CN105693857A - Method for preparing cattle hair detection antibody by feature diagnosis sequence - Google Patents
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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Abstract
The invention relates to the technical field of historical relic detection and discloses a method for preparing a cattle hair detection antibody by a feature diagnosis sequence. The method includes steps: a, synthesis of the feature diagnosis sequence; b, coupling of the feature diagnosis sequence and a carrier; c, primary immunization; d, antiserum preparation; e, purification of the cattle hair detection antibody. The cattle hair detection antibody prepared according to the method has the advantages of high sensitivity and high specificity.
Description
Technical field
The present invention relates to historical relic detection technique field, particularly relate to a kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody。
Background technology
Chinese history is long, and being unearthed from the grave of China's early stage has substantial amounts of ox hair goods, and these, ox hair goods contained substantial amounts of archaeological information in ancient times。
Ox hair is a kind of natural animal wool fibre。Ox hair is made up of epidermis scales layer and cortical layer two parts, being a kind of azelon, wherein keratin is mainly made up of 18 kinds of a-amino acids, and is coupled to helical long-chain molecule, containing carboxyl, hydroxyl and amido etc. on it, at intermolecular formation hydrogen bond and salt bridged bond etc.。
Generally to the qualification of ox hair goods mainly to the keratic qualification of ox hair。Ox hair keratin is a kind of hardening, and the protein not readily dissolved, the discriminating of ox hair goods is caused certain difficulty by this。At present, protein detection techniques is a kind of wide variety of advanced technology such as the Enzyme-multiplied immune technique combined based on Ag-Ab, but possesses high sensitivity and specific antibody is prepared by a great problem。
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides a kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody。Ox hair detection antibody prepared by the inventive method has high sensitivity and high specific。
The concrete technical scheme of the present invention is: a kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody, adopts following steps:
A. the synthesis of feature diagnosing sequence: adopt Fmoc polypeptide solid-state reaction method composite character diagnosing sequence, and increase cysteine residues for coupling reaction at the C end of described feature diagnosing sequence。
B. the coupling of feature diagnosing sequence and carrier: select hemocyanin as carrier protein, carries out coupling by SPDP connection method by described feature diagnosing sequence and described hemocyanin and prepares complete antigen。
C. initial immunity: the complete antigen that step b prepares is diluted with normal saline, by the complete antigen after dilution and complete Freund's adjuvant 1:(0.8-1.1 by volume) mix, it is subsequently adding penicillin and streptomycin carries out emulsifying, obtain initial immunity antigen emulsion；Use initial immunity antigen emulsion that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity。
D. prepared by antiserum: the complete antigen obtained by step b with normal saline is diluted, by the complete antigen after dilution and incomplete Freund's adjuvant 1:(0.8-1.1 by volume) mix, it is subsequently adding penicillin and streptomycin emulsifying, obtains strong immunizing antigen emulsion；During after initial immunity the 3rd week and the 5th week, use strong immunizing antigen emulsion that rabbit is carried out subcutaneous multi-point injection respectively, carry out booster immunization；During after carrying out initial immunity the 7th week and the 9th week, repeat booster immunization, when the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood, and separate and prepare antiserum。
E. the purification of ox hair detection antibody: protein A filler is mixed with phosphate buffered solution, evacuation 20-25min after stirring, obtain bubble-free protein A filler liquid；Protein A filler liquid is joined in glass column under air bubble free conditions, obtains albumen post；Albumen post is carried out；Above-mentioned prepared antiserum is diluted, joins after dilution and albumen post carries out loading, repeat loading once；Albumen post is cleaned again, carries out eluting with glycine solution antagonist, clean albumen post after eluting, prepare antibody。
Antibody prepared by the present invention has stronger specificity, and this antibody can have obvious immunoreation with ox hair keratin, highly sensitive, it is possible to the ox hair keratin molecule that ancient times have been ruptured in ox hair goods makes detection。
