CN108680405A - A kind of method of alkali aging mulberry silk simulation silk relics sample - Google Patents
A kind of method of alkali aging mulberry silk simulation silk relics sample Download PDFInfo
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Abstract
Disclosed by the invention is a kind of method of alkali aging mulberry silk simulation silk relics sample, belong to historical relic simulation field, aging is carried out to mulberry silk using sodium hydroxide, PH is adjusted to neutrality by alkali aging after terminating, solution is filtered, dialysis, freeze-drying, after the operations such as grinding, indirect ELISA experiment is carried out using the powder of grinding gained, the alkali aging sample closest to historical relic sample has been filtered out according to experimental result, the sample is when being from now on detected unknown silk relics sample using different antibodies, it can be as the replacement sample of historical relic sample, screening for antibody optimum diluting multiple and antibody specificity, the experiments such as sensitivity test, historical relic sample is solved to a certain extent a large amount of different, repeat the problem of sample deficiency in testing.
Description
Technical field
The present invention relates to a kind of method of simulation silk relics sample more particularly to a kind of alkali aging mulberry silk simulation silk texts
The method of object sample belongs to historical relic simulation field.
Background technology
Mulberry silk is a kind of natural polymer protein fibre, and China is the country of earliest sericulture filature knit silk fabric, but silkworm
The Origin of silk shrouds in dense fog always.Solve the problems, such as that China mulberry silk origin is not only to explore Chinese civilization origin
Needs, and be also to provide the evidence more convinced for " China be generally acknowledged in the world silk area of origin " this saying.Mulberry
The common detection methods of silk mainly have FTIR spectrum, Raman spectrum, X-ray diffraction etc., but mulberry silk is chronically at
Can be influenced by factors in grave or ruins environment, to the problems such as protein degradation and macromolecular chain are broken occur, adopt
It is difficult to detect the presence of fibroin albumen with traditional certain methods.The advanced technology of current protein detection is to be based on antibody-
The ELISA method of antigen, there has been the polyclonal antibodies of Bombyx mori silk fibroin, can detect the smaller mulberry silk of content
Fibroin.But mainly based on argillization silk goods, this kind fabric has often been degraded to unearthed silk goods manifestation mode at present
Short chain fiber or peptide fragment, or even the small molecules such as amino acid are degraded to, their microscratch mark remnants is in soil.And laboratory energy
The soil sample of acquisition generally only has several grams, and the fibroin albumen extracted from these argillization samples can only often meet one twice
ELISA is tested.
Invention content
In order to solve the above technical problem, the present invention provides the sides that a kind of alkali aging mulberry silk simulates silk relics sample
Method, the method for the present invention carry out alkali aging to mulberry silk using sodium hydroxide, have filtered out the alkali aging sample closest to historical relic sample.It should
Sample can be used for when being detected from now on to unknown silk relics sample using different antibodies as the replacement sample of historical relic sample
The experiments such as the screening of antibody optimum diluting multiple and antibody specificity, sensitivity test solve historical relic sample and exist to a certain extent
A large amount of problems that are different, repeating sample deficiency in testing.
