CN108387435B - Trace silk fibroin enrichment method - Google Patents
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- CN108387435B CN108387435B CN201810086592.1A CN201810086592A CN108387435B CN 108387435 B CN108387435 B CN 108387435B CN 201810086592 A CN201810086592 A CN 201810086592A CN 108387435 B CN108387435 B CN 108387435B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
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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
Abstract
The invention discloses a trace silk fibroin enrichment method, which comprises the following steps: 1) mixing different silk fibroin monoclonal antibodies; 2) coupling reaction; 3) obtaining immunomagnetic beads; 4) identifying; 5) immobilizing the silk fibroin monoclonal antibody of the immunomagnetic beads after coupling; 6) combining the silk fibroin monoclonal antibody immunomagnetic beads with silk fibroin; 7) eluting silk fibroin; 8) detecting; 9) and (5) adding a label and recovering. The invention utilizes the composite monoclonal antibody of the silk fibroin to prepare the immunomagnetic beads, and utilizes the immunomagnetic beads to enrich trace silk fibroin, can better extract trace silk fibroin in the archaeological soil sample, and has the advantages of extracting different decomposed fragments, high enrichment efficiency, simple and convenient operation, short experiment time consumption and the like.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a trace silk fibroin enrichment method.
Background
Silk is one of the important inventions of Chinese civilization, bears extremely rich culture, technology and social connotation, and provides precious data for the research of archaeology, historians and technical history. Unearthed silk fragments indicate that the origin of silk should be no later than 5500 years ago. However, a long time of development is necessary from the discovery of silk fibres to the weaving thereof into elegant silk. Therefore, our ancestors began using silk fiber much earlier than 5500 years ago. However, the problem of origin of silk has always been encaged in haze. How to determine the silk origin by using scientific means and the silk comes from China has very important historical and cultural values.
Silk is made of silk, which is a natural protein fiber composed of 18 amino acids. In a long historical course, silk fabrics buried in graves or sites in the same year lose physical appearance, degrade into peptide fragments and amino acids, or become traces, or dissolve into soil, are not recognizable under the naked eye, and evidence of the earlier ages is more difficult to find. How to search for earlier evidence in the case of disappearance of the macroscopic morphology of silk becomes a key issue for studying early silk. Compared with the complete silk fabric, the amorphous fiber silk fibroin residues are more likely to be preserved and can also become new evidence for seeking the survival of early silk.
In a long river which lasts hundreds of years or even thousands of years, the content of the remaining silk in the archaeological material is very little, so that trace silk fibroin in the archaeological material is enriched before a sample is detected so as to improve the detection accuracy, and meanwhile, the problems of simplicity and convenience in operation and low time consumption need to be considered.
At present, there are various methods for detecting trace silk fibroin, wherein an ELISA detection method is a latest technology for biological detection of silk fibroin by utilizing specific binding of antigen and antibody, but the ELISA detection method needs to detect that the target silk fibroin can have a certain concentration, silk fibroin in an archaeological excavation sample is corroded by years and the environment for a long time, the content of residual silk fibroin in soil is very little and can be decomposed into different fragments, a common solution extraction method is difficult to obtain enough silk fibroin, and a monoclonal antibody mixture prepared by utilizing different antigenic determinants of the silk fibroin can be combined with different fragments decomposed by trace silk fibroin, so that the detection efficiency is improved.
Disclosure of Invention
In order to solve the technical problem, the invention provides a method for enriching trace silk fibroin in a soil sample by using immunomagnetic beads to enrich the trace silk fibroin.
