CN108387435A - A kind of trace fibroin albumen enrichment method - Google Patents
A kind of trace fibroin albumen enrichment method Download PDFInfo
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- CN108387435A CN108387435A CN201810086592.1A CN201810086592A CN108387435A CN 108387435 A CN108387435 A CN 108387435A CN 201810086592 A CN201810086592 A CN 201810086592A CN 108387435 A CN108387435 A CN 108387435A
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Abstract
The invention discloses a kind of trace fibroin albumen enrichment methods, include the following steps：1）The mixing of different fibroin albumen monoclonal antibodies；2）Coupled reaction；3）The acquisition of immunomagnetic beads；4）Identification；5）Couple the fibroin albumen monoclonal antibody immobilization of rear immunomagnetic beads；6）The combination of fibroin albumen monoclonal antibody immunity magnetic bead and fibroin albumen；7）The elution of fibroin albumen；8）Detection；9）Mark-on reclaims.The present invention prepares immunomagnetic beads using the compound monoclonal antibody of fibroin albumen, and utilize this immunomagnetic beads enriched with trace fibroin albumen, the extraction of trace fibroin albumen in archaeology soil sample can be preferably carried out, has that extraction different decomposition segment, bioaccumulation efficiency be high, easy to operate, experiment takes the various aspects advantage such as short.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of trace fibroin albumen enrichment method.
Background technology
Important invention one of of the silk as Chinese civilization, carries extremely rich culture, technology and society intension, is
The research of archaeology, history, the history of technique provides precious data.The silk relic being unearthed shows that the origin of silk should not
Before being later than 5500.However it is necessarily required to develop for a long time to exquisite silk is weaved into from silk fiber is found.Cause
This, before our ancestors begin to use time of silk fiber far earlier than 5500.But the Origin of silk shrouds always
In dense fog.How using the means of science to determine that silk originates from and is originated from China, there is highly important history and cultural valence
Value.
Silk is made by silk, and silk is by the natural protein fibre of 18 kinds of Amino acid profiles.Very long history into
Cheng Zhong, the silk goods that current year is embedded in grave or ruins lose entity looks, are degraded into peptide fragment and amino acid, Huo Zhecheng already
For trace, or soil is dissolved into, can not be recognized under naked eyes, and age more early evidence is more difficult to seek.How in silk
Macroscopic form seeks evidence earlier in the case of disappearing become the critical issue of research early stage silk.Relative to complete silk
Fabric, invisible fiber fibroin albumen residue more likely preserve, it is also possible to as early stage silk remnants is sought
Fresh evidence.
In the greatly long time up to centuries or even thousands of years, remaining silk content is very little in material of engaging in archaeological studies,
Therefore it carries out trace fibroin albumen in archaeology material before being detected sample to be enriched with, to improve the accurate of detection
Property, while the operational easy and time-consuming few problem considered again.
The detection of progress trace fibroin albumen at present is to utilize antigen-antibody there are many method, wherein ELISA detection method
Specific binding be detected, be the state-of-the-art technology of fibroin albumen biological detection, but ELISA detection method needs testing goal
Object fibroin albumen can have certain concentration, and the invading by long-term years and environment of the silk-fibroin in archaeological excavation sample
It loses, remaining fibroin albumen content is very little in soil, and may be broken down into different segments, common solution extraction method
Enough fibroin albumens are hardly resulted in, the Monoclonal Antibody Mixture prepared using fibroin albumen difference antigenic determinant can be with
In conjunction with the different fragments that trace fibroin albumen resolves into, the efficiency of detection is improved.
Invention content
In order to solve the above technical problems, immunomagnetic beads enriched with trace fibroin albumen is utilized the present invention provides a kind of, to
The method that trace fibroin albumen in soil sample is enriched with.
