CN1183387C - Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method - Google Patents
Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method Download PDFInfo
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- CN1183387C CN1183387C CNB031022278A CN03102227A CN1183387C CN 1183387 C CN1183387 C CN 1183387C CN B031022278 A CNB031022278 A CN B031022278A CN 03102227 A CN03102227 A CN 03102227A CN 1183387 C CN1183387 C CN 1183387C
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- staphylococcal enterotoxin
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Abstract
The present invention discloses an immune chip for detecting staphylococcal enterotoxin and papaverine, and a preparation method thereof. The immune chip comprises a glass slide, wherein the glass slide is provided with a surface linked with glutaraldehyde as a bifunctional cross-linking reagent after being processed by silane as an aldehydation reagent; the surface is fixedly connected with antibody molecules through covalent bonds; the antibody molecule is at least one of a staphylococcal enterotoxin A-type antibody, a staphylococcal enterotoxin B-type antibody, a staphylococcal enterotoxin C-type antibody and a papaverine antibody. The present invention can simply, conveniently and effectively detect the staphylococcal enterotoxin and the papaverine in food and save detection cost; the present invention greatly overcomes the defects of complicated process and unable high-flux detection of the existing detection technology.
Description
Technical field
The present invention relates to a kind of immuno-chip and preparation method thereof, particularly relate to a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine and preparation method thereof.
Background technology
The biological immune chip is meant high density antigen or the antibody microarray that is coated on the solid phase carrier, is a kind of novel biochip that proposes after genetic chip.Immuno-chip is made up of the antigen or the antibody microarray that are fixed on the variety classes supporting dielectric, the position of fixed member and composition are known in the array, react with label (fluorescent material, enzyme or chemiluminescent substance etc.) antibody of mark or the identification molecule on antigen and the chip, detect by specific scanister then, the result is handled by Computer Analysis.Compare with traditional food pollutant detection technique that is used for, immuno-chip has many advantages.Traditional detection method once can only detect a kind of pollutant, and immuno-chip can detect multiple pollutant simultaneously, and each detection only needs a spot of reagent and sample.This technology if can be applied in the food hygiene detection, the health supervision working level is significantly improved.
China all has many food poisonings to take place every year, cause very big economic loss and bad social influence, having brought great harm to people's life, how better these incidents to have been prevented, is the problem of needing solution in the food inspection research badly.
In production, processing and the storage process of food, there are many objectionable impuritiess can cause the pollution of food, use as nonstandard agricultural chemicals, cause the high residues of pesticides in the food; Food contacts with poisonous chemical substance, causes the chemical contamination of food: the various toxin that microorganism that exists in the food and metabolism thereof produce, cause the biotic pollution of food.What is particularly worth mentioning is that, a few peoples are arranged in order to pursue economic interests for the moment merely, in food, add poisonous and harmful substance, the serious threat people's life and health, as: some illegal food and drink owners add pappy shell, grievous injury consumer's interests in food.The method that is used for food inspection now has a lot, as high performance liquid chromatography, vapor-phase chromatography, chemical analysis, immunofluorescence technique, euzymelinked immunosorbent assay (ELISA) etc., advantages such as that these methods have is highly sensitive, high specificity have been brought into play vital role in food inspection.Yet these technology once can only detect an a kind of or pollutant, and the pollutant in the food all is unknown, is that multiple pollutant exists simultaneously sometimes, and above method will be difficult to satisfy the needs of multivariate detection.
Summary of the invention
In order to overcome the deficiencies in the prior art, the purpose of this invention is to provide a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
Another object of the present invention provides a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine.
Technical scheme of the present invention is summarized as follows:
A kind of immuno-chip that detects staphylococcal enterotoxin and papaverine, comprise microslide, described microslide has through after the aldehyde radical reagent silane treatment, and through with the surface of difunctional cross-linking reagent glutaraldehyde cross-linking, fixedly connected by covalent bond with antibody molecule in described surface, described antibody molecule is at least a in SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and the papaverine antibody.
A kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine, it comprises the steps:
(1) prepare one and have through after the aldehyde radical reagent silane aldehyde radicalization, and through with the microslide on the surface of difunctional cross-linking reagent glutaraldehyde cross-linking;
(2) preparation SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and papaverine antibody;
(3) antibody is fixing, and it comprises:
1. the phosphate buffer with pH5.7～8.0 dilutes SEA type antibody, making concentration is 0.01～2.0mg/ml dilution SEB type antibody, making concentration is 0.05～2.0mg/ml, dilution staphylococcal enterotoxin C type antibody, making concentration is 0.01～1.0mg/ml, and it is 0.5～2.0mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 36～38 ℃, saturated humidity was placed 0.5～4 hour, with the phosphate buffer flushing of pH5.7～8.0, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody on microslide, dripping 20 μ l percents by volume and be 24～28% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 2～3% ovalbumin seals, 36～38 ℃, saturated humidity was placed 0.5～4 hour, use distilled water flushing, dry, 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
The pH of described phosphate buffer is to select 7.2 to be advisable.