The method comprises the following steps:
1) mixing different silk fibroin monoclonal antibodies: mixing three silk fibroin monoclonal antibodies of which the heavy chains are IgG1, IgG2b and IgA respectively from a mouse with the same mass concentration;
2) coupling reaction: adding 1mg of protein G-coated magnetic beads into 1mL of 2- (N-morpholine) ethanesulfonic acid buffer solution with pH of 6.0 and concentration of 15mmol/L, then adding 70 mu G of the mixed antibody prepared in the step 1), and placing on an oscillator for coupling reaction at room temperature overnight;
3) obtaining of immunomagnetic beads: collecting the magnetic beads subjected to coupling reaction in the step 2) by using a magnetic frame, and removing supernatant to obtain silk fibroin monoclonal antibody immunomagnetic beads with heavy chains of IgG1, IgG2b and IgA respectively;
4) and (3) identification: drying another 1mg of uncoupled magnetic beads and the coupled magnetic beads in the step 3) at 55-65 ℃, carrying out Fourier infrared detection, and observing 1541cm-1Determining a coupling state;
5) and (3) immobilizing the silk fibroin monoclonal antibody of the coupled immunomagnetic beads: adding 1ml of freshly prepared triethanolamine solution with the concentration of 0.2mol/L, PH of 8.2 into the immunomagnetic beads of the silk fibroin monoclonal antibody obtained in the step 3), wherein the triethanolamine solution contains 20mmol/L dimethyl phthalate, placing the mixed solution on an oscillator to ensure that the magnetic beads are in a suspension state, and incubating at room temperature for 30 min; collecting the magnetic beads by using a magnetic rack after the incubation is finished, removing supernatant, washing the crosslinked magnetic beads for 2-4 times by using 1mL of phosphate buffer solution, then washing the magnetic beads for 1-3 times by using 1mL of phosphate Tween buffer solution, and finally washing the magnetic beads for 1mL of solution containing 0.02% NaN3Resuspending the magnetic beads in 0.1% bovine serum albumin phosphate buffered saline and storing in a refrigerator at 4 ℃;
6) combining the silk fibroin monoclonal antibody immunomagnetic beads with silk fibroin: placing the immunomagnetic beads finally obtained in the step 5) on an oscillator to ensure that the immunomagnetic beads are in a suspension state, and incubating for 1h at normal temperature; adding a silk fibroin sample containing 10-100 ng per milliliter of immunomagnetic beads, placing on an oscillator to ensure that the magnetic beads are in a suspension state, and reacting for 60min at 25 ℃; then collecting the magnetic beads by using a magnetic frame and removing supernatant, and then washing the crosslinked magnetic beads for 3 times by using 1mL of phosphate buffer solution;
7) and (3) eluting silk fibroin: adding 100uL of elution solution and uniformly mixing the elution solution and the magnetic beads obtained in the step 6), wherein the elution solution is glycine, the concentration of the glycine is 0.1mol/L, PH is 2, placing the magnetic beads on an oscillator to ensure that the magnetic beads are in a suspension state, and incubating for 10min at room temperature; then collecting the magnetic beads by using a magnetic frame, transferring the supernatant into another small tube, and repeatedly collecting the eluent once;
8) and (3) detection: combining the eluates obtained in the step 7), adjusting the pH value to 7 by using 0.1mol/L NaOH solution, and then carrying out ELISA detection;
9) adding a standard and recovering: when 10-100 ng of silk fibroin is added into each milliliter of immunomagnetic beads, the enrichment efficiency of the silk fibroin reaches more than 80%.
The three silk fibroin monoclonal antibodies of which the heavy chains are IgG1, IgG2b and IgA respectively from mice in the step 1) are mixed with the same mass concentration.
The trace silk fibroin enrichment method is characterized in that 1mg of protein G-coated magnetic beads are taken in the step 2) and added into 1mL of 2- (N-morpholine) ethanesulfonic acid buffer solution with the pH value of 6.0 and the concentration of 15mmol/L, and then 70 mu G of the mixed antibody prepared in the step 1) is added onto an oscillator and placed at room temperature for coupling reaction overnight.
The trace silk fibroin enrichment method is characterized in that 1ml of freshly prepared triethanolamine solution with the concentration of 0.2mol/L, PH of 8.2 is added into immunomagnetic beads of the silk fibroin monoclonal antibody in the step 5), the mixed solution is placed on an oscillator to ensure that the magnetic beads are in a suspension state, and the beads are incubated at room temperature for 30min for immobilization.
The trace silk fibroin enrichment method is characterized in that the eluent in the step 7) is glycine, the concentration of the glycine is 0.1mol/L, PH is 2, the magnetic beads are ensured to be in a suspension state, incubation is carried out at room temperature for 10min, and the eluent is collected repeatedly.
The trace silk fibroin enrichment method is characterized in that in the step 9), when 10-100 ng of silk fibroin is added into each milliliter of immunomagnetic beads, the enrichment efficiency of the silk fibroin reaches more than 80%.
The technical points of the invention are as follows: coupling and immobilizing three silk fibroin monoclonal antibodies with heavy chains of IgG1, IgG2b and IgA and protein G wrapped magnetic beads, wherein the coupling and immobilizing technology comprises the steps of mixing the three silk fibroin monoclonal antibodies with heavy chains of IgG1, IgG2b and IgA by equal mass, adding the three silk fibroin monoclonal antibodies, and coupling solution conditions, coupling the silk fibroin monoclonal antibodies of immunomagnetic beads, immobilizing the solution conditions, and performing the steps; the operation technology for enriching the trace silk fibroin in the archaeological material by utilizing the immunomagnetic beads comprises the adding amount of a silk fibroin sample, reaction conditions and steps, and solution conditions and steps for eluting the silk fibroin.