To achieve the goals above, the present invention is achieved by the following technical solutions：
A kind of method of alkali aging mulberry silk simulation silk relics sample, including following preparation process：
A it) takes 10-15g mulberry silk raw silks to be placed in beaker, the Na of 1000-1500ml a concentration of 0.5% is added2CO3Solution,
30min is heated in 94-98 DEG C of water-bath, outwells the solution in beaker, and repeats an aforesaid operations, it then will be in beaker
Remaining mulberry silk is rinsed 3 times with deionized water, is placed in 60 DEG C of baking oven and is dried for standby；
B 1-1.5g step A) are taken) obtained by mulberry silk be placed in beaker, after shredding be added 100-150ml it is a concentration of
The NaOH solution of 2.5mol/L is placed at 25 DEG C on 10 revolutions per seconds of magnetic stirring apparatus, and after at the uniform velocity stirring 1 hour, ice vinegar is added
Acid adjusts PH to 7；
C) by step B) obtained by solution filtering, with molecule interception be 8000 bag filter dialyse, it is small every 4-6
When change a water, after dialysis 3 days, surplus solution in bag filter is freeze-dried and is ground, the mulberry silk of alkali aging 1 hour is obtained
Fibroin powder；
D) repeat step B), step C), prepare alkali aging respectively 3 hours, 6 hours, 9 hours, 12 hours and 24
The Bombyx mori silk fibroin powder of hour；
E 1-2mg step C) are taken respectively), step D) obtained by Bombyx mori silk fibroin powder and ground silk text
Object sample, is respectively put into the centrifuge tube of 15ml, and the carbonate buffer solution that 10mlPH is 9.6 is added in every centrifuge tube,
Clear coating buffer is obtained after concussion uniformly；
F) take one piece of 96 hole elisa Plates, 100 μ l step E be added per hole) in gained coating buffer, each column in the ELISA Plate
Add five holes, which is added same coating buffer, different coating buffers is used between the column and the column, respectively according to sky
White control, negative control, positive control, alkali aging 1 hour, alkali aging 3 hours, alkali aging 6 hours, alkali aging 9 hours, alkali is old
Change 12 hours, alkali aging 24 hours, the sequence of silk relics sample completes fabric swatch；
G) to step F) in 96 orifice plates that arrange carry out indirect enzyme-linked immunosorbent test, the specific steps are：It is added per hole
The phosphate buffer solution of the pH7.4 of 200 μ l is washed, 2 minutes every time, is washed altogether 3 times, is then added 200 μ l's per hole
The bovine serum albumen solution of 1wt% is incubated 2h at 37 DEG C, carries out Seal treatment；The phosphate of the pH7.4 of 200 μ l is added per hole
Buffer solution is washed, 2 minutes every time, altogether washing 3 times, then the rabbit-anti fibroin albumen primary antibody of 100 μ l of every hole addition, 37 DEG C
Lower incubation 1h；The phosphate buffer solution that the pH7.4 of 200 μ l is added per hole is washed, 2 minutes every time, altogether washing 3 times, so
The goat anti-rabbit antibodies of the HRP labels of 100 μ l are added per hole afterwards, 1h is incubated at 37 DEG C；The pH7.4 bufferings of 200 μ l are added per hole
Solution is washed, 2 minutes every time, is washed altogether 5 times, and the agent A liquid of the TMB colour developings of 50 μ l is then added per hole, adds 50 μ l
TMB color developing agents B liquid, rock uniformly, be protected from light 10min, then per hole be added 100 μ l H2SO4Terminate reaction, test
Its absorbance at 450nm, is denoted as OD450nm。
As an improvement step E) described in PH9.6 carbonate buffer solution, preparation method is：It weighs
1.5g sodium carbonate and 2.9g sodium bicarbonates, are added in 800ml deionized waters, are then mixed until whole dissolvings, use
The volumetric flask of 1000ml carries out constant volume, and the pH for adjusting solution is 9.6.
As an improvement the blank control is to replace rabbit-anti fibroin albumen using the bovine serum albumen solution of 1wt%
Primary antibody is added, and the negative control is to be coated with using carbonate buffer solution, and the positive control is to use unaged silk
The solution that fibroin is matched is coated with.
As an improvement step G) described in the preparation method of phosphate buffer solution of pH7.4 be：It weighs
0.27g potassium dihydrogen phosphates, 1.42g disodium hydrogen phosphates, 8g sodium chloride and 0.2g potassium chloride, are added in 800mL deionized waters, so
After be mixed until whole dissolvings, carry out constant volume with the volumetric flask of 1000mL, adjust solution pH be 7.4.
