CN108845124A - The ELISA kit of anaphylactogen is passed based on IgG1 antibody test gas - Google Patents
The ELISA kit of anaphylactogen is passed based on IgG1 antibody test gas Download PDFInfo
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- CN108845124A CN108845124A CN201810414087.5A CN201810414087A CN108845124A CN 108845124 A CN108845124 A CN 108845124A CN 201810414087 A CN201810414087 A CN 201810414087A CN 108845124 A CN108845124 A CN 108845124A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
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Abstract
Gas provided by the invention passes 1 detection kit of anaphylactogen specific IgG, belongs to gas and passes anaphylactogen detection field.The kit, including following reagent are coated with the detection plate that gas passes anaphylactogen；Washing buffer；Sample diluting liquid；Standard items or positive quality control product；1 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 IgG1 to detect gas and pass anaphylactogen, and is the ELISA kit based on indirect method preparation, 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 10ng/ml.
Description
Technical field
The present invention relates to gas to pass anaphylactogen inspection technology field, and in particular to passes anaphylactogen based on IgG1 antibody test gas
Kit, i.e. gas pass 1 detection kit of anaphylactogen specific IgG.
Background technique
Anaphylactia disease incidence accounts for 30% of total world population or more, is classified as the four big of 21 century by the World Health Organization
One of noninfectious disease.The disease 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 anaphylaxis eye closing, asthma, conjunctivitis, eczema, food hypersenstivity, drug allergy and serious allergic reaction etc., serious shadow
Ring people's lives quality and life security.
Gas passes anaphylactogen such as pollen particles, fungal spore, dust mite, grain flour, animal hair and urine, feather of birds etc.
The major incentive of anaphylactia, wherein pollen is the main source that gas passes anaphylactogen, results in 10%~20% allergia
The generation of disease.Different pollen has different protein ingredients, and antigenicity is also inconsistent, and there may be friendships between different pollen
Fork reactivity.Dust mite mainly has dermatophagoides pteronyssinus, dust mite, euroglyphus maynei etc., is cause allergic rhinitis and bronchial asthma important
Anaphylactogen.Cat and dog are the sources of common zoo-anaphylactogen, and mouse, cavy, horse, rabbit also may be used in some specific working environments
As important allergenic source, the hair of the above animal, epithelium, scurf, urine, saliva can have very strong sensitization.Closely
Year over such disease it is increasing, become common disease, frequently-occurring disease, be China health and economic development field need solve it is great
Problem
Based on the definition of anaphylactia " being one group of disease mediated by mast cell/basophilic granulocyte ", for
For susceptible person through sensitization, any a large amount of mast cells/basophilic granulocyte activation substance that can induce can induce allergy
Reaction.In these substances, the allergic reaction that IgE is mediated is undoubtedly most study, understands most deep one, the clinic in more than 40 years
Practice display, the allergic reaction that IgE is mediated are the most important hypotypes in allergic reaction.Traditionally caused by gas transmissibility anaphylactogen
Allergic reaction is also the allergic reaction mediated by IgE, for example, the Chinese patent of Publication No. CN105403692A discloses detection
The kit and method of house dust mite allergen specific IgE antibody, the Chinese patent of Publication No. CN103983789A disclose
A kind of test strips for specific dust mite class anaphylactogen IgE screening.As it can be seen that currently available technology passes the research of anaphylactogen to gas
The allergic reaction mediated by IgE is rested on, this will lead to the generation that cannot timely and accurately determine anaphylactia.
Summary of the invention
In view of this, having the kit it is an object of the invention to pass the kit of anaphylactogen based on IgG1 detection gas
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 that anaphylactogen is passed based on IgG1 antibody test gas, including following components：
It is coated with the detection plate that gas passes anaphylactogen；
Washing buffer；
Sample diluting liquid；
Standard items or positive quality control product；
1 antibody-solutions of anti-human igg of biotin labeling；
The avidin solution of enzyme label；
Substrate developing solution；
Terminate liquid.
Preferably, it is 0.1~2000 μ g/ml that the gas, which passes the peridium concentration of anaphylactogen,.