The invention has the following beneficial effects: the invention utilizes the monoclonal antibody of the silk fibroin to prepare the immunomagnetic beads, and utilizes the immunomagnetic beads to enrich the trace silk fibroin, can better enrich and extract the trace silk fibroin and the decomposed fragments thereof in the soil sample, and has the advantages of extracting different decomposed fragments, high enrichment efficiency, simple and convenient operation, short experiment time consumption and the like.
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FIG. 1 is an infrared detection spectrum of immunomagnetic beads of the present invention;
figure 2 shows the recovery rates of different addition amounts of silk fibroin according to the present invention.
Detailed Description
The technical solutions of the present invention are described below with specific examples, but the scope of the present invention is not limited thereto.
The specific process steps are as follows:
1) mixing different silk fibroin monoclonal antibodies: mixing three silk fibroin monoclonal antibodies of which the heavy chains are IgG1, IgG2b and IgA respectively from a mouse with the same mass concentration;
2) coupling reaction: adding 1mg of protein G (protein G) -coated magnetic beads into 1mL of 2- (N-morpholine) ethanesulfonic acid buffer solution with the pH of 6.0 and the concentration of 15mmol/L, then adding 70 mu g of the mixed antibody prepared in the step 1), and placing the mixed antibody on a shaker for coupling reaction at room temperature overnight;
3) obtaining of immunomagnetic beads: collecting the magnetic beads subjected to coupling reaction in the step 2) by using a magnetic frame, and removing supernatant to obtain silk fibroin monoclonal antibody immunomagnetic beads with heavy chains of IgG1, IgG2b and IgA respectively;
4) and (3) identification: drying another 1mg of uncoupled magnetic beads and the coupled magnetic beads in the step 3) at 60 ℃, carrying out Fourier infrared detection, and observing 1541cm-1Determining a coupling state; 1634.99cm-1 in FIG. 1 is a characteristic peak of carboxyl vibration, and the characteristic peak is obviously larger in the position of the coupled magnetic beads compared with the uncoupled magnetic beads, which shows that IThe silk fibroin antibodies with different heavy chains of gG1, IgG2b and IgA are successfully coupled with magnetic beads;
5) and (3) immobilizing the silk fibroin monoclonal antibody of the coupled immunomagnetic beads: adding 1ml of freshly prepared triethanolamine solution with the concentration of 0.2mol/L, PH of 8.2 into the immunomagnetic beads of the silk fibroin monoclonal antibody obtained in the step 3), wherein the triethanolamine solution contains 20mmol/L dimethyl phthalate (DMP), placing the mixed solution on an oscillator to ensure that the magnetic beads are in a suspension state, and incubating at room temperature for 30 min; after the incubation is finished, collecting the magnetic beads by using a magnetic frame and removing supernatant, washing the crosslinked magnetic beads for 3 times by using 1mL of Phosphate Buffered Saline (PBS), then washing the magnetic beads for 2 times by using 1mL of phosphate Tween buffer (PBST), finally resuspending the magnetic beads by using 1mL of Phosphate Buffered Saline (PBS) containing 0.02% NaN3 and 0.1% bovine serum albumin, and storing the magnetic beads in a refrigerator at 4 ℃;
6) combining the silk fibroin monoclonal antibody immunomagnetic beads with silk fibroin: placing the immunomagnetic beads finally obtained in the step 5) on an oscillator to ensure that the immunomagnetic beads are in a suspension state, and incubating for 1h at normal temperature; adding a silk fibroin sample containing 10-100 ng per milliliter of immunomagnetic beads, placing on an oscillator to ensure that the magnetic beads are in a suspension state, and reacting for 60min at 25 ℃; then collecting the magnetic beads by using a magnetic frame and removing supernatant, and then washing the crosslinked magnetic beads for 3 times by using 1mL of phosphate buffer solution;
7) and (3) eluting silk fibroin: adding 100uL of eluent (the concentration of glycine is 0.1mol/L, PH is 2) and the magnetic beads obtained in the step 6), uniformly mixing, placing the magnetic beads on an oscillator to ensure that the magnetic beads are in a suspension state, and incubating at room temperature for 10min, wherein the eluent is glycine, and the concentration of glycine is 0.1mol/L, PH is 2; then collecting the magnetic beads by using a magnetic frame, transferring the supernatant into another small tube, and repeatedly collecting the eluent once;