It is compared with the prior art beneficial effects of the present invention：
1) it uses sodium hydroxide to carry out aging to mulberry silk and reaches identical aging compared with traditional hydrothermal aging method
Greatly shorten the time required to degree, from being reduced to a few hours several moons, and alkali aging terminate after PH is adjusted to neutrality at once,
Ageing time is accurately controlled, if not adjusting PH, will appear the case where sample is by secondary aging in dialysis procedure, make the standard of experiment
True property and convincingness decline, while at the uniform velocity being stirred using magnetic stirring apparatus in alkali ageing process, and the mulberry silk after degumming is first
It shreds and adds sodium hydroxide solution, the degree that sample is mutually wound in whipping process can be reduced, while improving aging
Uniformity coefficient is susceptible to sample aging non-uniform phenomenon if not shredding and stirring, make repeat experimental result deviation compared with
Greatly；
2) the silk relics sample used has been accredited out from Light of Mulberry Silk Fabrics, and related experiment is completed, and is residual
The historical relic sample stayed, utilizes this residual sample again, has fully excavated the surplus value of sample；
3) the alkali aging sample closest to historical relic sample is selected, which utilizes different antibodies to unknown silk relics from now on
, can be as the replacement sample of historical relic sample when sample is detected, the screening for antibody optimum diluting multiple and antibody specificity, spirit
The experiments such as sensitivity test solve the problems, such as that historical relic sample sample in a large amount of different, repetitions are tested is insufficient to a certain extent.
Description of the drawings
Fig. 1 is indirect enzyme-linked immunosorbent test result statistical chart of the present invention.
Specific implementation mode
Below by way of specific embodiment, the present invention is further illustrated, but embodiments of the present invention not by
The limitation of following embodiment.
It is as shown in Figure 1 the result statistical chart of indirect enzyme-linked immunosorbent of the present invention test, wherein HAPJ0449 is historical relic sample
Data select replacement of the sample of alkali aging 4 hours or so as historical relic sample according to indirect enzyme-linked immunosorbent test result statistical chart
Sample is best, can carry out related experiment.
Embodiment 1
A it) takes 10g mulberry silks raw silk in beaker, the 0.5%Na2CO3 solution of 1000ml is added, is heated in 94 DEG C of water-baths
30min outwells the solution in beaker, repeats an aforesaid operations, and remaining mulberry silk deionized water is rinsed 3 times,
It is placed in 60 DEG C of baking oven and is dried for standby；
B 1g step A) are taken) obtained by mulberry silk in beaker, the NaOH that a concentration of 2.5mol/L of 100ml are added after shredding is molten
Liquid is placed at 25 DEG C on 10 revolutions per seconds of magnetic stirring apparatus, after at the uniform velocity stirring 1 hour, glacial acetic acid is added and adjusts PH to 7；
C) by step B) obtained by solution filtering, with molecule interception be 8000 bag filter dialyse, every 4 hours
A water is changed, after dialysing 3 days, surplus solution in bag filter is freeze-dried and is ground, the Bombyx mori Silk Fibroin of alkali aging 1 hour is obtained
Protein powder；
D) according to step B) and step C) described, continue to prepare alkali aging respectively 3 hours, 6 hours, 9 hours, 12 hours,
24 hours Bombyx mori silk fibroin powder, the time during adding alkali to PH neutralizations to be 7 is alkali ageing time；
E 1mg step C) are taken respectively), step D) obtained by Bombyx mori silk fibroin powder and ground silk relics
Sample is respectively put into and is put into 15ml centrifuge tubes, and the carbonate buffer solution that 10ml PH are 9.6 is added in every centrifuge tube, shake
Clear coating buffer is obtained after swinging uniformly, silk relics sample used is provided by Zhejiang silk museum, which is reflected
It makes and comes from Light of Mulberry Silk Fabrics, and related experiment is completed, be remaining historical relic sample, the carbonate of the PH9.6 is slow
