CN103575907A - Method for detecting egg albumin in ancient binder cultural relic material - Google Patents
Method for detecting egg albumin in ancient binder cultural relic material Download PDFInfo
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- CN103575907A CN103575907A CN201310527117.0A CN201310527117A CN103575907A CN 103575907 A CN103575907 A CN 103575907A CN 201310527117 A CN201310527117 A CN 201310527117A CN 103575907 A CN103575907 A CN 103575907A
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- G—PHYSICS
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- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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Abstract
The invention discloses a method for detecting egg albumin in an ancient binder cultural relic material, which is characterized in that the method is used for detecting the egg albumin content in the ancient binder cultural relic material by using an enzyme-linked immunosorbent assay method, namely, combining a Millpore rabbit-anti-egg albumin polyclonal antibody to the surface of a polystyrene solid-phase carrier so as to form a solid-phase antibody; then, combining the solid-phase antibody with binder cultural relic eluent so as to form an immune complex; after the immune complex is washed, adding an alkaline phosphatase labeled goat-anti-rabbit IgG (H+L) antibody, so that the antibody is combined with antigens in the immune complex so as to form an enzyme labelled antibody-antigen-solid phase antibody complex; and adding a substrate for enzyme reaction, so that the substrate is converted into a colored product by enzyme catalysis, and a positive correlation is formed between the amount of the product and the amount of a detected material in samples, therefore, qualitative analysis can be performed according to the shown shade. The method disclosed by the invention is low in cost, simple in detection, and rapid in response speed.
Description
Technical field
The present invention relates to the detection method of egg protein in jointing compound historical relic material in a kind of ancient times, be mainly used in the detection of egg protein in jointing compound historical relic material in ancient times.
Background technology
Jointing compound is as a kind of functional material, paints, plays an important role in the technique such as dyeing and finishing at seal.Ancient times, the key component of jointing compound material was egg protein, therefore the detection of egg protein was had to important scientific value to the research of jointing compound material.In ancient records and relics material, the method for protein detection of jointing compound mainly contains microchemistry experimental method, Raman spectroscopy.Microchemistry experimental method is mainly to utilize the chemico-physical properties such as the dissolubility of egg protein in organic solvent and fusing point thereof to identify, this method is simple, but poor specificity.Raman spectroscopy has higher spatial resolution, can avoid the interference between different component.Yet,
Ancient records and relics material is of the remote past, is subject to for a long time the impact of the environment such as illumination, oxidation, makes consolidating material complicated component, content very micro-, is also difficult to analysis and characterization single kind albumen consolidating material.
Summary of the invention
The technical problem to be solved in the present invention is: the problem existing for above-mentioned prior art provides the detection method of egg protein in a kind of highly sensitive, simple jointing compound historical relic material in ancient times efficiently.
The technical solution adopted in the present invention is: utilize sandwich enzyme-linked immunization method to detect the egg protein composition in jointing compound historical relic material in ancient times, be about to the anti-egg protein polyclonal antibody of Millpore rabbit and be attached to polystyrene surface of solid phase carriers formation insolubilized antibody, then with jointing compound historical relic eluent in conjunction with forming immune complex, after washing, add alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) antibody, its antigen in immune complex is combined and is formed enzyme labelled antibody-antigen-insolubilized antibody compound again; Add after the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product becomes positive correlation with the amount of tested substance in sample, therefore can carry out qualitative analysis according to the depth of colour generation.