Preferably, it includes dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, America that the gas, which passes the type of anaphylactogen,
Big Lian, dog epithelium, cat epithelium, argy wormwood, artemisia annua, Sievers wormwood, humulus grass, Ambrosia trifida, short artemisiifolia, timothy grass, birch, poplar
Tree, willow, elm, Chinese wax, oriental plane tree, pine tree, cypress, soft leaf thorn certain herbaceous plants with big flowers, Alternaria, aspergillus fumigatus, penicillium notatum, black root
Mould, aspergillus niger, grain flour, feather of birds, bee venom, latex or lily.
Preferably, the washing buffer is PBS buffer solution.
Preferably, the mass concentration of 1 antibody of anti-igg of the biotin labeling stands alone as 0.01~8000 μ g/ml.
Preferably, the substrate developing solution is tetramethyl benzidine substrates solution or 2,2- hydrazine-bis- -3- ethyl benzo thiophene
Oxazoline -6- sulfonic acid substrate solution.
Preferably, the sample diluting liquid is PBS buffer solution.
Preferably, the PBS buffer solution comprises the following components in 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, the concentration of the avidin solution of the enzyme label is 1~5000 times of working solution when using.
The ELISA kit provided by the invention that anaphylactogen is passed based on IgG1 antibody test gas, including following reagent, coating
There is gas to pass the detection plate of anaphylactogen；Washing buffer；Sample diluting liquid；Standard items or positive quality control product；Biotin labeling resists
Human IgG1's antibody；The avidin solution of enzyme label；Substrate developing solution；Terminate liquid.The present invention is detected using IgG1 is selected for the first time
Gas passes anaphylactogen, and the ELISA kit based on indirect method preparation, has detection sensitivity strong, and accuracy is high, favorable reproducibility
The characteristics of, it being capable of the large batch of sample of single treatment.Embodiment proves that kit detection sensitivity provided by the invention reaches
10ng/ml。
Further, the ELISA kit provided by the invention that anaphylactogen is passed based on IgG1 antibody test gas, positive quality control
Product are human IgG1's albumen, and the ELISA Plate that gas is transmitted through quick primordial covering can detecte any through sky in the liquid such as detection blood plasma, serum
The gas that gas is propagated passes the gas that allergen-induced body generates and passes anaphylactogen specific IgG 1, and there is detection gas to pass the type of anaphylactogen
It is more, the characteristics of having a wide range of application.The sensitivity of testing result can be effectively improved using biotin-avidin system.
Detailed description of the invention
Fig. 1 is that kit of the present invention makes and uses flow chart；
Fig. 2 is the standard curve for the ELISA kit that IgG1 antibody test gas passes anaphylactogen in embodiment 1.
Specific embodiment
The present invention provides the ELISA kits that anaphylactogen is passed based on IgG1 antibody test gas, including following reagent：
It is coated with the detection plate that gas passes anaphylactogen；
Washing buffer；
Sample diluting liquid；
Standard items or positive quality control product；
1 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 the detection plate for being coated with gas and passing anaphylactogen.The gas passes anaphylactogen
Peridium concentration is preferably 0.1~2000 μ g/ml, more preferably 0.1~100 μ g/ml.In the present invention, the gas that is coated with is transmitted through
In the detection plate of quick original gas pass the type of anaphylactogen include dermatophagoides pteronyssinus, dust mite, tropical flukeless mite, Groton bug, American cockroach,
Dog epithelium, cat epithelium, argy wormwood, artemisia annua, Sievers wormwood, humulus grass, Ambrosia trifida, short artemisiifolia, timothy grass, birch, poplar, willow
Tree, elm, Chinese wax, oriental plane tree, pine tree, cypress, soft leaf thorn certain herbaceous plants with big flowers, Alternaria, aspergillus fumigatus, penicillium notatum, bread mold, black song
Mould, grain flour, feather of birds, bee venom, latex or lily.
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 used for quantitative detection；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 Na2HPO4, 0.20 part of KH2PO4With 1000 parts of water；The pH value of the PBS buffer solution is 7.0~7.6.
ELISA kit provided by the invention includes 1 antibody of anti-human igg of biotin labeling.The biotin labeling
The mass concentration of 1 antibody of anti-human igg is preferably 0.01~8000 μ g/ml, more preferably 0.1~1000 μ g/ml, most preferably
0.1~100 μ g/ml.The addition volume of 1 antibody of anti-human igg of the biotin labeling is preferably the hole 0.02~0.2ml/.It is described
1 antibody of anti-human igg is preferably the monoclonal antibody of anti-human igg 1.