Solution is rushed, preparation method is：1.5g sodium carbonate and 2.9g sodium bicarbonates are weighed, is added in 800ml deionized waters, then mixes
Stirring is closed until whole dissolvings, constant volume is carried out with the volumetric flask of 1000ml, the pH for adjusting solution is 9.6；
F) take one piece of 96 hole elisa Plates, 100 μ l step E be added per hole) in gained coating buffer, each column adds five holes, this five
Same coating buffer is added in a hole, uses different coating buffers between the column and the column, and according to blank control, negative control is positive right
According to, alkali aging 1 hour, alkali aging 3 hours, alkali aging 6 hours, alkali aging 9 hours, alkali aging 12 hours, alkali aging 24 hours,
The sequence of silk relics sample completes fabric swatch, and the blank control is to replace rabbit-anti silk using the bovine serum albumen solution of 1wt%
Fibroin primary antibody is added, and negative control is to be coated with using carbonate buffer solution, and positive control is to use unaged fibroin
The solution that albumen is matched is coated with；
G) to step F) in 96 orifice plates that arrange carry out indirect enzyme-linked immunosorbent test, the specific steps are：It is added per hole
The phosphate buffer solution of the pH 7.4 of 200 μ l is washed, 2 minutes every time, is washed altogether 3 times, is then added 200 μ l's per hole
The bovine serum albumen solution of 1wt% is incubated 2h at 37 DEG C, carries out Seal treatment；The phosphate of the pH 7.4 of 200 μ l is added per hole
Buffer solution is washed, 2 minutes every time, altogether washing 3 times, then the rabbit-anti fibroin albumen primary antibody of 100 μ l of every hole addition, 37 DEG C
Lower incubation 1h；The phosphate buffer solution that the pH 7.4 of 200 μ l is added per hole is washed, 2 minutes every time, altogether washing 3 times, so
The goat anti-rabbit antibodies of the HRP labels of 100 μ l are added per hole afterwards, 1h is incubated at 37 DEG C；The pH 7.4 that 200 μ l are added per hole is buffered
Solution is washed, 2 minutes every time, is washed altogether 5 times, and the agent A liquid of the TMB colour developings of 50 μ l is then added per hole, adds 50 μ l
TMB color developing agents B liquid, rock uniformly, be protected from light 10min, then per hole be added 100 μ l H2SO4 terminate reaction.Test
Its absorbance at 450nm, is denoted as OD450nm, and the indirect enzyme-linked immunosorbent test result selects the alkali closest to historical relic sample
Ageing time, as the reference of later simulation silk relics sample, the preparation method of the phosphate buffer solution of the pH7.4
For：0.27g potassium dihydrogen phosphates, 1.42g disodium hydrogen phosphates, 8g sodium chloride and 0.2g potassium chloride are weighed, 800mL deionizations are added to
It in water, is then mixed until whole dissolvings, constant volume is carried out with the volumetric flask of 1000mL, the pH for adjusting solution is 7.4.
Embodiment 2
A it) takes 13g mulberry silks raw silk in beaker, the 0.5%Na2CO3 solution of 1300ml is added, is heated in 96 DEG C of water-baths
30min outwells the solution in beaker, repeats an aforesaid operations.Remaining mulberry silk deionized water is rinsed 3 times,
It is placed in 60 DEG C of baking oven and is dried for standby；
B 1.3g step A) are taken) obtained by mulberry silk in beaker, the NaOH of a concentration of 2.5mol/L of 130ml is added after shredding
Solution is placed at 25 DEG C on 10 revolutions per seconds of magnetic stirring apparatus, after at the uniform velocity stirring 1 hour, glacial acetic acid is added and adjusts PH to 7；
C) by step B) obtained by solution filtering, with molecule interception be 8000 bag filter dialyse, every 5 hours
A water is changed, after dialysing 3 days, surplus solution in bag filter is freeze-dried and is ground, the Bombyx mori Silk Fibroin of alkali aging 1 hour is obtained
Protein powder；
D) according to step B) and step C) described, continue to prepare alkali aging respectively 3 hours, 6 hours, 9 hours, 12 hours,
24 hours Bombyx mori silk fibroin powder, the time during adding alkali to PH neutralizations to be 7 is alkali ageing time；
E 1.5mg step C) are taken respectively), step D) obtained by Bombyx mori silk fibroin powder and ground silk text
