CN109085341A - A kind of chip and preparation method thereof detecting three kinds of anti-allergen antibodies - Google Patents
A kind of chip and preparation method thereof detecting three kinds of anti-allergen antibodies Download PDFInfo
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- CN109085341A CN109085341A CN201810950977.8A CN201810950977A CN109085341A CN 109085341 A CN109085341 A CN 109085341A CN 201810950977 A CN201810950977 A CN 201810950977A CN 109085341 A CN109085341 A CN 109085341A
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- chip
- slide
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- point sample
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention discloses the protein chip that a kind of detection can detect three kinds of Typical allergic original antibody concentration simultaneously, the chip is made as follows: (1) black slide pretreatment；(2) point sample allergenic components solution；(3) chip is closed, the chip is made.Chip of the present invention is used to detect the concentration of anti-allergen antibodies in the blood of patient, thus judge patient to anaphylactogen to be measured whether allergy and degree just.The kit that this method is formed is easy to use, and recall rate is high, but also the full-automatic operation of anti-allergen antibodies detection may be implemented.
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of for detecting the chip of three kinds of anti-allergen antibodies concentration
And preparation method thereof.
Background technique
With the great variety of the fast development of global economy level, people's lives mode and housing conditions, make
Spectrum of disease is obtained also to change therewith.It is clearly proposed in the World Health Assembly held by the World Health Organization in 2013:
Using anaphylactia as the Chronic Non-Communicable Diseases (NCD) of representative, since onset is early, the state of an illness easily fluctuates repeatedly, clinical phenotypes
The features such as complicated multiplicity, it has also become the hot spot of global public health concern.
Anaphylactia is also known as allergic disease, refers to body to certain sensitizer primary response sensitization of contact
Afterwards, when being contacted again the stimulation of identical sensitizer, what is occurred is a kind of based on physiological dysfunction or tissue cell insult
The disease of abnormal immune response.Each age level of the anaphylactia from newborn to the elderly may all occur, and often have
There is apparent genetic predisposition.Children's anaphylactia mainly has anaphylactic shock, skin nettle rash, eczema, allergic rhinitis, mistake
Quick property asthma, enteritis anaphylactica etc..And the diagnosis and treatment for anaphylactia, it is more importantly detected compared with symptomatic treatment specific
Sensitizer, and avoid contacting, to achieve the purpose that prevention.Therefore, anti-allergen antibodies are detected, analyze its testing result pair
Diagnosis, treatment and the prevention of children's anaphylactia are of great significance.
Anti-allergen antibodies detection method includes vivo approaches and in-vitro method, and vivo detection method mainly includes skin prick
Test (SPT), intradermal skin test (IST), Allergen provocation, class's patch test.Wherein SPT and IST method is to patient
Painful big and have certain risk, one anaphylactogen patient of every detection will bear primary pain more, Allergen provocation and
Class's patch test is because test operation is complicated, easily induces strong allergic reaction, clinically less use；External detection method is general
That detect is total IgE (tIgE) in serum, specific IgE (sIgE) and IgG.The in-vitro diagnosis of anaphylactia be initially into
The detection of promoting circulation of blood liquid tIgE antibody, but the influence factor of tIgE level is more, other than anaphylactia, parasitic infection, kind
The factors such as race, age can influence tIgE level, and tIgE is also only capable of the probability of instruction patient's sensitization.SIgE detection is I
The most frequently used and most worthy obtains external detection method in allergic reaction type, and the critical value of serum sIgE level is 0.35kU/L, surpasses
Crossing the value is the positive, and body is prompted to be in sensitization.External detection method mainly includes radio-allergo-sorbent test
(RAST), Diagnosis of Sghistosomiasis notation, enzyme linked immunosorbent assay (ELISA) etc..Wherein RAST is at high cost, immunoblotting analysis and ELISA operation
Complexity has certain requirement to operator.These methods can only generally detect a kind of anti-allergen antibodies every time, inefficiency,
It is with high costs.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides a kind of disposably three kinds of common mistakes of detection
The chip and preparation method thereof of quick original antibody.Chip of the present invention is for detecting anti-allergen antibodies, and recall rate is high, no pain, no danger
It is dangerous, it is at low cost, it is easy to operate, only a small amount of blood is needed just to can detect many a projects, and also achieve anti-allergen antibodies detection
Full-automatic operation
Technical scheme is as follows:
Chip of the present invention is made as follows:
(1) black slide pretreatment；
(2) point sample allergenic components solution；
(3) chip is closed, the chip is made.