2. the detection method of egg protein in jointing compound historical relic material in ancient times according to claim 1, is characterized in that step is as follows:
A) solution preparation: the preparation of PBS buffer solution: KCl0.2g, KH
2pO
40.2g, NaCl8.0g, NaH
2pO
47H
2o2.16g, with deionized water dissolving constant volume 1000mL, regulates PH to 7.4,121 ℃ of sterilization treatment; The preparation of eluent: the edta solution of 5mL1mol/L trihydroxy methyl aminomethane-HCl buffer solution, 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate (containing 10g solute in 50mL deionized water), add deionized water dissolving constant volume 500mL, with NaOH, regulate pH to 7.4, after aseptic suction filtration, at room temperature store;
B) PBS buffer solution 80-100 μ L is made as to blank, sample egg protein 0.1-0.5mg is made as positive control; The historical relic material of the PBS buffer solution of 80-100 μ L, 0.1-0.5mg sample egg protein, 0.1-0.5mg is dissolved in respectively in the eluent of 80-120 μ L, under room temperature, place 2-4 days, form positive control sample egg protein eluent, laboratory sample historical relic material eluent;
C) getting coating buffer dilution 10000-12000 primary antibodie is doubly the anti-egg protein polyclonal antibody of Millpore rabbit 80-100 μ L, be added in each hole of polystyrene reactant plate, use preservative film shrouding, at 4 ℃ of temperature, keep 24h, inferior daily PBS buffer solution fully washs 3 times, dries;
D) in each hole, add 1% bovine serum albumin(BSA) (BSA) solution, 100 μ L, sealing 1h, then washs three times each three minutes with PBS buffer solution;
E) in each hole, add positive control sample egg protein sample eluent and the laboratory sample historical relic material eluent of the step b of 40 μ L-70 μ L, put in 37 ℃ of incubators one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, dry;
F) in each hole, adding the ELIAS secondary antibody of diluting between 10000-12000 times is alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) 80-100 μ L, put in the baking oven of 37 ℃ one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, and dry;
G) in each hole, add the tetramethyl biphenyl amine aqueous solution 100 μ L of substrate solution 1%, put dark place and make its reaction 20min;
H) in each hole, add 2mol/L H
2sO
4solution 0.05mL, cessation reaction;
I) step h liquid is taken out, be added in microplate reader, read in the absorbance at 450nm place;
J) the two absorbance numerical value OD of positive control sample egg protein and laboratory sample historical relic material relatively; Compare with the OD value that blank sample egg protein detects, if egg protein test experience OD value obviously increases in historical relic eluent, prove in laboratory sample historical relic material and really have egg protein; Compare with the OD value that blank sample egg protein detects, if egg protein test experience OD value, without marked change, proves in laboratory sample historical relic material and do not have egg protein in historical relic eluent.
The invention has the beneficial effects as follows: 1, the present invention utilizes the method for sandwich enzyme-linked immunity to detect the egg protein composition in jointing compound historical relic material, highly sensitive, simple to operate on the one hand.On the other hand, avoid the interference of other albumen, high specificity.2, compared with prior art, the method applied in the present invention cost is low, detection is simple, fast response time.
Embodiment
The sandwich enzyme-linked immunization method that the present invention adopts detects egg protein in ancient times jointing compound historical relic materials, and highly sensitive, simple to operate on the one hand, on the other hand, high specificity, can avoid the interference of other albumen.The anti-egg protein polyclonal antibody of Millpore rabbit is attached to polystyrene surface of solid phase carriers and forms insolubilized antibody, then with jointing compound historical relic eluent in conjunction with forming immune complex, after washing, add alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) antibody, its antigen in immune complex is combined and is formed enzyme labelled antibody-antigen-insolubilized antibody compound again.Add after the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product becomes positive correlation with the amount of tested substance in sample, therefore can enter qualitative analysis according to the depth of colour generation.Because the catalytic efficiency of enzyme is very high, indirectly amplified immunoreactive result, make test there is higher sensitivity.
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
A detection method for egg protein in ancient times jointing compound historical relic materials, it comprises the steps:
A) solution preparation: the preparation of PBS buffer solution: KCl0.2g, KH
2pO
40.2g, NaCl8.0g, NaH
2pO
47H
2o2.16g, with deionized water dissolving constant volume 1000mL, regulates PH to 7.4,121 ℃ of sterilization treatment.The preparation of eluent: the edta solution of 5mL1mol/L trihydroxy methyl aminomethane-HCl buffer solution, 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate (containing 10g solute in 50mL deionized water), add deionized water dissolving and be settled to 500mL, with NaOH, regulate pH to 7.4.After aseptic suction filtration, at room temperature store.