Object sample is distinguished in 15ml centrifuge tubes, and the carbonate buffer solution that 10ml PH are 9.6 is added in every centrifuge tube, and concussion is uniform
After obtain clear coating buffer, silk relics sample used is provided by Zhejiang silk museum, the historical relic sample be accredited out
From Light of Mulberry Silk Fabrics, and related experiment is completed, be remaining historical relic sample, the carbonate buffer solution of the PH9.6,
Its preparation method is：1.5g sodium carbonate and 2.9g sodium bicarbonates are weighed, is added in 800ml deionized waters, is then mixed
Until whole dissolvings, constant volume is carried out with the volumetric flask of 1000ml, the pH for adjusting solution is 9.6；
F) take one piece of 96 hole elisa Plates, 100 μ l step E be added per hole) in gained coating buffer, each column adds five holes, this five
Same coating buffer is added in a hole, uses different coating buffers between the column and the column, and according to blank control, negative control is positive right
According to, alkali aging 1 hour, alkali aging 3 hours, alkali aging 6 hours, alkali aging 9 hours, alkali aging 12 hours, alkali aging 24 hours,
The sequence of silk relics sample completes fabric swatch, and the blank control is to replace rabbit-anti silk using the bovine serum albumen solution of 1wt%
Fibroin primary antibody is added, and negative control is to be coated with using carbonate buffer solution, and positive control is to use unaged fibroin
The solution that albumen is matched is coated with；
G) to step F) in 96 orifice plates that arrange carry out indirect enzyme-linked immunosorbent test, the specific steps are：It is added per hole
The phosphate buffer solution of the pH 7.4 of 200 μ l is washed, 2 minutes every time, is washed altogether 3 times, is then added 200 μ l's per hole
The bovine serum albumen solution of 1wt% is incubated 2h at 37 DEG C, carries out Seal treatment；The phosphate of the pH 7.4 of 200 μ l is added per hole
Buffer solution is washed, 2 minutes every time, altogether washing 3 times, then the rabbit-anti fibroin albumen primary antibody of 100 μ l of every hole addition, 37 DEG C
Lower incubation 1h；The phosphate buffer solution that the pH 7.4 of 200 μ l is added per hole is washed, 2 minutes every time, altogether washing 3 times, so
The goat anti-rabbit antibodies of the HRP labels of 100 μ l are added per hole afterwards, 1h is incubated at 37 DEG C；The pH 7.4 that 200 μ l are added per hole is buffered
Solution is washed, 2 minutes every time, is washed altogether 5 times, and the agent A liquid of the TMB colour developings of 50 μ l is then added per hole, adds 50 μ l
TMB color developing agents B liquid, rock uniformly, be protected from light 10min, then per hole be added 100 μ l H2SO4 terminate reaction.Test
Its absorbance at 450nm, is denoted as OD450nm, and the indirect enzyme-linked immunosorbent test result selects the alkali closest to historical relic sample
Ageing time, as the reference of later simulation silk relics sample, the preparation method of the phosphate buffer solution of the pH7.4
For：0.27g potassium dihydrogen phosphates, 1.42g disodium hydrogen phosphates, 8g sodium chloride and 0.2g potassium chloride are weighed, 800mL deionizations are added to
It in water, is then mixed until whole dissolvings, constant volume is carried out with the volumetric flask of 1000mL, the pH for adjusting solution is 7.4.
Embodiment 3
A it) takes 15g mulberry silks raw silk in beaker, the 0.5%Na2CO3 solution of 1500ml is added, is heated in 98 DEG C of water-baths
30min outwells the solution in beaker, repeats an aforesaid operations.Remaining mulberry silk deionized water is rinsed 3 times,
It is placed in 60 DEG C of baking oven and is dried for standby；
B 1.5g step A) are taken) obtained by mulberry silk in beaker, the NaOH of a concentration of 2.5mol/L of 150ml is added after shredding
Solution is placed at 25 DEG C on 10 revolutions per seconds of magnetic stirring apparatus, after at the uniform velocity stirring 1 hour, glacial acetic acid is added and adjusts PH to 7；
C) by step B) obtained by solution filtering, with molecule interception be 8000 bag filter dialyse, every 6 hours
A water is changed, after dialysing 3 days, surplus solution in bag filter is freeze-dried and is ground, the Bombyx mori Silk Fibroin of alkali aging 1 hour is obtained
Protein powder；