Include the following steps：
1）The mixing of different fibroin albumen monoclonal antibodies：The heavy chain for being derived from mouse is respectively three kinds of fibroins of IgG1, IgG2b, IgA
Protein monoclonal antibody homogenous quantities concentration mixes；
2）Coupled reaction：Protein G package magnetic bead 1mg is taken to be added to the 2- (N- of 1mL, pH 6.0, a concentration of 15mmol/L
Coffee quinoline) in ethanesulfonic acid buffer, step 1 is then added）The 70 μ g of mixed antibody of middle preparation, are placed in shaker at room temperature and couple instead
It should stay overnight；
3）The acquisition of immunomagnetic beads：With magnetic frame collection step 2）Magnetic bead after middle coupling reaction simultaneously removes supernatant, obtains weight
Chain is respectively the fibroin albumen monoclonal antibody immunity magnetic bead of IgG1, IgG2b, IgA；
4）Identification：The another magnetic bead for taking 1mg not to be coupled and step 3）Magnetic bead after middle coupling is dried at 55-65 DEG C, is carried out in Fu
Leaf infrared detection observes 1541cm-1The characteristic peak at place, determines coupled state；
5）Couple the fibroin albumen monoclonal antibody immobilization of rear immunomagnetic beads：In step 3）In obtained fibroin albumen monoclonal
The triethanolamine solution that a concentration of 0.2mol/L, PH of 1ml Fresh are 8.2, triethanolamine are added in antibody immune magnetic beads
Repefral containing 20mmol/L in solution, mixed solution are placed on oscillator to ensure that magnetic bead is in outstanding
Floating state, in incubation at room temperature 30min；Magnetic bead is collected with magnetic frame after the completion of incubation and remove supernatant, then use 1mL phosphoric acid slow
Magnetic bead 2-4 times after brine crosslinking is rushed, then washs magnetic bead 1-3 times with the phosphate Tween buffer of 1mL again, finally
Contain 0.02% NaN with 1mL3, 0.1% bovine serum albumin phosphate buffered saline solution magnetic bead is resuspended, and be stored in 4 DEG C of refrigerators；
6）The combination of fibroin albumen monoclonal antibody immunity magnetic bead and fibroin albumen：Step 5）Finally obtained immunomagnetic beads are set
In, to ensure that magnetic bead is in suspended state, being incubated 1h at normal temperatures on oscillator；Be added in every milliliter of immunomagnetic beads containing 10~
The fibroin albumen sample of 100ng is placed on oscillator to ensure that magnetic bead is in suspended state, and reacts 60min at 25 DEG C；Then
Magnetic bead is collected with magnetic frame and removes supernatant, the magnetic bead after then washing crosslinking with 1mL phosphate buffered saline solutions 3 times；
7）The elution of fibroin albumen：100uL elution solution and step 6 is added）Obtained magnetic bead mixing, elution solution is sweet ammonia
Acid, a concentration of 0.1mol/L, PH 2 are placed in magnetic bead on oscillator to ensure that magnetic bead is in suspended state, are being incubated at room temperature
10min；Then magnetic bead is collected with magnetic frame, supernatant is transferred in another tubule, is repeated once collection eluent；
8）Detection：Merge step 7）In eluent, be used in combination 0.1mol/L NaOH solutions adjustment pH be 7 after carry out ELISA inspections
It surveys；
9）Mark-on reclaims：When 10~100ng fibroin albumens are added in every milliliter of immunomagnetic beads, the bioaccumulation efficiency of fibroin albumen
Reach 80% or more.
The step 1）The heavy chain for being derived from mouse is respectively that three kinds of fibroin albumen monoclonal antibodies of IgG1, IgG2b, IgA are same
Mass concentration mixes；
A kind of trace fibroin albumen enrichment method, it is characterised in that the step 2）Protein G package magnetic bead 1mg is taken to add
Enter to 1mL, pH 6.0, a concentration of 15mmol/L 2- (N- morpholines) ethanesulfonic acid buffer in, then be added step 1）In
The 70 μ g of mixed antibody of preparation are placed in shaker at room temperature coupled reaction and stay overnight；
A kind of trace fibroin albumen enrichment method, it is characterised in that the step 5）Middle fibroin albumen monoclonal antibody is exempted from
The triethanolamine solution that a concentration of 0.2mol/L, PH of 1ml Fresh are 8.2 is added in epidemic disease magnetic bead, mixed solution is set
In on oscillator to ensure that magnetic bead is in suspended state, incubation at room temperature 30min, immobilize；
A kind of trace fibroin albumen enrichment method, it is characterised in that the step 7）In eluent be glycine, institute
It is 0.1mol/L, PH 2 to state glycine concentration, ensures that magnetic bead is in suspended state, in incubation at room temperature 10min, and is repeated once
Collect eluent.
