CN103575907B - The detection method of egg protein in a kind of ancient times jointing compound Wen Tianxiang - Google Patents
The detection method of egg protein in a kind of ancient times jointing compound Wen Tianxiang Download PDFInfo
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- CN103575907B CN103575907B CN201310527117.0A CN201310527117A CN103575907B CN 103575907 B CN103575907 B CN 103575907B CN 201310527117 A CN201310527117 A CN 201310527117A CN 103575907 B CN103575907 B CN 103575907B
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- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 58
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 58
- 150000001875 compounds Chemical class 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- 239000003480 eluent Substances 0.000 claims abstract description 54
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 9
- 239000004793 Polystyrene Substances 0.000 claims abstract description 9
- 229920002223 polystyrene Polymers 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 238000005406 washing Methods 0.000 claims abstract description 9
- 239000003547 immunosorbent Substances 0.000 claims abstract description 5
- 239000000427 antigen Substances 0.000 claims abstract description 4
- 102000036639 antigens Human genes 0.000 claims abstract description 4
- 108091007433 antigens Proteins 0.000 claims abstract description 4
- 239000000969 carrier Substances 0.000 claims abstract description 4
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 4
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 4
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 4
- 239000007790 solid phase Substances 0.000 claims abstract description 4
- 239000000126 substance Substances 0.000 claims abstract description 4
- 239000007853 buffer solution Substances 0.000 claims description 40
- 239000000523 sample Substances 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 27
- 239000006101 laboratory sample Substances 0.000 claims description 26
- 239000013068 control sample Substances 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- 239000013641 positive control Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- WBODDOZXDKQEFS-UHFFFAOYSA-N 1,2,3,4-tetramethyl-5-phenylbenzene Chemical group CC1=C(C)C(C)=CC(C=2C=CC=CC=2)=C1C WBODDOZXDKQEFS-UHFFFAOYSA-N 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 claims description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- 238000002835 absorbance Methods 0.000 claims description 5
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000004202 carbamide Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 239000003755 preservative agent Substances 0.000 claims description 5
- 230000002335 preservative effect Effects 0.000 claims description 5
- 239000000376 reactant Substances 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 238000000967 suction filtration Methods 0.000 claims description 5
- 241000283707 Capra Species 0.000 abstract 1
- 229910019142 PO4 Inorganic materials 0.000 abstract 1
- 238000002372 labelling Methods 0.000 abstract 1
- 239000010452 phosphate Substances 0.000 abstract 1
- -1 phosphate ester Chemical class 0.000 abstract 1
- 239000000463 material Substances 0.000 description 7
- 238000001069 Raman spectroscopy Methods 0.000 description 2
- 239000004568 cement Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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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
- G01N33/6803—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
- 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
- G01N33/543—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 detection method of egg protein in a kind of ancient times jointing compound Wen Tianxiang, it is characterized in that utilizing sandwich enzyme-linked immunosorbent method to detect the egg protein composition in jointing compound Wen Tianxiang in ancient times, be attached to polystyrene surface of solid phase carriers by Millpore rabbit anti-egg protein polyclonal antibody and form insolubilized antibody, then combine with jointing compound historical relic eluent and form immune complex, add alkaline phosphate ester enzyme labeling goat anti-rabbit igg (H+L) antibody after washing again, its antigen in immune complex is combined and forms enzyme labelled antibody-antigen-insolubilized antibody compound; After adding the substrate of enzyme reaction, substrate is become color products 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; The method applied in the present invention cost is low, detection is simple, fast response time.
Description
Technical field
The present invention relates to the detection method of egg protein in jointing compound Wen Tianxiang in a kind of ancient times, be mainly used in the detection of egg protein in jointing compound Wen Tianxiang in ancient times.