The black pretreated method of slide described in step (1) are as follows:
1. by black slide be placed in the slide pretreatment fluid containing NaOH impregnate 16~for 24 hours, later using purified water cleaning 2
~8 times；
2. black slide is placed in the solution of silane that mass concentration is 0.05~1wt% and impregnates 20~60min；
3. 0.2 is toasted under the conditions of 100~180 DEG C by being put into baking oven after soaked black slide nitrogen purging~
0.6h。
Allergenic components solution described in step (2) includes dermatophagoides pteronyssinus, cockroach, cat/dog hair allergenic components solution.
The method of point sample described in step (2) is Machine automated point sample, on black slide o'clock at 4 × 7 matrix；Each column
The point formed for same allergenic components solution.
Closed process described in step (3) are as follows: the good black slide of point sample is submerged into 1~4h in confining liquid, is taken out later black
Slide, and it is centrifuged the remaining confining liquid of removal, the chip is made.
The confining liquid is the buffer solution containing closed protein；The closed protein is bovine serum albumin(BSA) or egg white egg
It is white；The buffer is one of PBS buffer solution, Tris buffer, HEPS buffer, MOPS buffer or a variety of.
A kind of application of the chip, is made kit for the chip.
The kit further includes the secondary antibody solution for being marked with HRP enzyme or alkali phosphorus enzyme, detection liquid A and detection liquid B.Institute
It states detection liquid A and contains 1% luminol and 2%Tris, detection liquid B contains 1% hydrogen peroxide.
Detection basic principle of the invention are as follows:
The immunological method that product of the present invention detects anti-allergen antibodies is indirect method, in the chip matrix that glass is carrier
Fixed allergenic components (antigen), these antigens can capture antibody specific in tested sample, captured antibody and mark
Note has HRP enzyme or the secondary antibody of alkali phosphorus enzyme to combine, enzymatic chemiluminescent substrate, generates chemiluminescence.Its optical signal is logical
CCD camera acquisition is crossed, and carries out intellectual analysis.According to signal concentration quantitative curve, it is tested by strong and weak calculate of optical signal
The concentration of allergen specificity antibody in sample further determines that testing result is negative or positive.
The present invention is beneficial to be had the technical effect that
This method is intensive style for the first time, the method for detecting multiple anti-allergen antibodies simultaneously with high throughput.This technology utilizes
Chemiluminescence obtains the sensitivity of height.Majority method different from the past can only qualitative determination.This method may be implemented
Quantitative determination, to be inferred to the allergy severity of patient.This method is available with automatically there are one great advantages
The chip reading instrument of change, realizes high speed, easy detection, needs a large amount of manually operated methods to be more suitable hospital than other big
Amount is carried out.
The present invention is compared with external import reagent (Phadia ImmunoCAP), and the recall rate height of anaphylactogen is consistent, property
It can be good.
Detailed description of the invention
Fig. 1 is allergenic components point sample schematic diagram in preparation method of the present invention.
In figure, column 1: positive quality control column；Column 2 and column 6: blank；Column 3: dermatophagoides pteronyssinus allergometry column；Column 4: cockroach allergy is surveyed
Fixed column；Column 5: cat/dog hair allergometry column；Column 7: negative Quality Control column.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1
A kind of chip detecting Typical allergic original, the chip are made as follows:
(1) black slide pretreatment；
1. black slide is placed in the slide pretreatment fluid containing NaOH and impregnates 16h, later using purified water cleaning 2~8
It is secondary；
2. black slide is placed in the solution of silane (medium is 25% ethyl alcohol) that mass concentration is 1wt% and impregnates 20min；
3. toasting 0.2h under the conditions of 180 DEG C for being put into baking oven after soaked black slide nitrogen purging.
(2) point sample allergenic components solution；
Referring to Fig.1, using Machine automated point sample, on black slide o'clock at 4 × 7 matrix；Often it is classified as same anaphylactogen
The point that component solution is formed；The allergenic components solution includes dermatophagoides pteronyssinus, cockroach, cat/dog hair allergenic components solution.