A kind of trace fibroin albumen enrichment method, it is characterised in that the step 9）In 10~100ng fibroin eggs
When being added in vain in every milliliter of immunomagnetic beads, the bioaccumulation efficiency of fibroin albumen reaches 80% or more.
Technical key point of the present invention is：Heavy chain is that the monoclonal antibody of three kinds of fibroin albumens of IgG1, IgG2b, IgA is same
Protein G package magnetic bead couple and immobilization technology, including three kinds of monoclonal antibodies that heavy chain is IgG1, IgG2b, IgA
Solution condition associated with equal mass mixings and addition, coupling, couples the fibroin albumen monoclonal antibody immobilization of rear immunomagnetic beads
Solution condition and step；The operating technology that trace fibroin albumen in archaeology material is enriched with using immunomagnetic beads, including silk
Fibroin sample additive amount, reaction condition and step elute the solution condition and step of fibroin albumen.
The invention has the advantages that：The present invention prepares immunomagnetic beads using the monoclonal antibody of fibroin albumen, and
Using this immunomagnetic beads enriched with trace fibroin albumen, it can preferably carry out trace fibroin albumen in soil sample and its decompose segment
Enrichment and extraction, have that extraction different decomposition segment, bioaccumulation efficiency be high, easy to operate, experiment takes the various aspects advantage such as short.
Description of the drawings
Fig. 1 is the infrared detection collection of illustrative plates of immunomagnetic beads of the present invention；
Fig. 2 is the rate of recovery of fibroin albumen Different adding amount of the present invention.
Specific implementation mode
Below the technical scheme of the invention is illustrated by a specific example, but the scope of the present invention is not limited thereto.
Specific process step is as follows：
1）The mixing of different fibroin albumen monoclonal antibodies：The heavy chain for being derived from mouse is respectively three kinds of fibroins of IgG1, IgG2b, IgA
Protein monoclonal antibody homogenous quantities concentration mixes；
2）Coupled reaction：Take Protein G（protein G）Package magnetic bead 1mg is added to 1mL, pH 6.0, a concentration of 15mmol/
In 2- (N- morpholines) ethanesulfonic acid buffer of L, step 1 is then added）The 70 μ g of mixed antibody of middle preparation, are placed on oscillator
Room temperature coupled reaction is stayed overnight；
3）The acquisition of immunomagnetic beads：With magnetic frame collection step 2）Magnetic bead after middle coupled reaction simultaneously removes supernatant, obtains weight
Chain is respectively the fibroin albumen monoclonal antibody immunity magnetic bead of IgG1, IgG2b, IgA；
4）Identification：The another magnetic bead for taking 1mg not couple and step 3）In couple after magnetic bead dried at 60 DEG C, carry out Fourier it is red
1541cm is observed in outer detection-1The characteristic peak at place, determines coupled state；The feature shaken for carboxyl at 1634.99cm-1 in Fig. 1
Peak, the magnetic bead after coupling is compared with the magnetic bead not coupled, and characteristic peak obviously differs larger at this, shows IgG1, IgG2b, IgA tri-
The different fibroin albumen antibody of a heavy chain is successfully coupled with magnetic bead；
5）Couple the fibroin albumen monoclonal antibody immobilization of rear immunomagnetic beads：In step 3）In obtained fibroin albumen monoclonal
The triethanolamine solution that a concentration of 0.2mol/L, PH of 1ml Fresh are 8.2, three second are added in antibody immune magnetic beads
Repefral containing 20mmol/L in alkanolamine solution（DMP）, mixed solution be placed on oscillator with ensure
Magnetic bead is in suspended state, in incubation at room temperature 30min；Magnetic bead is collected with magnetic frame after the completion of incubation and remove supernatant, then
With 1mL phosphate buffered saline solutions（PBS）Magnetic bead after washing crosslinking 3 times, then uses the phosphate Tween buffer of 1mL again
(PBST) washing magnetic bead 2 times, finally contains the phosphate buffered saline solution of 0.02% NaN3,0.1% bovine serum albumin with 1mL（PBS）Weight