8) and (3) detection: combining the eluates obtained in the step 7), adjusting the pH value to 7 by using 0.1mol/L NaOH solution, and then carrying out ELISA detection;
9) adding a standard and recovering: when the addition amount of the silk fibroin is 10-100 ng/mL of immunomagnetic beads, the enrichment efficiency reaches more than 80%, and as can be seen in fig. 2, when the addition amount of the silk fibroin is 10-50 ng/mL of immunomagnetic beads, the recovery rate reaches more than 97%, when the addition amount of the silk fibroin is 100ng, the recovery rate can also reach 80%, but when the addition amount of the silk fibroin reaches 200ng, the recovery rate is only 57.8%; the result shows that trace silk fibroin can be effectively enriched by immunomagnetic beads, and the enrichment efficiency can reach more than 80% at 10-100 ng/mL.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (1)
1. A trace silk fibroin enrichment method is characterized by comprising the following steps:
1) mixing different silk fibroin monoclonal antibodies: mixing three silk fibroin monoclonal antibodies of which the heavy chains are IgG1, IgG2b and IgA respectively from a mouse with the same mass concentration;
2) coupling reaction: adding 1mg of protein G-coated magnetic beads into 1mL of 2- (N-morpholine) ethanesulfonic acid buffer solution with pH of 6.0 and concentration of 15mmol/L, then adding 70 mu G of the mixed antibody prepared in the step 1), and placing on an oscillator for coupling reaction at room temperature overnight;
3) obtaining of immunomagnetic beads: collecting the magnetic beads subjected to coupling reaction in the step 2) by using a magnetic frame, and removing supernatant to obtain silk fibroin monoclonal antibody immunomagnetic beads with heavy chains of IgG1, IgG2b and IgA respectively;
4) and (3) identification: drying another 1mg of uncoupled magnetic beads and the coupled magnetic beads in the step 3) at 55-65 ℃, carrying out Fourier infrared detection, and observing 1541cm-1Determining a coupling state;
5) and (3) immobilizing the silk fibroin monoclonal antibody of the coupled immunomagnetic beads: adding 1ml of freshly prepared triethanolamine solution with the concentration of 0.2mol/L, PH of 8.2 into the immunomagnetic beads of the silk fibroin monoclonal antibody obtained in the step 3), wherein the triethanolamine solution isThe mixed solution is placed on an oscillator to ensure that the magnetic beads are in a suspension state, and the mixed solution is incubated for 30min at room temperature; collecting the magnetic beads by using a magnetic rack after the incubation is finished, removing supernatant, washing the crosslinked magnetic beads for 2-4 times by using 1mL of phosphate buffer solution, then washing the magnetic beads for 1-3 times by using 1mL of phosphate Tween buffer solution, and finally washing the magnetic beads for 1mL of solution containing 0.02% NaN3Resuspending the magnetic beads in 0.1% bovine serum albumin phosphate buffered saline and storing in a refrigerator at 4 ℃;
6) combining the silk fibroin monoclonal antibody immunomagnetic beads with silk fibroin: placing the immunomagnetic beads finally obtained in the step 5) on an oscillator to ensure that the immunomagnetic beads are in a suspension state, and incubating for 1h at normal temperature; adding a silk fibroin sample containing 10-100 ng per milliliter of immunomagnetic beads, placing on an oscillator to ensure that the magnetic beads are in a suspension state, and reacting for 60min at 25 ℃; then collecting the magnetic beads by using a magnetic frame and removing supernatant, and then washing the crosslinked magnetic beads for 3 times by using 1mL of phosphate buffer solution;
7) and (3) eluting silk fibroin: adding 100uL of elution solution and uniformly mixing the elution solution and the magnetic beads obtained in the step 6), wherein the elution solution is glycine, the concentration of the glycine is 0.1mol/L, PH is 2, placing the magnetic beads on an oscillator to ensure that the magnetic beads are in a suspension state, and incubating for 10min at room temperature; then collecting the magnetic beads by using a magnetic frame, transferring the supernatant into another small tube, and repeatedly collecting the eluent once;
8) and (3) detection: combining the eluates obtained in the step 7), adjusting the pH value to 7 by using 0.1mol/L NaOH solution, and then carrying out ELISA detection;
9) adding a standard and recovering: when 10-100 ng of silk fibroin is added into each mL of immunomagnetic beads, the enrichment efficiency of the silk fibroin reaches more than 80%.
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| CN109187699B (en) * | 2018-09-05 | 2020-06-23 | 浙江理工大学 | Method for detecting ancient silk fabric |
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