B) PBS buffer solution 100 μ L are made as to blank, sample egg protein 0.1mg is made as positive control.The historical relic material of the PBS buffer solution of 100 μ L, 0.1mg sample egg protein, 0.1mg is dissolved in respectively in the eluent of 100 μ L, under room temperature, places 2 days, form positive control sample egg protein eluent, laboratory sample historical relic material eluent.
C) get primary antibodie (the anti-egg protein polyclonal antibody of Millpore rabbit) the 100 μ L of 10000 times of coating buffer dilutions, be added in each hole of polystyrene reactant plate, use preservative film shrouding, 4 ℃ of refrigerator the insides keep 24h.Then with PBS buffer solution, fully wash 3 times, dry.
D) in each hole, add 1% bovine serum albumin(BSA) (BSA) solution, 100 μ L, sealing 1h.Then with PBS buffer solution, wash three times each three minutes.
E) in each hole, add respectively positive control sample egg protein sample eluent and the laboratory sample historical relic material eluent of 40 μ L step b, put in 37 ℃ of incubators one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, dry.
F) in each hole, add dilution ELIAS secondary antibody (alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L)) the 100 μ L of 10000 times, put in 37 ℃ of incubators one hour.Remove liquid, with PBS damping fluid washing three times, each three minutes, and dry.
G) in each hole, add 1% substrate solution tetramethyl biphenyl amine aqueous solution 100 μ L, put dark place and make its reaction 20min.
H) in each hole, add 2mol/LH
2sO
40. solution 0.05mL, cessation reaction.
I) step h liquid is taken out, join in microplate reader, read in the absorbance at 450nm place
J) compare the absorbance numerical value OD of positive control sample egg protein and laboratory sample historical relic material.
Embodiment 2
A detection method for egg protein in ancient times jointing compound historical relic materials, it comprises the steps:
A) solution preparation: the preparation of PBS buffer solution: KCl0.2g, KH
2pO
40.2g, NaCl8.0g, NaH
2pO
47H
2o2.16g, with deionized water dissolving constant volume 1000mL, regulates PH to 7.4,121 ℃ of sterilization treatment.The preparation of eluent: the edta solution of 5mL1mol/L trihydroxy methyl aminomethane-HCl buffer solution, 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate (containing 10g solute in 50mL deionized water), add deionized water dissolving and be settled to 500mL, with NaOH pH value of solution to 7.4.After aseptic suction filtration, at room temperature store.
B) PBS buffer solution 100 μ L are made as to blank, sample egg protein 0.1mg is made as positive control.The historical relic material of the PBS buffer solution of 100 μ L, 0.1mg sample egg protein, 0.3mg is dissolved in respectively in the eluent of 100 μ L, under room temperature, places 3 days, form positive control sample egg protein eluent, laboratory sample historical relic material eluent.
C) get primary antibodie (the anti-egg protein polyclonal antibody of Millpore rabbit) the 100 μ L of 11000 times of coating buffer dilutions, be added in each hole of polystyrene reactant plate, use preservative film shrouding, 4 ℃ of refrigerator the insides, keep 24h.Then with PBS buffer solution, fully wash 3 times, dry.
D) in each hole, add 1% bovine serum albumin (BSA) solution, 100 μ L, sealing 1h.Then with PBS buffer solution, wash three times each three minutes.
E) in each hole, add respectively positive control sample egg protein sample eluent and the laboratory sample historical relic material eluent of 50 μ L step b, put in 37 ℃ of incubators one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, dry.
F) in each hole, add dilution ELIAS secondary antibody (alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L)) the 100 μ L of 11000 times, put in 37 ℃ of incubators one hour.Remove liquid, with PBS buffer solution washing three times, each three minutes, and dry.