Background technology
Jointing compound, as a kind of functional material, is painted in print, is played an important role in the technique such as dyeing and finishing.Ancient times, the key component of cement materials was egg protein, therefore had important scientific value to the research of detection to cement materials of egg protein.In ancient cultural relic material, the method for protein detection of jointing compound mainly contains microchemistry experimental method, Raman spectroscopy.Microchemistry experimental method mainly utilizes the egg protein chemico-physical properties such as dissolubility and fusing point thereof in organic solvent to identify, this method is simple, but poor specificity.Raman spectroscopy has higher spatial resolution, can avoid the interference between different component.But ancient cultural relic material is of the remote past, for a long time by the impact of the environment such as illumination, oxidation, makes consolidating material complicated component, content very micro-, be 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 detection method providing egg protein in a kind of highly sensitive, simple jointing compound Wen Tianxiang in ancient times efficiently for above-mentioned prior art Problems existing.
The technical solution adopted in the present invention is: utilize sandwich enzyme-linked immunosorbent method to detect the egg protein composition in jointing compound Wen Tianxiang in ancient times, be attached to polystyrene surface of solid phase carriers by Millpore rabbit anti-egg protein polyclonal antibody and form insolubilized antibody, then combine with jointing compound historical relic eluent and form immune complex, add ELIAS secondary antibody again after washing, its antigen in immune complex is combined and forms enzyme labelled antibody-antigen-insolubilized antibody compound; After adding the substrate of enzyme reaction, substrate is become color products 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.
Concrete steps are 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 and constant volume 1000mL, regulates PH to 7.4,121 DEG C of sterilization treatment; The preparation of eluent: 5mL1mol/L trishydroxymethylaminomethane-HCl buffer solution, the edta solution of 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate, add deionized water dissolving and constant volume 500mL, regulate pH to 7.4 with NaOH, at room temperature store after aseptic suction filtration;
B) PBS buffer solution 80-100 μ L is set to blank, sample egg protein 0.1-0.5mg is set to positive control; The Wen Tianxiang of the PBS buffer solution of described 80-100 μ L, described 0.1-0.5mg sample egg protein, 0.1-0.5mg is dissolved in the eluent of 80-120 μ L respectively, ambient temperatare puts 2-4 days, forms blank control sample eluent, positive control sample egg protein eluent, laboratory sample Wen Tianxiang eluent;
C) primary antibodie and the Millpore rabbit anti-egg protein polyclonal antibody 80-100 μ L of coating buffer dilution 10000-12000 times is got, be added in each hole of polystyrene reactant plate, use preservative film shrouding, at temperature 4 DEG C, keep 24h, secondary daily PBS buffer solution fully washs 3 times, dries;
D) add 1% bovine serum albumin(BSA) (BSA) solution 100 μ L in each hole, close 1h, then carry out washing three times with PBS buffer solution, each three minutes;
E) the blank control sample eluent of the step b of 40 μ L-70 μ L, positive control sample egg protein sample eluent and laboratory sample Wen Tianxiang eluent is added in each hole, to put in 37 DEG C of incubators one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, dry;
F) add in each hole dilution 10000-12000 doubly between ELIAS secondary antibody 80-100 μ L, to put in the baking oven of 37 DEG C one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, and dry;
G) add the tetramethyl biphenyl amine aqueous solution 100 μ L of substrate solution 1% in each hole, put dark place and make it react 20min;
H) 2mol/LH is added in each hole
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) blank control sample and the absorption values OD both laboratory sample Wen Tianxiang is compared; Compared with the OD value detected with blank control sample, if egg protein test experience OD value obviously increases in historical relic eluent, prove really there is egg protein in laboratory sample Wen Tianxiang; Compared with the OD value detected with blank control sample, if egg protein test experience OD value is without marked change in historical relic eluent, prove to there is not egg protein in laboratory sample Wen Tianxiang.
