is washed detection plate again, in the present invention, the method washed again with
The method of once washing described in above-mentioned technical proposal is identical, and details are not described herein.
It is described wash again after, after the present invention preferably drains the moisture in detection plate naturally, with vacuum bag sealing detection plate,
Obtain the detection plate for being coated with food allergen.
In the present invention, the concentration of 4 antibody of anti-human igg of the biotin labeling is preferably 0.01~8000 μ g/ml, more excellent
It is selected as 0.1~1000 μ g/ml, most preferably 0.1~100 μ g/ml.
The detection method of ELISA kit based on IgG4 antibody test food allergens, includes the following steps：
1) sample to be tested is added in the detection plate hole for being coated with food allergen, carries out carrying out first after being once incubated
Secondary washing；
2) 4 antibody of anti-human igg of biotin labeling, secondary incubation are added in the detection plate into the step 1) after washing
It carries out washing for second afterwards；
3) the avidin solution solution that enzyme marks is added in the detection plate into the step 2) after washing, after being incubated three times
Carry out third time washing；
4) to addition substrate developing solution in washing detection plate will be obtained in the step 3), after four times are incubated, to detection plate
Middle addition terminate liquid, the detection plate to be developed the color；
5) the color developing detection plate obtained according to the step 4), obtains qualitative or quantitative result.
Sample to be tested is added in the detection plate for being coated with food allergen by the present invention, is carried out for the first time after primary incubation
Washing.In the present invention, the sample to be tested includes the liquid containing IgG4 antibody such as serum, blood plasma.The sample to be tested adds
Dosage is preferably per the hole holes 0.02~0.2ml/.The dilution of the liquid such as the serum or blood plasma is 1~32000 times.Diluted blood
Clearly, dilution of the blood plasma in the preferably described kit of solution.
In the present invention, in order to keep testing result accurately credible, blank control wells, negative control hole, standard are preferably also set up
Sample wells or positive quality control hole, and ensure that the amount of liquid of each reacting hole addition is consistent.It is preferably carbonic acid in the blank control wells
PBS buffer solution is added as sample in the coated ELISA Plate hole of salt buffer；It is preferably food allergen in the negative control hole
The fluid samples such as serum or the blood plasma without allergies crowd are added in coated ELISA Plate hole；The standard sample wells or positive quality control
Hole is preferably that PBS buffer solution is added in the carbonate buffer solution coated ELISA Plate hole for use 4 albumen containing human IgG as sample.
In the present invention, the temperature being once incubated is preferably 10~40 DEG C, more preferably 15~35 DEG C.It is described once to incubate
The time educated is preferably 10~200min.
In the present invention, the first time wash temperature is preferably 10~40 DEG C, more preferably 15~35 DEG C；The washing is slow
The additive amount of fliud flushing is preferably at least the volume of coating buffer；The number of the once washing is preferably 2~6 times；The single is washed
The incubation time washed is preferably every time 0~5min (similarly hereinafter)
4 antibody of anti-human igg of biotin labeling is added in detection plate after being washed to the first time, secondary incubation is laggard
Second of washing of row.
In the present invention, the temperature of the secondary incubation is preferably 10~40 DEG C, more preferably 15~35 DEG C.It is described secondary to incubate
The time educated is preferably 10~200min.
In the present invention, the method for second of washing is not particularly limited, and use is well-known to those skilled in the art
Washing methods.
After described second is washed, the avidin solution of enzyme label is added into the detection plate after the washing by the present invention,
Third time washing is carried out after being incubated three times.
In the present invention, the avidin solution of the enzyme label is diluted before use, and the diluted multiple preferably 1~
5000 times.In the present invention, the additive amount of the enzyme label avidin solution is preferably the holes 0.02~0.2ml/, more preferably
The holes 0.03~0.16ml/.
In the present invention, the temperature being incubated three times is preferably 10~40 DEG C, more preferably 15~35 DEG C.It is described to incubate three times
The time educated is preferably 10~200min.
In the present invention, the method for the third time washing is not particularly limited, and use is well-known to those skilled in the art
Washing methods.
After washing three times, substrate developing solution is added into obtained detection plate by the present invention, after four times are incubated, into detection plate
Add terminate liquid, the detection plate to be developed the color.
In the present invention, the additive amount of the substrate developing solution is preferably the holes 0.02~0.2ml/, more preferably 0.03~
The holes 0.16ml/.
In the present invention, the temperature of four incubations is preferably 10~40 DEG C, more preferably 15~35 DEG C.The incubation
Time is preferably 1~45min, more preferably 10~30min.