Outstanding magnetic bead, and it is stored in 4 DEG C of refrigerators；
6）The combination of fibroin albumen monoclonal antibody immunity magnetic bead and fibroin albumen：Step 5）Finally obtained immunomagnetic beads are set
In, to ensure that magnetic bead is in suspended state, being incubated 1h at normal temperatures on oscillator；Be added in every milliliter of immunomagnetic beads containing 10~
The fibroin albumen sample of 100ng is placed on oscillator to ensure that magnetic bead is in suspended state, and reacts 60min at 25 DEG C；Then
Magnetic bead is collected with magnetic frame and removes supernatant, the magnetic bead after then washing crosslinking with 1mL phosphate buffered saline solutions 3 times；
7）The elution of fibroin albumen：100uL eluents are added（Glycine concentration is 0.1mol/L, PH 2）With step 6）It obtains
Magnetic bead mixing, eluent is glycine, and glycine concentration 0.1mol/L, PH are that 2 magnetic beads are placed on oscillator to ensure
Magnetic bead is in suspended state, in incubation at room temperature 10min；Then magnetic bead is collected with magnetic frame, supernatant is transferred to another tubule
In, it is repeated once collection eluent；
8）Detection：Merge step 7）In eluent, be used in combination 0.1mol/L NaOH solutions adjustment pH be 7 after carry out ELISA inspections
It surveys；
9）Mark-on reclaims：For fibroin albumen additive amount in 10~100ng/mL immunomagnetic beads, bioaccumulation efficiency reaches 80% or more, Fig. 2
As can be seen that fibroin albumen additive amount, in 10~50ng/mL immunomagnetic beads, the rate of recovery reaches 97% or more, fibroin albumen addition
When amount is 100ng, the rate of recovery can also reach 80%, but when albumen additive amount reaches 200ng, the rate of recovery only has 57.8%；Knot
Fruit show by immunomagnetic beads can effective enriched with trace fibroin albumen, in 10~100ng/mL, bioaccumulation efficiency can reach
80% or more.
Finally it should be noted that above implement to be merely illustrative of the technical solution of the present invention to be not intended to limit the present invention, to the greatest extent
Pipe is described the invention in detail with reference to preferred embodiment, it will be understood by those of ordinary skill in the art that, it can be to hair
Bright technical solution is modified or replaced equivalently, and without departing from the spirit of the technical scheme of the invention and range, should all be contained
It covers in scope of the presently claimed invention.
Claims (6)
1. a kind of trace fibroin albumen enrichment method, it is characterised in that include the following steps：
The mixing of different fibroin albumen monoclonal antibodies：The heavy chain for being derived from mouse is respectively three kinds of fibroin eggs of IgG1, IgG2b, IgA
White monoclonal antibody homogenous quantities concentration mixing；
Coupled reaction：Protein G package magnetic bead 1mg is taken to be added to 2- (the N- morphines of 1mL, pH 6.0, a concentration of 15mmol/L
Quinoline) in ethanesulfonic acid buffer, step 1 is then added）The 70 μ g of mixed antibody of middle preparation, are placed in shaker at room temperature coupled reaction
Overnight；
The acquisition of immunomagnetic beads：With magnetic frame collection step 2）Magnetic bead after middle coupling reaction simultaneously removes supernatant, obtains heavy chain
The respectively fibroin albumen monoclonal antibody immunity magnetic bead of IgG1, IgG2b, IgA；
Identification：The another magnetic bead for taking 1mg not to be coupled and step 3）Magnetic bead after middle coupling is dried at 55-65 DEG C, carries out Fourier
Infrared detection observes 1541cm-1The characteristic peak at place, determines coupled state；
Couple the fibroin albumen monoclonal antibody immobilization of rear immunomagnetic beads：In step 3）In obtained fibroin albumen monoclonal it is anti-
The triethanolamine solution that a concentration of 0.2mol/L, PH of 1ml Fresh are 8.2 is added in body immunomagnetic beads, triethanolamine is molten
Repefral containing 20mmol/L in liquid, mixed solution is placed on oscillator to be suspended with ensureing that magnetic bead is in