ELISA kit provided by the invention includes the avidin solution of enzyme label.The avidin solution of the enzyme label
Concentration be 1~5000 times of working solution when using.The present invention is not particularly limited the source of enzyme label Avidin, adopts
Avidin solution is marked with enzyme well-known to those skilled in the art.The avidin solution of the enzyme label is preferably HRP
The solution of streptavidin of label.
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 component of the PBS buffer solution includes following parts by weight of component：8.0 parts of NaCl, 0.20 part of KCl, 1.16 parts
Na2HPO4, 0.20 part of KH2PO4With 1000 parts of water.
ELISA kit provided by the invention includes standard items or positive quality control product.The standard items or positive quality control product
For human IgG1's albumen.The mass concentration of the positive quality control product is preferably 0.01~1000 μ g/ml；The positive quality control product adds
Dosage is preferably the hole 0.02~0.2ml/.
ELISA kit provided by the invention includes substrate developing solution.The substrate developing solution is tetramethyl benzidine bottom
Object solution or 2,2- hydrazine-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid substrate solution.The quality of the tetramethyl benzidine is dense
Degree preferably 0.02~0.05%；The 2,2- hydrazine-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid mass concentration 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%.
In the present invention, the preparation method of the ELISA kit that anaphylactogen is passed based on IgG1 antibody capture gas, preferably
The preparation method for passing the detection plate of anaphylactogen including being coated with gas.
In the present invention, the preparation method for being coated with the detection plate that gas passes anaphylactogen preferably includes following steps：
1) anaphylactogen is passed with coating buffer carrier gas, obtains gas and is transmitted through quick primordial covering liquid coating buffer；
2) 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, washes again after incubation in step 2) described in once washing after overnight detection plate hole
It washs.
The present invention passes anaphylactogen with coating buffer carrier gas, obtains coating buffer.In the present invention, the dilution process does not have
Have it is specifically limited, using diluted technical solution well-known to those skilled in the art.In the present invention, the coating buffering
Liquid is preferably carbonate buffer solution.In the present invention, the concentration of the carbonate buffer solution is preferably 0.01~0.2mol/L.It is described
The pH value of 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 0.02~0.2ml coating buffer 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 gas biography anaphylactogen is firm
In detection plate.
Next day after overnight detection plate hole described in once washing, adds confining liquid, washs 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 PBS buffer solution for the balf serum albumin that mass concentration is 1%~5%.The closing
The additive amount of liquid is preferably at least the volume of coating buffer.In the present invention, the time of the incubation is preferably 10~200min.It is described
The temperature of incubation is preferably 10~40 DEG C, and more preferably 15~35 DEG C.
After the incubation, the present invention will test plate and be washed 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,
It obtains being coated with the detection plate that gas passes anaphylactogen.
In the present invention, the concentration of 1 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 that the ELISA kit of anaphylactogen is passed based on IgG1 antibody test gas, is included the following steps：
1) sample to be tested is added in the detection plate hole for being coated with gas biography anaphylactogen, PBS buffer solution is added to coating
Have in the detection plate hole of human IgG1's albumen, carries out carrying out first time washing after being once incubated for；
2) 1 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) avidin solution that enzyme marks is added in the detection plate into the step 2) after washing, is carried out after being incubated for three times
Third time is washed；
4) to addition substrate developing solution in washing detection plate will be obtained in the step 3), after four times are incubated for, 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 gas biography anaphylactogen by the present invention, carries out for the first time after primary incubation
Washing.In the present invention, the sample to be tested includes the liquid containing IgG1 antibody such as serum or blood plasma.The sample to be tested adds
Dosage is preferably 0.02~0.2ml.The dilution of the liquid such as the serum or blood plasma is 1~32000 times.
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 guarantee 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；It is preferably that food allergen is coated in the negative control hole
The fluid samples such as serum or the blood plasma without allergies crowd are added in ELISA Plate hole；The standard sample wells or positive quality control hole is excellent
It is selected as being added PBS buffer solution in the carbonate buffer solution coated ELISA Plate hole for using the albumen containing human IgG1 as sample.
In the present invention, the temperature being once incubated for is preferably 10~40 DEG C, and 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, and 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).
1 antibody of anti-human igg of biotin labeling is added into the detection plate after first time washing, secondary incubation is laggard
Second of washing of row.
In the present invention, the additive amount of 1 antibody of anti-human igg of the biotin labeling is preferably the hole 0.02~0.2ml/.