G) in each hole, add 1% substrate solution tetramethyl biphenyl amine aqueous solution 100 μ L, put dark place and make its reaction 20min.
H) in each hole, add 2mol/LH
2sO
4solution 0.05mL, cessation reaction.
I) step h liquid is taken out, be added in microplate reader, read in the absorbance at 450nm place
J) compare the absorbance numerical value OD of positive control sample egg protein and laboratory sample historical relic material.
Embodiment 3
A detection method for egg protein in ancient times jointing compound historical relic materials, it comprises the steps:
A) solution preparation: the preparation of PBS buffer solution: KCl0.2g, KH
2pO
40.2g, NaCl8.0g, NaH
2pO
47H
2o2.16g, with deionized water dissolving constant volume 1000mL, regulates PH to 7.4,121 ℃ of sterilization treatment.The preparation of eluent: the edta solution of 5mL1mol/L trihydroxy methyl aminomethane-HCl buffer solution, 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate (containing 10g solute in 50mL deionized water), add deionized water dissolving and be settled to 500mL, with NaOH, regulate pH to 7.4.After aseptic suction filtration, at room temperature store.
B) PBS buffer solution 100 μ L are made as to blank, sample egg protein 0.1mg is made as positive control.The PBS buffer solution of 100 μ L, 0.1mg sample egg protein, 0.5mg laboratory sample historical relic material are dissolved in respectively in 100 μ L eluents, under room temperature, place 4 days, form positive control sample egg protein eluent, laboratory sample historical relic material eluent.
C) get primary antibodie (the anti-egg protein polyclonal antibody of Millpore rabbit) the 100 μ L of 12000 times of coating buffer dilutions, be added in each hole of polystyrene reactant plate, use preservative film shrouding, 4 ℃ of refrigerator the insides, keep 24h.Inferior daily PBS buffer solution fully washs 3 times, dries.
D) in each hole, add 1% bovine serum albumin(BSA) (BSA) solution, 100 μ L, sealing 1h.Then with PBS buffer solution, wash three times each three minutes.
E) in each hole, add respectively positive control sample egg protein sample eluent and the laboratory sample historical relic material eluent of 60 μ L step b, put in 37 ℃ of incubators one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, dry.
F) (alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) 100 μ L put in 37 ℃ of incubators one hour in each hole, to add the ELIAS secondary antibody of 12000 times of dilutions.Remove liquid, with PBS buffer solution washing three times, each three minutes, and dry.
G) in each hole, add 1% substrate solution tetramethyl biphenyl amine aqueous solution 100 μ L, put dark place and make its reaction 20min.
H) in each hole, add 2mol/LH
2sO
4solution 0.05mL, cessation reaction.
I) step h liquid is taken out, be added in microplate reader, read in the absorbance at 450nm place
J) compare the absorbance numerical value OD of positive control sample egg protein and laboratory sample historical relic material.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (2)
1. the detection method of egg protein in an ancient times jointing compound historical relic material, it is characterized in that utilizing sandwich enzyme-linked immunization method to detect the egg protein composition in jointing compound historical relic material in ancient times, be about to the anti-egg protein polyclonal antibody of Millpore rabbit and be attached to polystyrene surface of solid phase carriers formation insolubilized antibody, then with jointing compound historical relic eluent in conjunction with forming immune complex, after washing, add alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) antibody, its antigen in immune complex is combined and is formed enzyme labelled antibody-antigen-insolubilized antibody compound again; Add after the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product becomes positive correlation with the amount of tested substance in sample, therefore can carry out qualitative analysis according to the depth of colour generation.