The concentration of described SEA type antibody is preferably 1.0mg/ml.
The concentration of described SEB type antibody is preferably 0.5mg/ml.
The concentration of described staphylococcal enterotoxin C type antibody is preferably 0.5mg/ml.
The concentration of described papaverine antibody is preferably 1mg/ml.
The described laying temperature that four kinds of antibody diluents are added drop-wise to the zones of different of slide surface is 37 ℃, and be 0.5 hour standing time.
The percent by volume of described calf serum is 25%, and the percent by volume of ovalbumin is 2%, and the time of sealing with calf serum, ovalbumin is 0.5 hour.
A kind of immuno-chip that detects staphylococcal enterotoxin and papaverine provided by the invention, it is easy, detect staphylococcal enterotoxin and papaverine in the food effectively, and save to detect cost, it is loaded down with trivial details and can not carry out the deficiency of high throughput testing that it has improved the pre-treatment process of existing detection technique greatly.
Embodiment
A kind of immuno-chip that detects staphylococcal enterotoxin and papaverine, comprise microslide, described microslide is a glass slide, have through making the glass sheet surface amination after the aldehyde radical reagent silane treatment, amino passes through the surface with difunctional cross-linking reagent glutaraldehyde cross-linking, fixedly connected by covalent bond with antibody molecule in described surface, described antibody molecule is SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and papaverine antibody.
A kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine, embodiment is as follows:
Embodiment 1:
1. prepare one and have through after the aldehyde radical reagent silane aldehyde radicalization, and through with the microslide on the surface of difunctional cross-linking reagent glutaraldehyde cross-linking;
2. extract SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and papaverine antibody;
3. antibody is fixing, and it comprises:
1. the phosphate buffer with pH7.2 dilutes SEA type antibody, making concentration is 1.0mg/ml dilution SEB type antibody, making concentration is 0.5mg/ml, dilution staphylococcal enterotoxin C type antibody, making concentration is 0.5mg/ml, and it is 1mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 37 ℃, saturated humidity was placed 0.5 hour, with the phosphate buffer flushing of pH7.2, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody on microslide, dripping 20 μ l percents by volume and be 25% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 2% ovalbumin seals, 37 ℃, saturated humidity was placed 0.5 hour, use distilled water flushing, dry, 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
Embodiment 2:
Step 1 and step 2 are with embodiment 1.
3. antibody is fixing, and it comprises:
1. the phosphate buffer with pH5.7 dilutes SEA type antibody, making concentration is 0.01mg/ml dilution SEB type antibody, making concentration is 0.05mg/ml, dilution staphylococcal enterotoxin C type antibody, making concentration is 0.05mg/ml, and it is 0.5mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 36 ℃, saturated humidity was placed 1 hour, with the phosphate buffer flushing of pH5.7, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody on microslide, dripping 20 μ l percents by volume and be 24% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 2% ovalbumin seals, 36 ℃, saturated humidity was placed 1 hour, use distilled water flushing, dry, 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
Embodiment 3:
Step 1 and step 2 are with embodiment 1.
3. antibody is fixing, and it comprises:
1. the phosphate buffer with pH8.0 dilutes SEA type antibody, making concentration is 2.0mg/ml dilution SEB type antibody, making concentration is 2.0mg/ml, dilution staphylococcal enterotoxin C type antibody, making concentration is 1.0mg/ml, and it is 2mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 38 ℃, saturated humidity was placed 4 hours, with the phosphate buffer flushing of pH8.0, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody on microslide, dripping 20 μ l percents by volume and be 28% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 3% ovalbumin seals
38 ℃, saturated humidity was placed 4 hours, used distilled water flushing, dried, and 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
Embodiment 4:
Step 1 and step 2 are with embodiment 1.
3. antibody is fixing, and it comprises:
1. the phosphate buffer with pH7.4 dilutes SEA type antibody, making concentration is 0.5mg/ml dilution SEB type antibody, making concentration is 1.0mg/ml, dilution staphylococcal enterotoxin C type antibody, making concentration is 0.1mg/ml, and it is 1mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 37 ℃, saturated humidity was placed 2 hours, with the phosphate buffer flushing of pH7.4, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody on microslide, dripping 20 μ l percents by volume and be 25% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 2% ovalbumin seals, 37 ℃, saturated humidity was placed 2 hours, use distilled water flushing, dry, 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
Embodiment 5:
Step 1 and step 2 are with embodiment 1.
3. antibody is fixing, and it comprises:
1. the phosphate buffer with pH6.0 dilutes SEA type antibody, making concentration is 0.1mg/ml dilution SEB type antibody, making concentration is 0.1g/ml, dilution staphylococcal enterotoxin C type antibody, making concentration is 0.01mg/ml, and it is 1mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 37 ℃, saturated humidity was placed 1 hour, with the phosphate buffer flushing of pH6.0, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody on microslide, dripping 20 μ l percents by volume and be 25% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 2% ovalbumin seals, 37 ℃, saturated humidity was placed 1 hour, use distilled water flushing, dry, 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
What detect the staphylococcal enterotoxin employing is sandwich method, and what adopt when detecting papaverine is competition law.
