CN103575907B - The detection method of egg protein in a kind of ancient times jointing compound Wen Tianxiang - Google Patents

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

A detection method for egg protein in ancient cement cultural relic materials

technical field

The invention relates to a method for detecting egg protein in ancient cement cultural relic materials, which is mainly used for detecting egg protein in ancient cement cultural relic materials.

Background technique

As a functional material, binder plays an important role in printing, dyeing and finishing processes. The main component of ancient cement materials is egg protein, so the detection of egg protein has important scientific value for the study of cement materials. The protein detection methods of cementing agents in ancient cultural relic materials mainly include microchemical experiment method and Raman spectroscopy. The microchemical experiment mainly uses the chemical and physical properties of egg protein such as solubility in organic solvents and its melting point to identify. This method is simple and easy to implement, but its specificity is poor. Raman spectroscopy has high spatial resolution and can avoid interference between different components. However, ancient cultural relic materials have a long history and have been affected by light, oxidation and other environments for a long time, which makes the cementing materials complex and small in content, and it is difficult to analyze and characterize a single type of protein cementing material.

Contents of the invention

The technical problem to be solved by the present invention is to provide a high-sensitivity, simple and efficient detection method for egg protein in ancient cement cultural relic materials in view of the problems existing in the above-mentioned prior art.

The technical scheme adopted in the present invention is: use the sandwich enzyme-linked immunosorbent method to detect the egg protein component in the ancient cement cultural relic materials, that is, combine the Millpore rabbit anti-oval protein polyclonal antibody to the surface of the polystyrene solid phase carrier to form a solid phase antibody , and then combine with the eluent of cement cultural relics to form an immune complex, and then add the enzyme-labeled secondary antibody after washing, which combines with the antigen in the immune complex to form an enzyme-labeled antibody-antigen-solid-phase antibody complex; add the substrate of the enzyme reaction After the reaction, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is positively correlated with the amount of the test substance in the sample, so qualitative analysis can be performed according to the depth of the color.

Specific steps are as follows:

a) Solution preparation: preparation of PBS buffer solution: KCl 0.2g, KH ₂ PO ₄ 0.2g, NaCl 8.0g, NaH ₂ PO ₄ 7H ₂ O 2.16g, dissolved in deionized water and fixed to 1000mL, adjusted pH to 7.4, sterilized at 121°C; eluent preparation: 5mL 1mol/L tris-HCl buffer solution, 1mL 0.5mol/L EDTA solution, 180g urea, 25mL 20% dodecyl Sodium sulfate, add deionized water to dissolve and set the volume to 500mL, adjust the pH to 7.4 with NaOH, store at room temperature after sterile suction filtration;

b) Set 80-100 μL of PBS buffer solution as a blank control, and 0.1-0.5 mg of sample egg protein as a positive control; mg of cultural relic materials were dissolved in 80-120 μL eluent, and placed at room temperature for 2-4 days to form the eluent of the blank control sample, the eluate of the positive control sample egg protein, and the eluent of the cultural relic material of the experimental sample;

c) Take 80-100 μL of the primary antibody diluted 10,000-12,000 times in the coating solution, that is, Millpore rabbit anti-ovalbumin polyclonal antibody, and add it to each well of the polystyrene reaction plate, seal the plate with plastic wrap, and keep it at 4°C for 24 hours , the next day, fully wash 3 times with PBS buffer solution, and spin dry;

d) Add 100 μL of 1% bovine serum albumin (BSA) solution to each well, block for 1 hour, and then wash with PBS buffer solution three times, each time for three minutes;

e) Add 40μL-70μL of the blank control sample eluent, the positive control sample egg protein sample eluent and the experimental sample cultural relic material eluent of step b to each well, put it in a 37°C incubator for one hour, and remove the liquid , wash three times with PBS buffer solution, three minutes each time, and spin dry;