As preferably, the feature diagnosing sequence described in step a is the aminoacid sequence GLLDSEDCKLPCNPCATTNAYGK as shown in sequence table SEQ IDNo.1。
As preferably, the concentration of the complete antigen after diluting in step c is 0.8-1.2mg/mL。
As preferably, the concentration of the complete antigen after step d dilution is 0.25-0.75mg/mL。
As preferably, when carrying out initial immunity, booster immunization, the injection volume of each injection point is 100 μ L。
As preferably, in step d, separate from blood and obtain sero-fast method and be: the blood collected is placed in set at room temperature, then solidificating blood is placed in the incubator of 36-38 DEG C and places 25-35min, it is placed in 11-13h in the refrigerator of 4 DEG C again, the thick antiserum making clot fully shrink and being precipitated out completely；Collecting thick antiserum, in 4 DEG C of environment, when 3000g, centrifugal 14-16min, takes supernatant, is positioned over-80 DEG C and stores for future use。
As preferably, the detailed process of step e is: mixed by the proportioning of 1.5g/ (6-7) mL with phosphate buffered solution by protein A filler, evacuation 20-25min after stirring, obtains bubble-free protein A filler liquid；Then the protein A filler liquid of 6-7 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post；By the phosphate buffered solution of 20 parts by volume, albumen post being carried out, cleaning flow velocity is 60mL/h；By phosphate buffered solution, the antiserum of 15 parts by volume is diluted to 20 parts by volume, joins after dilution and albumen post carries out loading, repeat loading once；By the phosphate buffered solution of 30 parts by volume, albumen post being cleaned again, cleaning flow velocity is 60mL/h；With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out eluting；Wash albumen post by phosphate buffered solution after eluting, prepare antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations。
As preferably, the concentration of phosphate buffered solution used in step e is 0.05mol/L, pH is 7.4。
It is compared with the prior art, the invention has the beneficial effects as follows:
(1) utilize feature diagnosing sequence to prepare ox hair detection antibody and there is the features such as highly sensitive, high specificity, it is possible to targets identification bovine keratin, it is possible to be applied to MBP enzyme linked immuno-adsorbent assay, immunofluorescence technique etc.。
(2) adopt feature diagnosing sequence to prepare ox hair detection antibody and the detection of ox hair fabric in early stage grave can be provided a kind of sensitive, special, discrimination method efficiently, provide new scientific basis for ox hair archaeology and the Origin。
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described。In the following example, the coupling of feature diagnosing sequence and synthesis and feature diagnosing sequence and carrier all entrusts Hangzhou Huaan Bio-Tech. Co., Ltd. to complete。
Embodiment 1
A kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody, adopts following steps:
A. the synthesis of feature diagnosing sequence: adopt Fmoc polypeptide solid-state reaction method composite character diagnosing sequence, and increase cysteine residues for coupling reaction at the C end of described feature diagnosing sequence。The aminoacid sequence such as sequence table SEQ IDNo.1 of described feature diagnosing sequence show GLLDSEDCKLPCNPCATTNAYGK。
B. the coupling of feature diagnosing sequence and carrier: select hemocyanin as carrier protein, carries out coupling by SPDP connection method by described feature diagnosing sequence and described hemocyanin and prepares complete antigen。
C. initial immunity: with normal saline by the step b complete antigen diluted concentration prepared to 1mg/mL, complete antigen after dilution is mixed with complete Freund's adjuvant 1:0.95 by volume, it is subsequently adding penicillin and streptomycin carries out emulsifying, obtain initial immunity antigen emulsion；Use initial immunity antigen emulsion that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity。When carrying out initial immunity, the injection volume of each injection point is 100 μ L。
D. prepared by antiserum: the complete antigen diluted concentration obtained by step b with normal saline is to 0.5mg/mL, complete antigen after dilution is mixed with incomplete Freund's adjuvant 1:0.95 by volume, is subsequently adding penicillin and streptomycin emulsifying, obtains strong immunizing antigen emulsion；During after initial immunity the 3rd week and the 5th week, use strong immunizing antigen emulsion that rabbit is carried out subcutaneous multi-point injection respectively, carry out booster immunization；During after carrying out initial immunity the 7th week and the 9th week, repeating booster immunization, when carrying out booster immunization, the injection volume of each injection point is 100 μ L。