After finishing with the reaction of reaction of the present invention or antigenic competition, ScanArray3000 detects with the laser confocal scanning instrument, and scanning resolution is 10 μ m.Excite and detect wavelength and decide according to fluorescent marker.Cy3, the Cy5 excitation wavelength is 530nm, detects wavelength and is respectively 585nm, 650nm.Fluorescence signal quantitatively adopts Imagene3.0, and (Biodiscovery, Inc) software is analyzed.
Claims (9)
1. immuno-chip that detects staphylococcal enterotoxin and papaverine in the food, comprise microslide, described microslide has through after the aldehyde radical reagent silane treatment, and through with the surface of difunctional cross-linking reagent glutaraldehyde cross-linking, it is characterized in that described surface fixedlys connected by covalent bond with antibody molecule, described antibody molecule is at least a in SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and the papaverine antibody.
2. a preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food is characterized in that it comprises the steps:
(1) prepare one and have through after the aldehyde radical reagent silane aldehyde radicalization, and through with the microslide on the surface of difunctional cross-linking reagent glutaraldehyde cross-linking;
(2) preparation SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and papaverine antibody;
(3) antibody is fixing, and it comprises:
1. making its concentration with the phosphate buffer of pH5.7～8.0 dilution SEA type antibody is 0.01～2.0mg/ml, it is 0.05～2.0mg/ml that dilution SEB type antibody makes its concentration, it is 0.01～1.0mg/ml that dilution staphylococcal enterotoxin C type antibody makes its concentration, and it is 0.5～2.0mg/ml that dilution papaverine antibody makes its concentration;
2. with point sample instrument four kinds of antibody diluents are added drop-wise to the zones of different of slide surface, 36～38 ℃, saturated humidity was placed 0.5～4 hour, with the phosphate buffer flushing of pH5.7～8.0, used distilled water flushing again, dried;
3. the zone of containing SEA type antibody, SEB type antibody, staphylococcal enterotoxin C type antibody and papaverine antibody on microslide, dripping 20 μ l percents by volume and be 24～28% calf serum seals, the zone of containing papaverine antibody on microslide, dripping 20 μ l percents by volume and be 2～3% ovalbumin seals, 36～38 ℃, saturated humidity was placed 0.5～4 hour, use distilled water flushing again, dry, 4 ℃ of preservations have obtained a kind of immuno-chip that detects staphylococcal enterotoxin and papaverine.
3. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2, the pH that it is characterized in that described phosphate buffer is 7.2.
4. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2, the concentration that it is characterized in that described SEA type antibody is 1.0mg/ml.
5. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2, the concentration that it is characterized in that described SEB type antibody is 0.5mg/ml.
6. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2 is characterized in that the concentration of described staphylococcal enterotoxin C type antibody is 0.5mg/ml.
7. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2, the concentration that it is characterized in that described papaverine antibody is 1mg/ml.
8. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2, it is characterized in that the described laying temperature that four kinds of antibody diluents are added drop-wise to the zones of different of slide surface is 37 ℃, be 0.5 hour standing time.
9. a kind of preparation method who detects the immuno-chip of staphylococcal enterotoxin and papaverine in the food according to claim 2, the percent by volume that it is characterized in that described calf serum is 25%, the percent by volume of ovalbumin is 2%, and the timeliness of sealing with calf serum, ovalbumin is 0.5 hour.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB031022278A CN1183387C (en) | 2003-01-30 | 2003-01-30 | Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method |
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| CNB031022278A CN1183387C (en) | 2003-01-30 | 2003-01-30 | Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method |
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| CN1183387C true CN1183387C (en) | 2005-01-05 |
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Families Citing this family (5)
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| CN100494960C (en) * | 2005-04-20 | 2009-06-03 | 中国科学院上海微系统与信息技术研究所 | Minitype gathering chip for biological sample, and preparation method |
| CN101535811B (en) * | 2006-10-18 | 2016-05-04 | 生物梅里埃公司 | For the method for in-vitro diagnosis PVL-producing staphylococcus aureus |
| CN102455356A (en) * | 2010-10-29 | 2012-05-16 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immunochip test method of staphylococcus enterotoxins and fumonisin |
| CN102928599A (en) * | 2012-10-30 | 2013-02-13 | 中国人民解放军第四军医大学 | Staphylococcal enterotoxin A chemiluminiscence enzyme-linked immunoassay detection kit |
| CN114088861A (en) * | 2021-10-27 | 2022-02-25 | 上海市食品药品检验研究院 | Method for detecting enterotoxin C in milk by using multi-dimensional liquid chromatography-mass spectrometry |
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