In the present invention, the sulfuric acid solution or mass concentration 30% of a concentration of 0.5~2.0mol/L of the terminate liquid~
40% sodium hydroxide solution.The additive amount of the terminate liquid is preferably the holes 0.02~0.2ml/, more preferably 0.03~
The holes 0.16ml/.
The detection plate of the colour developing obtained described in observation or detection, obtains qualitative or quantitative result.
In the present invention, the qualitative results are colors in the reacting hole for observe by the naked eye detection plate, in blank well and the moon
Property control wells do not develop the color, positive quality control hole develop the color in the case of, sample well colour developing is so judged as measuring samples be the positive；Instead
It, in the case where blank well and negative control hole do not develop the color the colour developing of positive quality control hole, sample well does not develop the color, then to be checked
Sample is feminine gender.
In the present invention, the quantitative result is judged by detecting the OD values of solution in detection plate reacting hole, with blank
The OD values in each hole are surveyed after control wells zeroing, and quantitative detection is carried out based on standard curve.It is described detection preferably when with
ABTS develops the color, then Detection wavelength is set as 405nm；When TMB develops the color, then Detection wavelength is set as 450nm.
In the present invention, using the preparation method of standard curve known in the art.The standard curve is four ginsengs
Curve fit equation:Y=(A-D)/[1+ (X/C)B]+D；Wherein A=2.66460；B=-1.81121；C=49.03420；D
=0.17936；R2=0.99721.X indicates that the concentration of IgG4 antibody, Y indicate OD values.IgG4 antibody indicates that the range of linearity is 0.3
~300ng/ml.
The kit provided by the invention that food allergen is detected based on IgG4 is carried out with reference to embodiment detailed
Illustrate, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Coating：Wheat and milk foodstuff anaphylactogen are diluted to a concentration of 10 μ g/ of total protein quality with carbonate buffer solution
The food allergen coating buffer of ml；Obtaining standard items coating buffer with carbonate buffer solution dilution 4 albumen of human IgG, (albumen concentration is shown in
Table 1), 0.05ml coating buffers are separately added into each reacting hole of polystyrene board, 4 DEG C are overnight.Next day discards solution in hole,
0.35ml washing buffers are added per hole, wash 3 times, are incubated 30s (referred to as washing, similarly hereinafter) every time；
Closing：PBS buffer solution (the closing containing 2% balf serum albumin of 0.05ml is separately added into each reacting hole
Liquid), pH：7.2,15~35 DEG C of incubation 120min, discard solution in hole, washing is same as above；
Sample is added：Suspicious Wheat Dood Allergy patients serum and milk allergy patients serum (test serum), normal human serum
(negative control hole) dilutes 100 times with PBS buffer solution, and the serum after 0.05ml dilutions is added per hole in corresponding allergy primordial covering
ELISA Plate hole in；0.05ml PBS, 15~35 DEG C of incubations are added per hole for standard sample wells/positive quality control hole, blank control wells
60min is washed out；
Add detection antibody：The biotin that the mass concentration that 0.05ml is added into each reacting hole by IgG4 is 0.5 μ g/ml is even
4 antibody-solutions of anti-human igg of connection, 15~35 DEG C be incubated 60min after wash；
The avidin solution of enzyme label：The Avidin that 0.05ml horseradish peroxidase-labeleds are separately added into per hole is molten
Liquid, 15~35 DEG C of incubation 30min, is washed out；
Substrate solution is added to develop the color：The tmb substrate solution 0.05ml of Fresh is separately added into per hole, 15~35 DEG C are protected from light incubation
16min；
Terminate reaction：It is separately added into 2mol/L sulfuric acid solutions 0.05ml per hole；
Detect OD values：ELISA Plate is placed in microplate reader, the absorbance value (OD at Detection wavelength 450nm450), with blank
The OD in each hole is surveyed after control wells zeroing450It is worth (table 1), and draws standard curve (attached drawing 2), food mistake is calculated according to standard curve
The concentration (table 2) of quick original specific IgG 4 antibody.The each Kong Jun of this experiment does multiple holes, and experiment is repeated 3 times.