When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood, the blood collected is placed in set at room temperature, then solidificating blood is placed in the incubator of 37 DEG C and places 30min, it is placed in 12h in the refrigerator of 4 DEG C again, the thick antiserum making clot fully shrink and being precipitated out completely；Collecting thick antiserum, in 4 DEG C of environment, when 3000g, centrifugal 15min, takes supernatant, prepares antiserum, is positioned over-80 DEG C and stores for future use。
E. the purification of ox hair detection antibody: protein A filler is mixed by the proportioning of 1.5g/6.5mL with phosphate buffered solution, evacuation 22.5min after stirring, obtain bubble-free protein A filler liquid；Then the protein A filler liquid of 6.5mL is joined in glass column under air bubble free conditions, obtain albumen post；By the phosphate buffered solution of 20mL, albumen post being carried out, cleaning flow velocity is 60mL/h；By phosphate buffered solution, the antiserum of 15mL is diluted to 20mL, joins after dilution and albumen post carries out loading, repeat loading once；By the phosphate buffered solution of 30mL, albumen post being cleaned again, cleaning flow velocity is 60mL/h；With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out eluting；Wash albumen post by phosphate buffered solution after eluting, prepare antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations。The concentration of above-mentioned phosphate buffered solution is 0.05mol/L, pH is 7.4。
F. specific detection: take the bovine serum albumin solution that ox hair keratin mass concentration is 1% and be configured to the concentration of 1 μ g/mL and be coated in ELISA Plate 12h, wash three times by the phosphate buffered solution of pH7.4；Every hole adds 200 μ L, and mass concentration is that the bovine serum albumin solution of 1% closes 2h at 37 DEG C；Wash 3 times by the phosphate buffered solution of pH7.4, then ox hair is detected antibody respectively with the bovine serum albumin solution dilution 1:240000 that mass concentration is 1%, 1:60000,1:20000,1:10000,1:5000,1:1000,1:200 times, every hole adds 100 μ L ox hair detection antibody, hatch 1h at 37 DEG C, wash 5 times by the phosphate buffered solution of pH7.4；Every hole adds the horseradish peroxidase mark two of 100 μ L and resists, and hatches 1h at 37 DEG C, and described two anti-employing mass concentrations are the bovine serum albumin dilution 5000 times of 1%；The tetramethyl biphenyl amine aqueous solution that mass concentration is 1% that last every hole adds 100 μ L develops the color in dark place, after 10min, adds the H of 100 μ L2mol/L2SO4Terminate reaction；OD is measured by microplate reader450nm, according to OD450nmValue, judges situations such as the associativities of antigen-antibody；It is assured that in sample when OD value reaches positive criteria containing bovine keratin；If not containing bovine keratin in sample, then OD value will be lower than positive criteria, according to this standard, it can be determined that the existence of ox hair。
Embodiment 2
A kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody, adopts following steps:
A. the synthesis of feature diagnosing sequence: adopt Fmoc polypeptide solid-state reaction method composite character diagnosing sequence, and increase cysteine residues for coupling reaction at the C end of described feature diagnosing sequence。The aminoacid sequence such as sequence table SEQ IDNo.1 of described feature diagnosing sequence show GLLDSEDCKLPCNPCATTNAYGK。
B. the coupling of feature diagnosing sequence and carrier: select hemocyanin as carrier protein, carries out coupling by SPDP connection method by described feature diagnosing sequence and described hemocyanin and prepares complete antigen。
C. initial immunity: with normal saline by the step b complete antigen diluted concentration prepared to 0.8mg/mL, complete antigen after dilution is mixed with complete Freund's adjuvant 1:0.8 by volume, it is subsequently adding penicillin and streptomycin carries out emulsifying, obtain initial immunity antigen emulsion；Use initial immunity antigen emulsion that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity。When carrying out initial immunity, the injection volume of each injection point is 100 μ L。
D. antiserum and preparation: the complete antigen diluted concentration obtained by step b with normal saline is to 0.25mg/mL, complete antigen after dilution is mixed with incomplete Freund's adjuvant 1:0.8 by volume, is subsequently adding penicillin and streptomycin emulsifying, obtains strong immunizing antigen emulsion；During after initial immunity the 3rd week and the 5th week, use strong immunizing antigen emulsion that rabbit is carried out subcutaneous multi-point injection respectively, carry out booster immunization；During after carrying out initial immunity the 7th week and the 9th week, repeating booster immunization, when carrying out booster immunization, the injection volume of each injection point is 100 μ L。
