CN108593932A - The ELISA kit of anaphylactogen is passed based on IgM antibody detection gas - Google Patents
The ELISA kit of anaphylactogen is passed based on IgM antibody detection gas 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
- G01N33/6854—Immunoglobulins
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The ELISA kit provided by the invention that anaphylactogen is passed based on IgM antibody detection gas, is belonged to gas and passes anaphylactogen detection technique field.The kit includes following reagent, is coated with the detection plate that gas passes anaphylactogen；Washing buffer；Sample diluting liquid；Standard items or positive quality control product；The anti-human IgM antibodies of biotin labeling；The avidin solution of enzyme label；Substrate developing solution；Terminate liquid.The characteristics of present invention selects IgM and passes anaphylactogen to detect gas, 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 10ng/ml.
Description
Technical field
The present invention relates to anaphylactogen inspection technology fields, and in particular to the reagent of anaphylactogen is passed based on IgM antibody detection gas
Box, i.e. gas pass anaphylactogen specific IgM 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, gas are transmitted through quick, 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 are the main sources that gas passes anaphylactogen, result 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 substance that can induce a large amount of mast cells/basophilic granulocyte activation 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 screenings.As it can be seen that people pass gas the research stop of anaphylactogen at present
In the allergic reaction mediated by IgE, this can lead to the generation that cannot timely and accurately determine anaphylactia.
Invention content
In view of this, it is an object of the invention to pass the kit of anaphylactogen based on IgM detection gas, the kit is made to have
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 IgM antibody detection 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；
The anti-human IgM antibodies of biotin labeling；
The avidin solution of enzyme label；
Substrate developing solution；
Terminate liquid.
Preferably, the peridium concentration that the gas passes anaphylactogen is 0.1~2000 μ g/ml.
Preferably, the type for being coated with gas biography anaphylactogen in the detection plate of gas biography anaphylactogen includes dermatophagoides pteronyssinus, dust
Mite, tropical flukeless mite, Groton bug, American cockroach, dog epithelium, cat epithelium, argy wormwood, artemisia annua, Sievers wormwood, humulus grass, trilobated leaf
Artemisiifolia, short artemisiifolia, timothy grass, birch, willow, willow, elm, Chinese wax, oriental plane tree, pine tree, cypress, soft leaf thorn certain herbaceous plants with big flowers, interlinkage
Spore is mould, aspergillus fumigatus, penicillium notatum, bread mold, 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 the anti-IgM 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.
The ELISA kit provided by the invention that anaphylactogen is passed based on IgM antibody detection 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 IgM antibody；The avidin solution of enzyme label；Substrate developing solution；Terminate liquid.The present invention detects gas using IgM is selected for the first time
Anaphylactogen, and the ELISA kit prepared based on indirect method are passed, has the characteristics that detection sensitivity is strong, it being capable of single treatment
Large batch of sample.Embodiment proves that kit detection sensitivity provided by the invention reaches 10ng/ml.
The ELISA kit provided by the invention that anaphylactogen is passed based on IgM antibody detection gas, positive quality control product is people IgM
Albumen, the ELISA Plate that gas is transmitted through quick primordial covering can detect the gas for detecting any air-borne transmission in the liquid such as blood plasma, serum
It passes the gas that allergen-induced body generates and passes anaphylactogen specific IgM, the type that anaphylactogen is passed with detection gas is more, application range
Wide feature.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 that IgM antibody detects the standard curve that gas passes the ELISA kit of anaphylactogen in embodiment 1.