f) Add 80-100 μL of enzyme-labeled secondary antibody diluted 10000-12000 times to each well, place in an oven at 37°C for one hour, remove the liquid, wash with PBS buffer solution three times, three minutes each time, and spin dry ;

g) Add 100 μL of 1% tetramethylbenzidine solution in the substrate solution to each well, and let it react for 20 minutes in a dark place;

h) Add 0.05 mL of 2mol/L H ₂ SO ₄ solution to each well to terminate the reaction;

i) Take out the liquid in step h, add it to a microplate reader, and read the absorbance at 450nm;

j) Compare the absorbance value OD of the blank control sample and the experimental sample cultural relic material; compared with the OD value detected by the blank control sample, if the OD value of the egg protein detection experiment in the cultural relic eluate increases significantly, it proves that the experimental sample cultural relic material Oval protein does exist; compared with the OD value detected by the blank control sample, if there is no significant change in the OD value of the egg protein detection experiment in the cultural relic eluate, it proves that there is no egg protein in the cultural relic material of the experimental sample.

The beneficial effects of the present invention are as follows: 1. The present invention uses the method of sandwich ELISA to detect the egg protein component in the cement cultural relic material, on the one hand, it has high sensitivity and simple operation. On the other hand, it avoids the interference of other proteins and has strong specificity. 2. Compared with the prior art, the method adopted by the present invention has low cost, simple detection and fast response.

Detailed ways

The sandwich enzyme-linked immunosorbent method adopted in the present invention detects the egg protein in the ancient cement cultural relic material. On the one hand, it has high sensitivity and simple operation; on the other hand, it has strong specificity and can avoid the interference of other proteins. Combine Millpore rabbit anti-ovalin polyclonal antibody to the surface of polystyrene solid-phase carrier to form a solid-phase antibody, and then combine with the eluent of cement cultural relics to form an immune complex. After washing, add the enzyme-labeled secondary antibody, which is complexed with the immune The antigen in the substance is combined to form an enzyme-labeled antibody-antigen-solid-phase antibody complex. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is positively correlated with the amount of the tested substance in the sample, so qualitative analysis can be performed according to the depth of the color. Due to the high catalytic efficiency of the enzyme, the results of the immune response are indirectly amplified, so that the test has a high sensitivity.

The present invention will be further described below in conjunction with embodiment:

Example 1

A method for detecting egg protein in ancient cement cultural relic materials, comprising the steps of:

a) Solution preparation: preparation of PBS buffer solution: KCl 0.2g, KH ₂ PO ₄ 0.2g, NaCl 8.0g, NaH ₂ PO ₄ 7H ₂ O 2.16g, dissolved in deionized water and fixed to 1000mL, adjusted pH to 7.4, sterilized at 121°C. Preparation of eluent: 5mL1mol/L Tris-HCl buffer solution, 1mL0.5mol/L EDTA solution, 180g urea, 25mL20% sodium lauryl sulfate, add deionized water to dissolve And the volume was adjusted to 500mL, and the pH was adjusted to 7.4 with NaOH. Store at room temperature after sterile suction filtration.

b) 100 μL of PBS buffer solution was set as a blank control, and 0.1 mg of egg protein sample was set as a positive control. The 100 μL PBS buffer solution, the 0.1 mg sample egg protein, and the 0.1 mg cultural relic material were respectively dissolved in 100 μL eluent, and left at room temperature for 2 days to form a blank control sample eluate and a positive control sample egg. Protein eluent, experimental sample cultural relic material eluent.

c) Take 100 μL of the primary antibody (Millpore rabbit anti-egg protein polyclonal antibody) diluted 10,000 times in the coating solution, add it to each well of the polystyrene reaction plate, seal the plate with plastic wrap, and keep it in the refrigerator at 4°C for 24 hours. Then wash thoroughly with PBS buffer solution 3 times and spin dry.