When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood, the blood collected is placed in set at room temperature, then solidificating blood is placed in the incubator of 36 DEG C and places 25min, it is placed in 11h in the refrigerator of 4 DEG C again, the thick antiserum making clot fully shrink and being precipitated out completely；Collecting thick antiserum, in 4 DEG C of environment, when 3000g, centrifugal 14min, takes supernatant, prepares antiserum, is positioned over-80 DEG C and stores for future use。
E. the purification of ox hair detection antibody: protein A filler is mixed by the proportioning of 1.5g/6mL with phosphate buffered solution, evacuation 20min after stirring, obtain bubble-free protein A filler liquid；Then the protein A filler liquid of 6mL is joined in glass column under air bubble free conditions, obtain albumen post；By the phosphate buffered solution of 20mL, albumen post being carried out, cleaning flow velocity is 60mL/h；By phosphate buffered solution, the antiserum of 15mL is diluted to 20mL, joins after dilution and albumen post carries out loading, repeat loading once；By the phosphate buffered solution of 30mL, albumen post being cleaned again, cleaning flow velocity is 60mL/h；With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out eluting；Wash albumen post by phosphate buffered solution after eluting, prepare antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations。The concentration of above-mentioned phosphate buffered solution is 0.05mol/L, pH is 7.4。
Embodiment 3
A kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody, adopts following steps:
A. the synthesis of feature diagnosing sequence: adopt Fmoc polypeptide solid-state reaction method composite character diagnosing sequence, and increase cysteine residues for coupling reaction at the C end of described feature diagnosing sequence。The aminoacid sequence such as sequence table SEQ IDNo.1 of described feature diagnosing sequence show GLLDSEDCKLPCNPCATTNAYGK。
B. the coupling of feature diagnosing sequence and carrier: select hemocyanin as carrier protein, carries out coupling by SPDP connection method by described feature diagnosing sequence and described hemocyanin and prepares complete antigen。
C. initial immunity: with normal saline by the step b complete antigen diluted concentration prepared to 1.2mg/mL, complete antigen after dilution is mixed with complete Freund's adjuvant 1:1.1 by volume, it is subsequently adding penicillin and streptomycin carries out emulsifying, obtain initial immunity antigen emulsion；Use initial immunity antigen emulsion that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity。When carrying out initial immunity, the injection volume of each injection point is 100 μ L。
D. prepared by antiserum: the complete antigen diluted concentration obtained by step b with normal saline is to 0.75mg/mL, complete antigen after dilution is mixed with incomplete Freund's adjuvant 1:1.1 by volume, is subsequently adding penicillin and streptomycin emulsifying, obtains strong immunizing antigen emulsion；During after initial immunity the 3rd week and the 5th week, use strong immunizing antigen emulsion that rabbit is carried out subcutaneous multi-point injection respectively, carry out booster immunization；During after carrying out initial immunity the 7th week and the 9th week, repeating booster immunization, when carrying out booster immunization, the injection volume of each injection point is 100 μ L。
When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood, the blood collected is placed in set at room temperature, then solidificating blood is placed in the incubator of 38 DEG C and places 35min, it is placed in 13h in the refrigerator of 4 DEG C again, the thick antiserum making clot fully shrink and being precipitated out completely；Collecting thick antiserum, in 4 DEG C of environment, when 3000g, centrifugal 16min, takes supernatant, prepares antiserum, is positioned over-80 DEG C and stores for future use。
E. the purification of ox hair detection antibody: protein A filler is mixed by the proportioning of 1.5g/7mL with phosphate buffered solution, evacuation 25min after stirring, obtain bubble-free protein A filler liquid；Then the protein A filler liquid of 7mL is joined in glass column under air bubble free conditions, obtain albumen post；By the phosphate buffered solution of 20mL, albumen post being carried out, cleaning flow velocity is 60mL/h；By phosphate buffered solution, the antiserum of 15mL is diluted to 20mL, joins after dilution and albumen post carries out loading, repeat loading once；By the phosphate buffered solution of 30mL, albumen post being cleaned again, cleaning flow velocity is 60mL/h；With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out eluting；Wash albumen post by phosphate buffered solution after eluting, prepare antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations。The concentration of above-mentioned phosphate buffered solution is 0.05mol/L, pH is 7.4。