(3) allergenic components solution is closed；
The good black slide of point sample is submerged into 3h, Zhi Houqu in confining liquid (the Tris buffer containing 3% bovine serum albumin(BSA))
Black slide out, and it is centrifuged the remaining confining liquid of removal, the chip is made.
(4) kit
By chip be marked with the secondary antibody solution (mouse anti human IgE) of HRP enzyme, detection liquid A (containing 1% luminol and
2%Tris), detection liquid B (1% hydrogen peroxide) is packaged into kit jointly.
Embodiment 2
A kind of chip detecting Typical allergic original, the chip are made as follows:
(1) black slide pretreatment；
It is impregnated for 24 hours 1. being placed in black slide in the slide pretreatment fluid containing NaOH, later using purified water cleaning 2~8
It is secondary；
It is impregnated 2. black slide is placed in the solution of silane (medium is 25% ethyl alcohol) that mass concentration is 0.05wt%
60min；
3. toasting 0.4h under the conditions of 140 DEG C for being put into baking oven after soaked black slide nitrogen purging.
(2) point sample allergenic components solution；
Referring to Fig.1, using Machine automated point sample, on black slide o'clock at 4 × 7 matrix；Often it is classified as same anaphylactogen
The point that component solution is formed；The allergenic components solution includes dermatophagoides pteronyssinus, cockroach, cat/dog hair allergenic components solution.
(3) allergenic components solution is closed；
The good black slide of point sample is submerged into 4h in confining liquid (PBS buffer solution containing 6% ovalbumin), takes out black glass later
Piece, and it is centrifuged the remaining confining liquid of removal, the chip is made.
(4) kit
By chip be marked with the secondary antibody solution (mouse anti human IgE) of HRP enzyme, detection liquid A (containing 1% luminol and
2%Tris), it detects liquid B (containing 1% hydrogen peroxide) and is packaged into kit jointly.
Embodiment 3
A kind of chip detecting Typical allergic original, the chip are made as follows:
(1) black slide pretreatment；
1. black slide is placed in the slide pretreatment fluid containing NaOH and impregnates 20h, later using purified water cleaning 2~8
It is secondary；
2. black slide is placed in the solution of silane (medium is 25% ethyl alcohol) that mass concentration is 0.5wt% and impregnates 30min；
3. toasting 0.6h under the conditions of 100 DEG C for being put into baking oven after soaked black slide nitrogen purging.
(2) point sample allergenic components solution；
Referring to Fig.1, using Machine automated point sample, on black slide o'clock at 4 × 7 matrix；Often it is classified as same anaphylactogen
The point that component solution is formed；The allergenic components solution includes dermatophagoides pteronyssinus, cockroach, cat/dog hair allergenic components solution.
(3) allergenic components solution is closed；
The good black slide of point sample is submerged into 1h, Zhi Houqu in confining liquid (the MOPS buffer containing 4% bovine serum albumin(BSA))
Black slide out, and it is centrifuged the remaining confining liquid of removal, the chip is made.
(4) kit
By chip be marked with the secondary antibody solution (mouse anti human IgE) of HRP enzyme, detection liquid A (containing 1% luminol and
2%Tris), it detects liquid B (containing 1% hydrogen peroxide) and is packaged into kit jointly.
Test case:
Clinical serum is detected using the SLXP-001 type biological chip reading apparatus that our company produces, SLXP-001 type
The course of work of biological chip reading apparatus is as follows:
The test serum sample of instrument automatic sucking 200ul is into reaction cup, and instrument is by egg made from the embodiment of the present invention
White chip is automatically put into test serum, and 37 DEG C are incubated for 40 minutes, and subsequent instrument clamping jaw takes out chip, rushes automatically through instrument
The secondary antibody solution (200ul, instrument are inhaled in advance automatically) for being marked with HRP enzyme is put into after washing, and is incubated for again after forty minutes,
Instrument clamping jaw takes out chip again, is put into luminous substrate solution after instrument auto-flushing (by the detection liquid A of 100ul
Mixed with the detection liquid B of 100ul, by instrument automatic sucking and mixing), imaging of taking pictures finally is carried out to protein chip, it is soft
Part automatically analyzes picture, provides analysis result.Using the testing result of external import reagent (Phadia ImmunoCAP) as reference
Value.Testing result is as shown in table 1.