The standard concentration and its OD of the ELISA kit of 1 human IgG of table, 4 antibody test food allergen450
Value
| Serial number | 4 albumen concentration (ng/ml) of human IgG | OD450It is worth (mean value) |
| 1 | 300.0 | 2.5547 |
| 2 | 100.0 | 2.1651 |
| 3 | 30.0 | 0.8511 |
| 4 | 10.0 | 0.4114 |
| 5 | 3.0 | 0.2225 |
| 6 | 1.0 | 0.1662 |
| 7 | 0.3 | 0.1536 |
| 8 | 0 | 0.1283 |
Standard curve is drawn according to the OD Value Datas of serial number 1,2,3,4,5,6,7 and 8 to be obtained according to standard curve such as attached drawing 2
To four parameter curve equations:Y=(A-D)/[1+ (X/C)B]+D；Wherein A=2.66460；B=-1.81121；C=
49.03420；D=0.17936；R2=0.99721.It can be obtained by embodiment 1：(1) IgG4 antibody tests food allergen
ELISA kit detection range is 0.3~300ng/ml；(2) spirit of the ELISA kit of IgG4 antibody tests food allergen
Sensitivity is 1ng/ml.The case where 2 human IgG of table, 4 antibody test suspected allergies patients serum's food anaphylactogen specificity IgG 4 antibody
As seen from the above embodiment, the ELISA kit provided by the invention based on IgG4 antibody test food allergens,
IgG4 is selected to detect food allergen, and the ELISA kit prepared based on indirect method, has detection sensitivity strong, it is accurate
The characteristics of exactness is high, favorable reproducibility, and being capable of the large batch of sample of single treatment.Embodiment proves kit provided by the invention
Detection sensitivity reaches 1ng/ml.Type with detection food allergen is more, the characteristics of having a wide range of application.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. the ELISA kit based on IgG4 antibody test food allergens, which is characterized in that including following components：
It is coated with the detection plate of food allergen；
Washing buffer；
Sample diluting liquid；
Standard items or positive quality control product；
4 antibody-solutions of anti-human igg of biotin labeling；
The avidin solution of enzyme label；
Substrate developing solution；
Terminate liquid.
2. kit according to claim 1, which is characterized in that the peridium concentration of the food allergen be 0.1~
2000μg/ml。
3. kit according to claim 1 or 2, which is characterized in that described to be coated in the detection plate of food allergen
The type of food allergen includes aquatic products category, fowl poultry kind, egg-milk, greengrocery, fruits, cereals or nut fruits.
4. kit according to claim 1, which is characterized in that the washing buffer is PBS buffer solution.
5. kit according to claim 1, which is characterized in that 4 antibody-solutions of anti-human igg of the biotin labeling
Mass concentration is 0.1~2000 μ g/ml.
6. kit according to claim 1, which is characterized in that the substrate developing solution is that tetramethyl benzidine substrates are molten
Liquid or 2,2- hydrazines-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid substrate solutions.
7. kit according to claim 1, which is characterized in that the sample diluting liquid is PBS buffer solution.
8. the kit according to claim 4 or 7, which is characterized in that the PBS buffer solution includes the group of following parts by weight
Point：8.0 parts of NaCl, 0.20 part of KCl, 1.16 parts of Na2HPO4, 0.20 part of KH2PO4With 1000 parts of water；The pH of the PBS buffer solution
Value is 7.0~7.6.
9. kit according to claim 1, which is characterized in that the terminate liquid is that the sulfuric acid of 0.5~2.0mol/L is molten
The sodium hydroxide solution that liquid or mass concentration are 30%~40%.
10. kit according to claim 1, which is characterized in that a concentration of of avidin solution of the enzyme label makes
1~5000 times of used time working solution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004099785A1 (en) * | 2003-05-08 | 2004-11-18 | Yorktest Laboratories Limited | Assay panel comprising food allergens |
| CN101256186A (en) * | 2008-03-28 | 2008-09-03 | 杭州浙大生科生物技术有限公司 | Food anaphylactogen specificity IgG4 antibody ELISA detection kit and preparation method thereof |
-
2018
- 2018-05-03 CN CN201810413986.3A patent/CN108646027A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004099785A1 (en) * | 2003-05-08 | 2004-11-18 | Yorktest Laboratories Limited | Assay panel comprising food allergens |
| CN101256186A (en) * | 2008-03-28 | 2008-09-03 | 杭州浙大生科生物技术有限公司 | Food anaphylactogen specificity IgG4 antibody ELISA detection kit and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| E.E.GUHSL等: "IgE, IgG4 and IgA specific to Bet v1-related food allergens do not predict oral allergy syndrome", 《ALLERGY》 * |
| JOHN M. JAMES, HUGH A.SAMPSON: "Immunologic changes associated with the development of tolerance in children with cow milk allergy", 《THE JOURNAL OF PEDIATRICS》 * |
| ROSA M. CODINA等: "Specific immunoglobulins to soybean hull allergens in soybean asthma", 《CHEST》 * |
| 朱立平、陈学清主编: "《免疫学常用实验方法》", 31 March 2000, 人民军医出版社 * |
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