D) according to step B) and step C) described, continue to prepare alkali aging respectively 3 hours, 6 hours, 9 hours, 12 hours,
24 hours Bombyx mori silk fibroin powder, the time during adding alkali to PH neutralizations to be 7 is alkali ageing time；
E 2mg step C) are taken respectively), step D) obtained by Bombyx mori silk fibroin powder and ground silk relics
Sample is respectively put into 15ml centrifuge tubes, and the carbonate buffer solution that 10ml PH are 9.6 is added in every centrifuge tube, and concussion is equal
Clear coating buffer is obtained after even, silk relics sample used is provided by Zhejiang silk museum, which is accredited out
From Light of Mulberry Silk Fabrics, and related experiment is completed, and is remaining historical relic sample, and the carbonate buffer of the PH9.6 is molten
Liquid, preparation method are：1.5g sodium carbonate and 2.9g sodium bicarbonates are weighed, is added in 800ml deionized waters, then mixing is stirred
It mixes until whole dissolvings, constant volume is carried out with the volumetric flask of 1000ml, the pH for adjusting solution is 9.6；
F) take one piece of 96 hole elisa Plates, 100 μ l step E be added per hole) in gained coating buffer, each column adds five holes, this five
Same coating buffer is added in a hole, uses different coating buffers between the column and the column, and according to blank control, negative control is positive right
According to, alkali aging 1 hour, alkali aging 3 hours, alkali aging 6 hours, alkali aging 9 hours, alkali aging 12 hours, alkali aging 24 hours,
The sequence of silk relics sample completes fabric swatch, and the blank control is to replace rabbit-anti silk using the bovine serum albumen solution of 1wt%
Fibroin primary antibody is added, and negative control is to be coated with using carbonate buffer solution, and positive control is to use unaged fibroin
The solution that albumen is matched is coated with；
G) to step F) in 96 orifice plates that arrange carry out indirect enzyme-linked immunosorbent test, the specific steps are：It is added per hole
The phosphate buffer solution of the pH 7.4 of 200 μ l is washed, 2 minutes every time, is washed altogether 3 times, is then added 200 μ l's per hole
The bovine serum albumen solution of 1wt% is incubated 2h at 37 DEG C, carries out Seal treatment；The phosphate of the pH 7.4 of 200 μ l is added per hole
Buffer solution is washed, 2 minutes every time, altogether washing 3 times, then the rabbit-anti fibroin albumen primary antibody of 100 μ l of every hole addition, 37 DEG C
Lower incubation 1h；The phosphate buffer solution that the pH 7.4 of 200 μ l is added per hole is washed, 2 minutes every time, altogether washing 3 times, so
The goat anti-rabbit antibodies of the HRP labels of 100 μ l are added per hole afterwards, 1h is incubated at 37 DEG C；The pH 7.4 that 200 μ l are added per hole is buffered
Solution is washed, 2 minutes every time, is washed altogether 5 times, and the agent A liquid of the TMB colour developings of 50 μ l is then added per hole, adds 50 μ l
TMB color developing agents B liquid, rock uniformly, be protected from light 10min, then per hole be added 100 μ l H2SO4 terminate reaction, test
Its absorbance at 450nm, is denoted as OD450nm, and the indirect enzyme-linked immunosorbent test result selects the alkali closest to historical relic sample
Ageing time, as the reference of later simulation silk relics sample, the preparation method of the phosphate buffer solution of the pH7.4
For：0.27g potassium dihydrogen phosphates, 1.42g disodium hydrogen phosphates, 8g sodium chloride and 0.2g potassium chloride are weighed, 800mL deionizations are added to
It in water, is then mixed until whole dissolvings, constant volume is carried out with the volumetric flask of 1000mL, the pH for adjusting solution is 7.4.
Raw materials used in the present invention, equipment is unless otherwise noted the common raw material, equipment of this field；In the present invention
Method therefor is unless otherwise noted the conventional method of this field.
The above is only presently preferred embodiments of the present invention, is not imposed any restrictions to the present invention, every according to the present invention
Technical spirit still falls within the technology of the present invention side to any simple modification, change and equivalent transformation made by above example
The protection domain of case.