Specific implementation mode
The present invention provides the ELISA kits that anaphylactogen is passed based on IgM antibody detection 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；
The anti-human IgM antibodies 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
The type that gas passes anaphylactogen in the detection plate of quick original 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, willow, 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 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 PBS buffer solution preferably includes the component of following parts by weight：8.0 parts of NaCl, 0.20 part of KCl, 1.16 parts
Na2HPO4With 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 anti-human IgM antibodies' solution of biotin labeling.The biotin labeling
The mass concentration of anti-human IgM antibodies' preparation be preferably 0.01~8000 μ g/ml.The anti-IgM of the biotin labeling is molten
The addition volume of liquid is preferably the holes 0.02~0.2ml/.The anti-human IgM antibodies are preferably the monoclonal antibody of anti-human IgM.
ELISA kit provided by the invention includes the avidin solution of enzyme label.The avidin solution of the enzyme label
A concentration of use when 1~5000 times of working solution.The present invention marks the source of Avidin to be not particularly limited the enzyme, adopts
Avidin solution is marked with enzyme well-known to those skilled in the art.
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
Na2HPO4With 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 people's IgM 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 holes 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- 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 of mass concentration 30%~40%.
In the present invention, the preparation method of the ELISA kit that anaphylactogen is passed based on IgM 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 solution 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, is washed again after incubation in step 2) described in once washing after overnight detection plate hole
It washs, obtains being coated with the detection plate that gas passes anaphylactogen.
The present invention passes anaphylactogen with coating buffer solution 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 buffers 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, 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 equal to coating buffer；The number of the once washing is preferred
It is 2~6 times；The 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, 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,
It obtains being coated with the detection plate that gas passes anaphylactogen.
In the present invention, the concentration of anti-human IgM antibodies' preparation of the biotin labeling 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 detection method that the ELISA kit of anaphylactogen is passed based on IgM antibody detection gas, is included the following steps：
1) sample to be tested is added to and is coated in the detection plate hole that gas passes anaphylactogen, PBS is added to coating someone IgM
In the detection plate hole of albumen, carry out carrying out first time washing after being once incubated；
2) anti-human IgM antibodies' preparation of biotin labeling is added in the detection plate into the step 1) after washing, it is secondary
It carries out washing for second after incubation；
3) avidin solution that enzyme marks is added in the detection plate into the step 2) after washing, is carried out after being incubated 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, 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 to by the present invention to be coated in the detection plate that gas passes anaphylactogen, is carried out for the first time after primary incubation
Washing.In the present invention, the sample to be tested includes the liquid containing IgM 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.Dilute serum or blood plasma
It is preferably the dilution in mentioned reagent box with 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；It is preferably that gas is transmitted through quick primordial covering 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 using in the coated ELISA Plate hole of the carbonate buffer solution of the albumen of IgM containing people and PBS buffer solution is added 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).
Anti-human IgM antibodies' preparation of biotin labeling, secondary incubation are added in detection plate after being washed to the first time
It carries out washing for second afterwards.
In the present invention, the additive amount of anti-human IgM antibodies' preparation solution of the biotin labeling is preferably 0.02~
The holes 0.2ml/.
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 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 the 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 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/.The substrate developing solution is preferably tetramethyl benzidine (TMB) substrate solution or 2,2- hydrazines-bis- -3- ethylo benzenes
And thiazoline -6- sulfonic acid (ABTS) substrate solution.
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 of a concentration of 0.5~2.0mol/L of the terminate liquid be 30%~
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 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 that measuring samples are sun
Property, conversely, in the case where blank control wells and negative control hole do not develop the color the colour developing of positive quality control hole, sample well does not develop the color,
So measuring samples are 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, standard curve is drawn using routine side.The standard curve is fitting a straight line equation:Y=A+B × X；
Wherein A=0.27122；B=0.00171；R2=0.99818.X indicates that the concentration of IgM, Y indicate OD values.IgM antibody detects gas
The ELISA kit detection range for passing anaphylactogen is 10~1000ng/ml.