The invention has the beneficial effects as follows: 1, the present invention utilizes the method for sandwich enzyme-linked immunosorbent to detect the egg protein composition in jointing compound Wen Tianxiang, highly sensitive, simple to operate on the one hand.On the other hand, the interference of other albumen is avoided, 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 immunosorbent method that the present invention adopts detects egg protein in ancient times jointing compound Wen Tianxiang, and highly sensitive, simple to operate on the one hand, on the other hand, high specificity, can avoid the interference of other albumen.Millpore rabbit anti-egg protein polyclonal antibody is attached to polystyrene surface of solid phase carriers and forms insolubilized antibody, then combine with jointing compound historical relic eluent and form immune complex, add ELIAS secondary antibody again after washing, its antigen in immune complex is combined and forms enzyme labelled antibody-antigen-insolubilized antibody compound.After adding the substrate of enzyme reaction, substrate is become color products 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, be indirectly exaggerated immunoreactive result, make test have 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 Wen Tianxiang, 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 and constant volume 1000mL, regulates PH to 7.4,121 DEG C of sterilization treatment.The preparation of eluent: 5mL1mol/L trishydroxymethylaminomethane-HCl buffer solution, the edta solution of 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate, add deionized water dissolving and be settled to 500mL, regulating pH to 7.4 with NaOH.At room temperature store after aseptic suction filtration.
B) PBS buffer solution 100 μ L is set to blank, sample egg protein 0.1mg is set to positive control.The Wen Tianxiang of the PBS buffer solution of described 100 μ L, described 0.1mg sample egg protein, 0.1mg is dissolved in the eluent of 100 μ L respectively, ambient temperatare puts 2 days, forms blank control sample eluent, positive control sample egg protein eluent, laboratory sample Wen Tianxiang eluent.
C) get primary antibodie (Millpore rabbit anti-egg protein polyclonal antibody) the 100 μ L that coating buffer dilutes 10000 times, be added in each hole of polystyrene reactant plate, use preservative film shrouding, inside 4 DEG C of refrigerators, keep 24h.Then fully wash 3 times with PBS buffer solution, dry.
D) add 1% bovine serum albumin(BSA) (BSA) solution 100 μ L in each hole, close 1h.Then washing is carried out three times with PBS buffer solution, each three minutes.
E) the blank control sample eluent of 40 μ L step b, positive control sample egg protein sample eluent and laboratory sample Wen Tianxiang eluent is added in each hole respectively, to put in 37 DEG C of incubators one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, dry.
F) add the ELIAS secondary antibody 100 μ L of dilution 10000 times in each hole, to put in 37 DEG C of incubators one hour.Remove liquid, with PBS buffer solution three times, each three minutes, and dry.
G) add the substrate solution tetramethyl biphenyl amine aqueous solution 100 μ L of 1% in each hole, put dark place and make it react 20min.
H) 2mol/LH is added in each hole
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) blank control sample and the absorption values OD both laboratory sample Wen Tianxiang is compared; Compared with the OD value detected with blank control sample, if egg protein test experience OD value obviously increases in historical relic eluent, prove really there is egg protein in laboratory sample Wen Tianxiang; Compared with the OD value detected with blank control sample, if egg protein test experience OD value is without marked change in historical relic eluent, prove to there is not egg protein in laboratory sample Wen Tianxiang.
Embodiment 2
A detection method for egg protein in ancient times jointing compound Wen Tianxiang, 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 and constant volume 1000mL, regulates PH to 7.4,121 DEG C of sterilization treatment.The preparation of eluent: the edta solution of 5mL1mol/L trishydroxymethylaminomethane-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 solution pH to 7.4.At room temperature store after aseptic suction filtration.
B) PBS buffer solution 100 μ L is set to blank, sample egg protein 0.1mg is set to positive control.The Wen Tianxiang of the PBS buffer solution of described 100 μ L, described 0.1mg sample egg protein, 0.3mg is dissolved in the eluent of 100 μ L respectively, ambient temperatare puts 3 days, forms blank control sample eluent, positive control sample egg protein eluent, laboratory sample Wen Tianxiang eluent.