Claims (4)
1. a kind of method of alkali aging mulberry silk simulation silk relics sample, it is characterised in that including following preparation process：
A it) takes 10-15g mulberry silk raw silks to be placed in beaker, the Na of 1000-1500ml a concentration of 0.5% is added2CO3Solution,
30min is heated in 94-98 DEG C of water-bath, outwells the solution in beaker, and repeats an aforesaid operations, then will be remained in beaker
Remaining mulberry silk is rinsed 3 times with deionized water, is placed in 60 DEG C of baking oven and is dried for standby；
B 1-1.5g step A) are taken) obtained by mulberry silk be placed in beaker, 100-150ml a concentration of 2.5mol/L are added after shredding
NaOH solution, be placed at 25 DEG C on 10 revolutions per seconds of magnetic stirring apparatus, at the uniform velocity stir 1 hour after, be added glacial acetic acid adjust PH
To 7；
C) by step B) obtained by solution filtering, with molecule interception be 8000 bag filter dialyse, changed every 4-6 hours
Surplus solution in bag filter is freeze-dried and is ground, obtain the Bombyx mori Silk Fibroin egg of alkali aging 1 hour after dialysing 3 days by water
White powder end；
D) repeat step B), step C), prepare alkali aging respectively 3 hours, 6 hours, 9 hours, 12 hours and 24 hours
Bombyx mori silk fibroin powder；
E 1-2mg step C) are taken respectively), step D) obtained by Bombyx mori silk fibroin powder and ground silk relics sample,
It is respectively put into the centrifuge tube of 15ml, the carbonate buffer solution that 10mlPH is 9.6 is added in every centrifuge tube, concussion is equal
Clear coating buffer is obtained after even；
F) take one piece of 96 hole elisa Plates, 100 μ l step E be added per hole) in gained coating buffer, each column in the ELISA Plate adds five
A hole, five Kong Zhongjun are added same coating buffer, different coating buffers are used between the column and the column, respectively according to blank pair
According to, negative control, positive control, alkali aging 1 hour, alkali aging 3 hours, alkali aging 6 hours, alkali aging 9 hours, alkali aging 12
Hour, the sequence of alkali aging 24 hours, silk relics sample completes fabric swatch；
G) to step F) in 96 orifice plates that arrange carry out indirect enzyme-linked immunosorbent test, the specific steps are：200 μ l are added per hole
The phosphate buffer solution of pH7.4 washed, 2 minutes every time, wash altogether 3 times, the 1wt% of 200 μ l be then added per hole
Bovine serum albumen solution, 2h is incubated at 37 DEG C, carries out Seal treatment；The phosphate-buffered that the pH7.4 of 200 μ l is added per hole is molten
Liquid is washed, 2 minutes every time, is washed altogether 3 times, and the rabbit-anti fibroin albumen primary antibody of 100 μ l is then added per hole, is incubated at 37 DEG C
1h；The phosphate buffer solution that the pH7.4 of 200 μ l is added per hole is washed, 2 minutes every time, is washed altogether 3 times, then per hole
The goat anti-rabbit antibodies of the HRP labels of 100 μ l are added, 1h is incubated at 37 DEG C；Per hole be added 200 μ l pH7.4 buffer solutions into
Row washing 2 minutes every time, is washed 5 times altogether, and the agent A liquid of the TMB colour developings of 50 μ l is then added per hole, and the TMB for adding 50 μ l is aobvious
The B liquid of toner rocks uniformly, is protected from light 10min, and the H of 100 μ l is then added per hole2SO4Terminate reaction, test its
Absorbance at 450nm, is denoted as OD450nm。
2. a kind of method of alkali aging mulberry silk simulation silk relics sample as described in claim 1, which is characterized in that step E)
Described in PH9.6 carbonate buffer solution, preparation method is：1.5g sodium carbonate and 2.9g sodium bicarbonates are weighed, is added
It into 800ml deionized waters, is then mixed until whole dissolvings, carries out constant volume with the volumetric flask of 1000ml, adjust solution
PH be 9.6.
3. a kind of method of alkali aging mulberry silk simulation silk relics sample as described in claim 1, which is characterized in that the sky
To be added instead of rabbit-anti fibroin albumen primary antibody using the bovine serum albumen solution of 1wt%, the negative control is to use for white control
Carbonate buffer solution is coated with, and the positive control is that the solution matched using unaged fibroin albumen is coated with.
4. a kind of method of alkali aging mulberry silk simulation silk relics sample as described in claim 1, which is characterized in that step G)
Described in the preparation method of phosphate buffer solution of pH7.4 be：Weigh 0.27g potassium dihydrogen phosphates, 1.42g phosphoric acid hydrogen two
Sodium, 8g sodium chloride and 0.2g potassium chloride, are added in 800mL deionized waters, are then mixed until whole dissolvings, use
The volumetric flask of 1000mL carries out constant volume, and the pH for adjusting solution is 7.4.
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