d) Add 100 μL of 1% bovine serum albumin (BSA) solution to each well, and block for 1 h. Then wash with PBS buffer solution three times, each for three minutes.

e) Add 40 μL of the blank control sample eluent, the positive control sample egg protein sample eluent, and the experimental sample cultural relic material eluent of step b to each well, place in a 37°C incubator for one hour, remove the liquid, and use Wash with PBS buffer solution three times, each time for three minutes, and spin dry.

f) Add 100 μL of enzyme-labeled secondary antibody diluted 10,000 times to each well, and place in a 37°C incubator for one hour. Remove the liquid, wash three times with PBS buffer for three minutes each, and spin dry.

g) Add 100 μL of 1% substrate solution tetramethylbenzidine solution to each well, and place in a dark place to react for 20 minutes.

h) Add 0.05 mL of 2 mol/L H ₂ SO ₄ 0. solution to each well to terminate the reaction.

i) Take out the liquid in step h, add it to a microplate reader, and read the absorbance at 450nm

j) Compare the absorbance value OD of the blank control sample and the experimental sample cultural relic material; compared with the OD value detected by the blank control sample, if the OD value of the egg protein detection experiment in the cultural relic eluate increases significantly, it proves that the experimental sample cultural relic material Oval protein does exist; compared with the OD value detected by the blank control sample, if there is no significant change in the OD value of the egg protein detection experiment in the cultural relic eluate, it proves that there is no egg protein in the cultural relic material of the experimental sample.

Example 2

A method for detecting egg protein in ancient cement cultural relic materials, comprising the steps of:

a) Solution preparation: preparation of PBS buffer solution: KCl 0.2g, KH ₂ PO ₄ 0.2g, NaCl 8.0g, NaH ₂ PO ₄ 7H ₂ O 2.16g, dissolved in deionized water and fixed to 1000mL, adjusted pH to 7.4, sterilized at 121°C. Preparation of eluent: 5mL1mol/L Tris-HCl buffer solution, 1mL0.5mol/L EDTA solution, 180g urea, 25mL20% sodium lauryl sulfate (50mL deionized water contains 10 g of solute), add deionized water to dissolve and set the volume to 500 mL, and use NaOH solution to pH 7.4. Store at room temperature after sterile suction filtration.

b) 100 μL of PBS buffer solution was set as a blank control, and 0.1 mg of egg protein sample was set as a positive control. The 100 μL PBS buffer solution, the 0.1 mg sample egg protein, and the 0.3 mg cultural relic material were dissolved in 100 μL eluent respectively, and left at room temperature for 3 days to form a blank control sample eluate and a positive control sample egg. Protein eluent, experimental sample cultural relic material eluent.

c) Take 100 μL of the primary antibody (Millpore rabbit anti-egg protein polyclonal antibody) diluted 11,000 times in the coating solution, add it to each well of the polystyrene reaction plate, seal the plate with plastic wrap, and keep it in a refrigerator at 4°C for 24 hours. Then wash thoroughly with PBS buffer solution 3 times and spin dry.

d) 100 μL of 1% bovine serum albumin (BSA) solution was added to each well, and blocked for 1 h. Then wash with PBS buffer solution three times, each for three minutes.

e) Add 50 μL of the eluent of the blank control sample, the eluent of the positive control sample egg protein sample, and the eluent of the cultural relic material of the experimental sample in step b to each well, put it in a 37°C incubator for one hour, remove the liquid, and use Wash with PBS buffer solution three times, each time for three minutes, and spin dry.

f) Add 100 μL of enzyme-labeled secondary antibody diluted 11,000 times to each well, and place in a 37°C incubator for one hour. Remove the liquid, wash three times with PBS buffer solution for three minutes each, and spin dry.

g) Add 100 μL of 1% substrate solution tetramethylbenzidine solution to each well, and let it react for 20 minutes in a dark place.

h) Add 0.05 mL of 2mol/L H ₂ SO ₄ solution to each well to terminate the reaction.