C) get primary antibodie (Millpore rabbit anti-egg protein polyclonal antibody) the 100 μ L that coating buffer dilutes 11000 times, be added in each hole of polystyrene reactant plate, use preservative film shrouding, inside 4 DEG C of refrigerators, keep 24h.Then fully wash 3 times with PBS buffer solution, dry.
D) add 1% bovine serum albumin (BSA) solution 100 μ L in each hole, close 1h.Then washing is carried out three times with PBS buffer solution, each three minutes.
E) the blank control sample eluent of 50 μ L step b, positive control sample egg protein sample eluent and laboratory sample Wen Tianxiang eluent is added in each hole respectively, to put in 37 DEG C of incubators one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, dry.
F) add the ELIAS secondary antibody 100 μ L of dilution 11000 times in each hole, to put in 37 DEG C of incubators one hour.Remove liquid, wash three times with PBS buffer solution, each three minutes, and dry.
G) add the substrate solution tetramethyl biphenyl amine aqueous solution 100 μ L of 1% in each hole, put dark place and make it react 20min.
H) 2mol/LH is added in each hole
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) blank control sample and the absorption values OD both laboratory sample Wen Tianxiang is compared; Compared with the OD value detected with blank control sample, if egg protein test experience OD value obviously increases in historical relic eluent, prove really there is egg protein in laboratory sample Wen Tianxiang; Compared with the OD value detected with blank control sample, if egg protein test experience OD value is without marked change in historical relic eluent, prove to there is not egg protein in laboratory sample Wen Tianxiang.
Embodiment 3
A detection method for egg protein in ancient times jointing compound Wen Tianxiang, 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 and constant volume 1000mL, regulates PH to 7.4,121 DEG C of sterilization treatment.The preparation of eluent: the edta solution of 5mL1mol/L trishydroxymethylaminomethane-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, regulating pH to 7.4 with NaOH.At room temperature store after aseptic suction filtration.
B) PBS buffer solution 100 μ L is set to blank, sample egg protein 0.1mg is set to positive control.The PBS buffer solution of described 100 μ L, described 0.1mg sample egg protein, 0.5mg laboratory sample Wen Tianxiang are dissolved in 100 μ L eluents respectively, ambient temperatare puts 4 days, forms blank control sample eluent, positive control sample egg protein eluent, laboratory sample Wen Tianxiang eluent.
C) get primary antibodie (Millpore rabbit anti-egg protein polyclonal antibody) the 100 μ L that coating buffer dilutes 12000 times, be added in each hole of polystyrene reactant plate, use preservative film shrouding, inside 4 DEG C of refrigerators, keep 24h.Secondary daily PBS buffer solution fully washs 3 times, dries.
D) add 1% bovine serum albumin(BSA) (BSA) solution 100 μ L in each hole, close 1h.Then washing is carried out three times with PBS buffer solution, each three minutes.
E) the blank control sample eluent of 60 μ L step b, positive control sample egg protein sample eluent and laboratory sample Wen Tianxiang eluent is added in each hole respectively, to put in 37 DEG C of incubators one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, dry.
F) add the ELIAS secondary antibody 100 μ L of dilution 12000 times in each hole, to put in 37 DEG C of incubators one hour.Remove liquid, wash three times with PBS buffer solution, each three minutes, and dry.
G) add the substrate solution tetramethyl biphenyl amine aqueous solution 100 μ L of 1% in each hole, put dark place and make it react 20min.
H) 2mol/LH is added in each hole
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) blank control sample and the absorption values OD both laboratory sample Wen Tianxiang is compared; Compared with the OD value detected with blank control sample, if egg protein test experience OD value obviously increases in historical relic eluent, prove really there is egg protein in laboratory sample Wen Tianxiang; Compared with the OD value detected with blank control sample, if egg protein test experience OD value is without marked change in historical relic eluent, prove to there is not egg protein in laboratory sample Wen Tianxiang.
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 application claims.