State, in incubation at room temperature 30min；Magnetic bead is collected with magnetic frame after the completion of incubation and remove supernatant, then use 1mL phosphoric acid buffers
Magnetic bead after brine crosslinking 2-4 times, then washs magnetic bead 1-3 times with the phosphate Tween buffer of 1mL again, finally uses
1mL contains 0.02% NaN3, 0.1% bovine serum albumin phosphate buffered saline solution magnetic bead is resuspended, and be stored in 4 DEG C of refrigerators；
The combination of fibroin albumen monoclonal antibody immunity magnetic bead and fibroin albumen：Step 5）Finally obtained immunomagnetic beads are placed in
To ensure that magnetic bead is in suspended state on oscillator, it is incubated 1h at normal temperatures；Be added in every milliliter of immunomagnetic beads containing 10~
The fibroin albumen sample of 100ng is placed on oscillator to ensure that magnetic bead is in suspended state, and reacts 60min at 25 DEG C；Then
Magnetic bead is collected with magnetic frame and removes supernatant, the magnetic bead after then washing crosslinking with 1mL phosphate buffered saline solutions 3 times；
The elution of fibroin albumen：100uL elution solution and step 6 is added）Obtained magnetic bead mixing, elution solution is glycine,
A concentration of 0.1mol/L, PH 2 is placed in magnetic bead on oscillator to ensure that magnetic bead is in suspended state, is being incubated at room temperature
10min；Then magnetic bead is collected with magnetic frame, supernatant is transferred in another tubule, is repeated once collection eluent；
Detection：Merge step 7）In eluent, be used in combination 0.1mol/L NaOH solutions adjustment pH be 7 after carry out ELISA detections；
Mark-on reclaims：When 10~100ng fibroin albumens are added in every mL immunomagnetic beads, the bioaccumulation efficiency of fibroin albumen reaches
80% or more.
2. a kind of trace fibroin albumen enrichment method according to claim 1, it is characterised in that the step 1）It is derived from mouse
Heavy chain be respectively IgG1, IgG2b, IgA three kinds of fibroin albumen monoclonal antibody homogenous quantities concentration mixing.
3. a kind of trace fibroin albumen enrichment method according to claim 1, it is characterised in that the step 2）Take albumen
G packages magnetic bead 1mg is added in 2- (N- morpholines) ethanesulfonic acid buffer of 1mL, pH 6.0, a concentration of 15mmol/L, so
Step 1 is added afterwards）The 70 μ g of mixed antibody of middle preparation are placed in shaker at room temperature coupled reaction and stay overnight.
4. a kind of trace fibroin albumen enrichment method according to claim 1, it is characterised in that the step 5）Middle fibroin
The triethanolamine that a concentration of 0.2mol/L, PH that 1ml Fresh is added in protein monoclonal antibody immunomagnetic beads are 8.2 is molten
Liquid, mixed solution are placed on oscillator to ensure that magnetic bead is in suspended state, in incubation at room temperature 30min, immobilize.
5. a kind of trace fibroin albumen enrichment method according to claim 1, it is characterised in that the step 7）In wash
De- liquid is glycine, and the glycine concentration is 0.1mol/L, PH 2, ensures that magnetic bead is in suspended state, is being incubated at room temperature
10min, and it is repeated once collection eluent.
6. a kind of trace fibroin albumen enrichment method described in claim 1, it is characterised in that the step 9）In 10~100ng
When fibroin albumen is added in every milliliter of immunomagnetic beads, the bioaccumulation efficiency of fibroin albumen reaches 80% or more.
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Cited By (3)
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| CN113061157A (en) * | 2021-03-31 | 2021-07-02 | 浙江理工大学 | Protein extraction and enrichment method of silk proteomics |
| WO2024119493A1 (en) * | 2022-12-05 | 2024-06-13 | 中国丝绸博物馆 | Preparation method for immunomagnetic bead test strip for rapidly detecting trace protein in cultural relics, and application thereof |
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| WO2024119493A1 (en) * | 2022-12-05 | 2024-06-13 | 中国丝绸博物馆 | Preparation method for immunomagnetic bead test strip for rapidly detecting trace protein in cultural relics, and application thereof |
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