2. the detection method of egg protein in jointing compound historical relic material in ancient times according to claim 1, is characterized in that step is as follows:
A) solution preparation: the preparation of PBS buffer solution: KCl0.2g, KH
2pO
40.2g, NaCl8.0g, NaH
2pO
47H
2o2.16g, with deionized water dissolving constant volume 1000mL, regulates PH to 7.4,121 ℃ of sterilization treatment; The preparation of eluent: the edta solution of 5mL1mol/L trihydroxy methyl aminomethane-HCl buffer solution, 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate (containing 10g solute in 50mL deionized water), add deionized water dissolving constant volume 500mL, with NaOH, regulate pH to 7.4, after aseptic suction filtration, at room temperature store;
B) PBS buffer solution 80-100 μ L is made as to blank, sample egg protein 0.1-0.5mg is made as positive control; The historical relic material of the PBS buffer solution of 80-100 μ L, 0.1-0.5mg sample egg protein, 0.1-0.5mg is dissolved in respectively in the eluent of 80-120 μ L, under room temperature, place 2-4 days, form positive control sample egg protein eluent, laboratory sample historical relic material eluent;
C) getting coating buffer dilution 10000-12000 primary antibodie is doubly the anti-egg protein polyclonal antibody of Millpore rabbit 80-100 μ L, be added in each hole of polystyrene reactant plate, use preservative film shrouding, at 4 ℃ of temperature, keep 24h, inferior daily PBS buffer solution fully washs 3 times, dries;
D) in each hole, add 1% bovine serum albumin(BSA) (BSA) solution, 100 μ L, sealing 1h, then washs three times each three minutes with PBS buffer solution;
E) in each hole, add positive control sample egg protein sample eluent and the laboratory sample historical relic material eluent of the step b of 40 μ L-70 μ L, put in 37 ℃ of incubators one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, dry;
F) in each hole, adding the ELIAS secondary antibody of diluting between 10000-12000 times is alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) 80-100 μ L, put in the baking oven of 37 ℃ one hour, remove liquid, with PBS buffer solution washing three times, each three minutes, and dry;
G) in each hole, add the tetramethyl biphenyl amine aqueous solution 100 μ L of substrate solution 1%, put dark place and make its reaction 20min;
H) in each hole, add 2mol/L H
2sO
4solution 0.05mL, cessation reaction;
I) step h liquid is taken out, be added in microplate reader, read in the absorbance at 450nm place;
J) the two absorbance numerical value OD of blank sample egg protein and laboratory sample historical relic material relatively; Compare with the OD value that blank sample egg protein detects, if egg protein test experience OD value obviously increases in historical relic eluent, prove in laboratory sample historical relic material and really have egg protein; Compare with the OD value that blank sample egg protein detects, if egg protein test experience OD value, without marked change, proves in laboratory sample historical relic material and do not have egg protein in historical relic eluent.
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| CN201310527117.0A CN103575907B (en) | 2013-10-30 | 2013-10-30 | The detection method of egg protein in a kind of ancient times jointing compound Wen Tianxiang |
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| EP0460102A1 (en) * | 1989-02-27 | 1991-12-11 | Reaads Medical Products, Inc. | Method and diagnostic test kit for detection of autoimmune antibody |
| US6368873B1 (en) * | 1998-04-09 | 2002-04-09 | Applied Biotech, Inc. | Identification of human urine for drug testing |
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| CN102279259A (en) * | 2010-06-10 | 2011-12-14 | 中国疾病预防控制中心寄生虫病预防控制所 | Diagnostic reagent kit for paragonimiasis detection and preparation method thereof |
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| Title |
|---|
| LAURA CARTECHINI ET AL: "Immunodetection of Proteins in Ancient Paint Media", 《ACCOUNTS OF CHEMICAL RESEARCH》, vol. 43, no. 6, 30 June 2010 (2010-06-30), pages 867 - 876 * |
| 洪川等: "酶联免疫吸附测定法在古代牛奶残留物检测中的应用", 《酶联免疫吸附测定法在古代牛奶残留物检测中的应用》, vol. 23, no. 1, 28 February 2011 (2011-02-28), pages 25 - 28 * |
| 郑军: "免疫检测技术在文物胶结材料检测中的应用", 《中国文物报》, 25 September 2009 (2009-09-25), pages 007 * |
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