In the present invention, the temperature of the secondary incubation is preferably 10~40 DEG C, and 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 that the present invention marks enzyme is added in the detection plate after washing, three times
Third time washing is carried out after incubation.
In the present invention, the avidin solution of enzyme label is diluted before use, and the diluted multiple preferably 1~
5000 times.In the present invention, the additive amount of the avidin solution of the enzyme label is preferably the hole 0.02~0.2ml/, more preferably
The hole 0.03~0.16ml/.
In the present invention, the temperature being incubated for three times is preferably 10~40 DEG C, and 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 for, 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 hole 0.02~0.2ml/, more preferably 0.03~
The hole 0.16ml/.The substrate developing solution is preferably tetramethyl benzidine (TMB) substrate solution or 2,2- hydrazine-bis- -3- ethylo benzene
And thiazoline -6- sulfonic acid (ABTS) substrate solution.
In the present invention, the temperature of four incubations is preferably 10~40 DEG C, and 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 that the concentration of the terminate liquid is 0.5~2.0mol/L be 30%~
40% sodium hydroxide solution.The additive amount of the terminate liquid is preferably the hole 0.02~0.2ml/, more preferably 0.03~
The hole 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 control wells
It does not develop the color with negative control hole, in the case that positive quality control hole is developed the color, sample well colour developing is so judged as measuring samples for sun
Property, conversely, sample well does not develop the color in the case where blank control wells and negative control hole do not develop the color the colour developing of positive quality control hole,
So measuring samples are feminine gender.
In the present invention, the quantitative result is judged by the OD value of solution in detection detection plate reacting hole, with blank
The OD value in each hole is surveyed after control wells zeroing, and carries out quantitative detection based on standard curve.It is described detection preferably when with
ABTS colour developing, then Detection wavelength is set as 405nm；When TMB colour developing, then Detection wavelength is set as 450nm.
In the present invention, standard curve is prepared using conventional method.The standard curve is straight line fitting equation:Y=A+B ×
X；Wherein A=0.39292；B=0.00211；R2=0.99906.IgG1 antibody test gas passes the ELISA kit inspection of anaphylactogen
Survey range is 3~1000ng/ml.
The kit provided by the invention for passing anaphylactogen based on IgG1 detection gas is carried out below with reference to embodiment detailed
Illustrate, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Coating：It is 10 μ that dermatophagoides pteronyssinus, dust mite and pollen humuli scandentis anaphylactogen, which are diluted to mass concentration, with carbonate buffer solution
The gas of g/ml is transmitted through quick primordial covering liquid；Standard items coating buffer (protein concentration is obtained with carbonate buffer solution dilution 3 albumen of human IgG
It is shown in Table 1).0.05ml coating buffer is separately added into each reacting hole of polystyrene board, 4 DEG C overnight.Next day discards molten in hole
0.35ml washing buffer is added in liquid, every hole, washs 3 times, is incubated for 0.5min (referred to as washing, similarly hereinafter) every time；
Closing：The PBS buffer solution that 0.05ml contains 2% balf serum albumin, pH are separately added into each reacting hole：7.2
(confining liquid), 15~35 DEG C of incubation 120min, discards solution in hole, washing is same as above；
Sample is added：Suspicious dust mite allergy patients serum and pollen humuli scandentis allergic patients sera (test serum), normal person
Serum (negative control hole) PBS buffer solution dilutes 50 times, and the serum after 0.05ml dilution is added in corresponding anaphylactogen in every hole
In coated ELISA Plate hole；Standard sample wells/positive quality control hole, the every hole of blank control wells are added 0.05ml PBS, and 15~35 DEG C
It is incubated for 60min, is washed out；
Add detection antibody：It is anti-that the anti-human igg 1 that 0.05ml mass concentration is 2.83 μ g/ml biotin labelings is added to every hole
Body washs after 15~35 DEG C of incubation 60min；
The Avidin of enzyme label：Every hole is separately added into the avidin solution of 0.05ml horseradish peroxidase-labeled, and 15
~35 DEG C of incubation 30min, are washed out；
Substrate solution is added to develop the color：Every hole is separately added into the tmb substrate solution 0.05ml of Fresh, and 15~35 DEG C are protected from light incubation
20min；
Terminate reaction：Every hole is separately added into 2mol/L sulfuric acid solution 0.05ml；
Detect OD value：ELISA Plate is placed in microplate reader, the absorbance (OD at Detection wavelength 450nm450) value, with blank
The OD value (table 1) in each hole is surveyed after control wells zeroing, and draws standard curve (attached drawing 2), is transmitted through according to standard curve calculating gas quick
The concentration (table 2) of former 1 antibody of specific IgG.The each Kong Jun of this experiment does multiple holes, and experiment is repeated 3 times.