The kit provided by the invention that anaphylactogen is passed based on IgM detection gas is carried out specifically with reference to embodiment
It is bright, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Coating：It is 10 μ g/ that pollen humuli scandentis and Sievers wormwood pollen allergens, which are diluted to mass concentration, with carbonate buffer solution
The gas of ml is transmitted through quick primordial covering liquid；Obtaining standard items coating buffer with carbonate buffer solution dilution people's IgM albumen, (albumen concentration is shown in Table
1).0.05ml coating buffers are separately added into each reacting hole of polystyrene board, 4 DEG C overnight.Next day discards solution in hole, often
0.35ml washing buffers are added in hole, wash 3 times, are incubated 0.5min every time.(referred to as washing, similarly hereinafter)；
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 pollen humuli scandentis allergic patients sera and mugwort pollen allergic patients sera (test serum), just
Ordinary person's serum (negative control hole) dilutes 50 times with PBS buffer solution, and the serum after 0.05ml dilutions is added per hole in corresponding mistake
In the ELISA Plate hole of quick primordial covering；0.05ml PBS are added per hole in standard sample wells/positive quality control hole, blank control wells, 15~
35 DEG C of incubation 60min, are washed out；
Add detection antibody：IgM is added the anti-human IgM that 0.05ml mass concentrations are 0.5 μ g/ml biotin labelings to every hole and resists
Liquid solution, 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
15min；
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 (OD at Detection wavelength 450nm450) value, with blank
The OD values (table 1) in each hole are surveyed after control wells zeroing, and draw standard curve (attached drawing 2), are transmitted through according to standard curve calculating gas quick
The concentration (table 2) of former specific IgM antibodies.The each Kong Jun of this experiment does multiple holes, and experiment is repeated 3 times.
1 human IgM antibody of table detects the standard concentration and its OD that gas passes the ELISA kit of anaphylactogen450Value
| Serial number | People's IgM albumen concentration (ng/ml) | OD450It is worth (mean value) |
| 1 | 1000.0 | 1.966 |
| 2 | 300.0 | 0.805 |
| 3 | 100.0 | 0.4865 |
| 4 | 30.0 | 0.3191 |
| 5 | 10.0 | 0.262 |
| 6 | 3.0 | 0.2196 |
| 7 | 1.0 | 0.2444 |
| 8 | 0 | 0.2447 |
Standard curve is drawn according to the OD Value Datas of serial number 1,2,3,4,5 and 8 to be obtained directly according to standard curve such as attached drawing 2
Line fit equation:Y=A+B × X；Wherein A=0.27122；B=0.00171；R2=0.99818.It can be obtained by embodiment 1：
(1) the ELISA kit detection range of IgM antibody detection food allergen is 10~1000ng/ml；(2) IgM antibody detects gas
The sensitivity for passing the ELISA kit of anaphylactogen is 10ng/ml.
2 human IgM antibody of table detects the case where suspected allergies patients serum's gas passes anaphylactogen specific IgM antibodies
As seen from the above embodiment, the ELISA kit provided by the invention that anaphylactogen is passed based on IgM antibody detection gas,
It selects IgM and passes anaphylactogen, and the ELISA kit prepared based on indirect method to detect gas, with the strong spy of detection sensitivity
Point, 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.The type that anaphylactogen is passed 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 anti-IgM detection gas, 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；
The anti-human IgM antibodies 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 that the gas passes anaphylactogen is 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 that gas passes anaphylactogen
The type that gas passes 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, willow, willow, elm, Chinese wax, method
State Chinese parasol tree, pine tree, cypress, soft leaf thorn certain herbaceous plants with big flowers, Alternaria, aspergillus fumigatus, penicillium notatum, bread mold, aspergillus niger, grain flour, bird
Feather, 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 the anti-human IgM antibodies of the biotin labeling
A concentration of 0.01~8000 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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| 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 |
| 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 |
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| CN101109750A (en) * | 2007-07-27 | 2008-01-23 | 杭州浙大生科生物技术有限公司 | Reagent kit for detecting food allergen specificity IgG ELISA and preparing method thereof |
| 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 |
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Application publication date: 20180928 |