i) Take out the liquid in step h, add it to a microplate reader, and read the absorbance at 450nm

j) Compare the absorbance value OD of the blank control sample and the experimental sample cultural relic material; compared with the OD value detected by the blank control sample, if the OD value of the egg protein detection experiment in the cultural relic eluate increases significantly, it proves that the experimental sample cultural relic material Oval protein does exist; compared with the OD value detected by the blank control sample, if there is no significant change in the OD value of the egg protein detection experiment in the cultural relic eluate, it proves that there is no egg protein in the cultural relic material of the experimental sample.

Example 3

A method for detecting egg protein in ancient cement cultural relic materials, comprising the steps of:

a) Solution preparation: preparation of PBS buffer solution: KCl 0.2g, KH ₂ PO ₄ 0.2g, NaCl 8.0g, NaH ₂ PO ₄ 7H ₂ O 2.16g, dissolved in deionized water and fixed to 1000mL, adjusted pH to 7.4, sterilized at 121°C. Preparation of eluent: 5mL1mol/L Tris-HCl buffer solution, 1mL0.5mol/L EDTA solution, 180g urea, 25mL20% sodium lauryl sulfate (50mL deionized water contains 10 g of solute), add deionized water to dissolve and adjust the volume to 500 mL, and adjust the pH to 7.4 with NaOH. Store at room temperature after sterile suction filtration.

b) 100 μL of PBS buffer solution was set as a blank control, and 0.1 mg of egg protein sample was set as a positive control. The 100 μL PBS buffer solution, the 0.1 mg sample egg protein, and the 0.5 mg experimental sample cultural relic material were respectively dissolved in 100 μL eluent, and left at room temperature for 4 days to form a blank control sample eluate and a positive control sample egg. Protein eluent, experimental sample cultural relic material eluent.

c) Take 100 μL of the primary antibody (Millpore rabbit anti-egg protein polyclonal antibody) diluted 12000 times in the coating solution, add it to each well of the polystyrene reaction plate, seal the plate with plastic wrap, and keep it in the refrigerator at 4°C for 24 hours. The next day, wash thoroughly with PBS buffer solution 3 times and spin dry.

d) Add 100 μL of 1% bovine serum albumin (BSA) solution to each well, and block for 1 h. Then wash with PBS buffer solution three times, each for three minutes.

e) Add 60 μL of the eluent of the blank control sample, the eluent of the positive control sample egg protein sample, and the eluent of the cultural relic material of the experimental sample in step b to each well, put it in a 37°C incubator for one hour, remove the liquid, and use Wash with PBS buffer solution three times, each time for three minutes, and spin dry.

f) Add 100 μL of enzyme-labeled secondary antibody diluted 12,000 times to each well, and place in a 37°C incubator for one hour. Remove the liquid, wash three times with PBS buffer solution for three minutes each, and spin dry.

g) Add 100 μL of 1% substrate solution tetramethylbenzidine solution to each well, and let it react for 20 minutes in a dark place.

h) Add 0.05 mL of 2mol/L H ₂ SO ₄ solution to each well to terminate the reaction.

i) Take out the liquid in step h, add it to a microplate reader, and read the absorbance at 450nm

j) Compare the absorbance value OD of the blank control sample and the experimental sample cultural relic material; compared with the OD value detected by the blank control sample, if the OD value of the egg protein detection experiment in the cultural relic eluate increases significantly, it proves that the experimental sample cultural relic material Oval protein does exist; compared with the OD value detected by the blank control sample, if there is no significant change in the OD value of the egg protein detection experiment in the cultural relic eluate, it proves that there is no egg protein in the cultural relic material of the experimental sample.

In addition to the above-mentioned embodiments, the present invention can also have other implementations. All technical solutions formed by equivalent replacement or equivalent transformation fall within the scope of protection required by the present invention.

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.