1 human IgG1's antibody test gas of table passes the standard concentration and its OD value of the ELISA kit of anaphylactogen
| Serial number | Human IgG1's protein concentration (ng/ml) | OD value (mean value) |
| 1 | 1000 | 2.5169 |
| 2 | 300.0 | 0.9773 |
| 3 | 100.0 | 0.6013 |
| 4 | 30.0 | 0.4645 |
| 5 | 10.0 | 0.4404 |
| 6 | 3.0 | 0.3976 |
| 7 | 0 | 0.3967 |
Standard curve is drawn according to the OD Value Data of serial number 1,2,3,4,5,6 and 7 to be obtained such as attached drawing 1 according to standard curve
Straight line fitting equation:Y=A+B × X；Wherein A=0.39292；B=0.00211；R2=0.99906.It can be obtained by embodiment 1
It arrives：(1) it is 3~1000ng/ml that IgG1 antibody test gas, which passes the ELISA kit detection range of anaphylactogen,；(2) IgG1 antibody is examined
The sensitivity for surveying the ELISA kit that gas passes anaphylactogen is 10ng/ml.
2 human IgG1's antibody test suspected allergies patients serum's gas of table passes the case where 1 antibody of anaphylactogen specific IgG
As seen from the above embodiment, the ELISA kit provided by the invention that anaphylactogen is passed based on IgG1 antibody test gas,
IgG1 is selected to detect gas and pass anaphylactogen, and the ELISA kit based on indirect method preparation, has detection sensitivity strong, it is quasi-
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 10ng/ml.The type for passing anaphylactogen with detection gas 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. passing the ELISA kit of anaphylactogen based on 1 antibody test gas of anti-igg, which is characterized in that including following components：
It is coated with the detection plate that gas passes anaphylactogen；
Washing buffer；
Sample diluting liquid；
Standard items or positive quality control product；
1 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 gas pass the peridium concentration of anaphylactogen be 0.1~
2000μg/ml。
3. kit according to claim 1 or 2, which is characterized in that the gas pass anaphylactogen type include dermatophagoides pteronyssinus,
Dust mite, tropical flukeless mite, Groton bug, American cockroach, dog epithelium, cat epithelium, argy wormwood, artemisia annua, Sievers wormwood, humulus grass, three
Decomposite leaf artemisiifolia, short artemisiifolia, timothy grass, birch, poplar, willow, elm, Chinese wax, oriental plane tree, pine tree, cypress, soft leaf thorn certain herbaceous plants with big flowers,
Alternaria, aspergillus fumigatus, penicillium notatum, bread mold, aspergillus niger, grain flour, feather of birds, bee venom, latex or lily.
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 the quality of 1 antibody of anti-human igg of the biotin labeling
Concentration stands alone as 0.01~8000 μ g/ml.
6. kit according to claim 1, it is characterised in that the substrate developing solution is that tetramethyl benzidine substrates are molten
Liquid or 2,2- hydrazine-bis- -3- ethyl benzo thiazole phenanthroline -6- sulfonic acid substrate solution.
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 the concentration of the avidin solution of the enzyme label is to make
1~5000 times of used time working solution.
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Citations (3)
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| CN106662578A (en) * | 2014-04-03 | 2017-05-10 | 詹尼斯费尔公司 | Peptides, reagents and methods for detecting food allergy |
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2018
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| WO1998028624A1 (en) * | 1996-12-20 | 1998-07-02 | Pharmacia & Upjohn Diagnostics Ab | Methods for diagnosis of allergic bronchopulmonary aspergillosis |
| WO2009082798A1 (en) * | 2008-01-02 | 2009-07-09 | Fundação De Amparo À Pesquisa Do Estado De Minas Gerais - Fapemig | Method and kit for quantifying of allergen-especific human igg subclasses for the control and attendance of specific immunotherapy |
| CN106662578A (en) * | 2014-04-03 | 2017-05-10 | 詹尼斯费尔公司 | Peptides, reagents and methods for detecting food allergy |
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Application publication date: 20181120 |