Table 1
As seen from the above table, the chip and internationally famous manufacturer Phadia Immuno of detection anaphylactogen provided by the present invention
CAP comparison result is similar (relative error majority is within 5%), and in sensitivity, the range of linearity etc. is also without notable difference.
The reference reagent that this experiment uses is world-famous import brand.It can be with using the chip of detection anaphylactogen provided by the invention
Realize high efficiency, letter operation, low cost, multiple advantages such as the used time is short, with good application prospect.
Claims (8)
1. a kind of chip for detecting three kinds of Typical allergic original antibodies, it is characterised in that the chip is made as follows:
(1) black slide pretreatment；
(2) point sample allergenic components solution；
(3) chip is closed, the chip is made.
2. chip according to claim 1, it is characterised in that the black pretreated method of slide described in step (1) are as follows:
1. by black slide be placed in the slide pretreatment fluid containing NaOH impregnate 16 ~ for 24 hours, later using purified water clean 2 ~ 8 times；
2. black slide is placed in the solution of silane that mass concentration is 0.05 ~ 1wt% and impregnates 20 ~ 60min；
3. toasting 0.2 ~ 0.6h under the conditions of 100 ~ 180 DEG C for being put into baking oven after soaked black slide nitrogen purging.
3. chip according to claim 1, it is characterised in that allergenic components solution described in step (2) includes family dirt
Mite, cockroach, cat/dog hair allergenic components solution.
4. chip according to claim 1, it is characterised in that the method for point sample described in step (2) is Machine automated point
Sample, on black slide o'clock at 4 × 7 matrix；Often it is classified as the point that same allergenic components solution is formed.
5. chip according to claim 1, it is characterised in that closed process described in step (3) are as follows: by good black of point sample
Slide submerges 1 ~ 4h in confining liquid, takes out black slide later, and is centrifuged the remaining confining liquid of removal, and the chip is made.
6. chip according to claim 5, it is characterised in that the confining liquid is the buffer solution containing closed protein；Institute
Stating closed protein is bovine serum albumin(BSA) or ovalbumin；The buffer is PBS buffer solution, Tris buffer, HEPS buffering
One of liquid, MOPS buffer are a variety of.
7. a kind of application of any one of claim 1 ~ 6 chip, it is characterised in that kit is made in the chip.
8. application according to claim 7, it is characterised in that the kit further includes being marked with HRP enzyme or alkali phosphorus enzyme
Secondary antibody solution, the chemiluminescent substrate sensitive to marker.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810950977.8A CN109085341A (en) | 2018-08-21 | 2018-08-21 | A kind of chip and preparation method thereof detecting three kinds of anti-allergen antibodies |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810950977.8A CN109085341A (en) | 2018-08-21 | 2018-08-21 | A kind of chip and preparation method thereof detecting three kinds of anti-allergen antibodies |
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|---|---|
| CN109085341A true CN109085341A (en) | 2018-12-25 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109613271A (en) * | 2019-01-10 | 2019-04-12 | 江苏三联生物工程有限公司 | A kind of chip and preparation method thereof detecting common indoor sucking anti-allergen antibodies |
| CN109725161A (en) * | 2019-01-10 | 2019-05-07 | 江苏三联生物工程有限公司 | A kind of chip and preparation method thereof detecting common aquatic product anti-allergen antibodies |
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| CN1485619A (en) * | 2002-09-26 | 2004-03-31 | 缪金明 | Allergen (sensitinogen) protein chip detecting process |
| CN101738469A (en) * | 2008-11-19 | 2010-06-16 | 中国海洋大学 | Method for detecting food allergen based on visible protein chip |
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| CN109613271A (en) * | 2019-01-10 | 2019-04-12 | 江苏三联生物工程有限公司 | A kind of chip and preparation method thereof detecting common indoor sucking anti-allergen antibodies |
| CN109725161A (en) * | 2019-01-10 | 2019-05-07 | 江苏三联生物工程有限公司 | A kind of chip and preparation method thereof detecting common aquatic product anti-allergen antibodies |
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