Claims (1)
1. the detection method of egg protein in an ancient times jointing compound Wen Tianxiang, it is characterized in that utilizing sandwich enzyme-linked immunosorbent method to detect the egg protein composition in jointing compound Wen Tianxiang in ancient times, be attached to polystyrene surface of solid phase carriers by Millpore rabbit anti-egg protein polyclonal antibody and form insolubilized antibody, then combine with jointing compound historical relic eluent and form immune complex, add ELIAS secondary antibody again after washing, its antigen in immune complex is combined and forms enzyme labelled antibody-antigen-insolubilized antibody compound; After adding the substrate of enzyme reaction, substrate is become color products 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;
Concrete steps are 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 and constant volume 1000mL, regulates PH to 7.4,121 DEG C of sterilization treatment; The preparation of eluent: 5mL1mol/L trishydroxymethylaminomethane-HCl buffer solution, the edta solution of 1mL0.5mol/L, 180g urea, 25mL20% lauryl sodium sulfate, add deionized water dissolving and constant volume 500mL, regulate pH to 7.4 with NaOH, at room temperature store after aseptic suction filtration;
B) PBS buffer solution 80-100 μ L is set to blank, sample egg protein 0.1-0.5mg is set to positive control; The Wen Tianxiang of the PBS buffer solution of described 80-100 μ L, described 0.1-0.5mg sample egg protein, 0.1-0.5mg is dissolved in the eluent of 80-120 μ L respectively, ambient temperatare puts 2-4 days, forms blank control sample eluent, positive control sample egg protein eluent, laboratory sample Wen Tianxiang eluent;
C) primary antibodie and the Millpore rabbit anti-egg protein polyclonal antibody 80-100 μ L of coating buffer dilution 10000-12000 times is got, be added in each hole of polystyrene reactant plate, use preservative film shrouding, at temperature 4 DEG C, keep 24h, secondary daily PBS buffer solution fully washs 3 times, dries;
D) add 1% bovine serum albumin(BSA) (BSA) solution 100 μ L in each hole, close 1h, then carry out washing three times with PBS buffer solution, each three minutes;
E) the blank control sample eluent of the step b of 40 μ L-70 μ L, positive control sample egg protein sample eluent and laboratory sample Wen Tianxiang eluent is added in each hole, to put in 37 DEG C of incubators one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, dry;
F) add in each hole dilution 10000-12000 doubly between ELIAS secondary antibody 80-100 μ L, to put in the baking oven of 37 DEG C one hour, remove liquid, wash three times with PBS buffer solution, each three minutes, and dry;
G) add the tetramethyl biphenyl amine aqueous solution 100 μ L of substrate solution 1% in each hole, put dark place and make it react 20min;
H) 2mol/LH is added in each hole
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) blank control sample and the absorption values OD both laboratory sample Wen Tianxiang is compared; Compared with the OD value detected with blank control sample, if egg protein test experience OD value obviously increases in historical relic eluent, prove really there is egg protein in laboratory sample Wen Tianxiang; Compared with the OD value detected with blank control sample, if egg protein test experience OD value is without marked change in historical relic eluent, prove to there is not egg protein in laboratory sample Wen Tianxiang.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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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| Publication Number | Publication Date |
|---|---|
| CN103575907A CN103575907A (en) | 2014-02-12 |
| CN103575907B true CN103575907B (en) | 2015-12-02 |
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| EP0460102A4 (en) * | 1989-02-27 | 1992-01-15 | Biostar Medical Products Incorporated | 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 |
| AU2005218391B2 (en) * | 2004-03-05 | 2008-11-20 | Prima Meat Packers, Ltd. | Method of detecting allergen |
| FR2940977B1 (en) * | 2009-01-13 | 2016-11-18 | Oreal | USE OF COMPLEX FORMS OF THE CALMODULIN-LIKE SKIN PROTEIN CLSP PROTEIN |
| 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 |
| CN102539788B (en) * | 2012-01-13 | 2013-12-11 | 吉林大学 | Double-antibody sandwich ELISA (enzyme linked immuno-sorbent assay) test method for ovalbumin |
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