Raw materials used, equipment in the present invention, unless otherwise noted, is the conventional raw material of this area, equipment；Method therefor in the present invention, unless otherwise noted, is the conventional method of this area。
The above, be only presently preferred embodiments of the present invention, not the present invention is imposed any restrictions, every any simple modification, change and equivalent transformation above example made according to the technology of the present invention essence, all still falls within the protection domain of technical solution of the present invention。
Claims (8)
1. one kind utilizes the method that feature diagnosing sequence prepares ox hair detection antibody, it is characterised in that adopt following steps:
A. the synthesis of feature diagnosing sequence: adopt Fmoc polypeptide solid-state reaction method composite character diagnosing sequence, and increase cysteine residues for coupling reaction at the C end of described feature diagnosing sequence；
B. the coupling of feature diagnosing sequence and carrier: select hemocyanin as carrier protein, carries out coupling by SPDP connection method by described feature diagnosing sequence and described hemocyanin and prepares complete antigen；
C. initial immunity: the complete antigen that step b prepares is diluted with normal saline, by the complete antigen after dilution and complete Freund's adjuvant 1:(0.8-1.1 by volume) mix, it is subsequently adding penicillin and streptomycin carries out emulsifying, obtain initial immunity antigen emulsion；Use initial immunity antigen emulsion that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity；
D. prepared by antiserum: the complete antigen obtained by step b with normal saline is diluted, by the complete antigen after dilution and incomplete Freund's adjuvant 1:(0.8-1.1 by volume) mix, it is subsequently adding penicillin and streptomycin emulsifying, obtains strong immunizing antigen emulsion；During after initial immunity the 3rd week and the 5th week, use strong immunizing antigen emulsion that rabbit is carried out subcutaneous multi-point injection respectively, carry out booster immunization；During after carrying out initial immunity the 7th week and the 9th week, repeat booster immunization, when the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood, and separate and prepare antiserum；
E. the purification of ox hair detection antibody: protein A filler is mixed with phosphate buffered solution, evacuation 20-25min after stirring, obtain bubble-free protein A filler liquid；Protein A filler liquid is joined in glass column under air bubble free conditions, obtains albumen post；Albumen post is carried out；Above-mentioned prepared antiserum is diluted, joins after dilution and albumen post carries out loading, repeat loading once；Albumen post is cleaned again, carries out eluting with glycine solution antagonist, clean albumen post after eluting, prepare antibody。
2. as claimed in claim 1 a kind of utilize feature diagnosing sequence prepare ox hair detection antibody method, it is characterised in that the feature diagnosing sequence described in step a is the aminoacid sequence GLLDSEDCKLPCNPCATTNAYGK as shown in sequence table SEQ IDNo.1。
3. a kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody as claimed in claim 1, it is characterised in that in step c, the concentration of complete antigen after dilution is 0.8-1.2mg/mL。
4. a kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody as claimed in claim 3, it is characterised in that the concentration of complete antigen after step d dilution is 0.25-0.75mg/mL。
5. as claimed in claim 1 a kind of utilize feature diagnosing sequence prepare ox hair detection antibody method, it is characterised in that when carrying out initial immunity, booster immunization, the injection volume of each injection point is 100 μ L。
6. as claimed in claim 1 a kind of utilize feature diagnosing sequence prepare ox hair detection antibody method, it is characterized in that, in step d, separate from blood and obtain sero-fast method and be: the blood collected is placed in set at room temperature, then solidificating blood is placed in the incubator of 36-38 DEG C and places 25-35min, it is placed in 11-13h in the refrigerator of 4 DEG C again, the thick antiserum making clot fully shrink and being precipitated out completely；Collecting thick antiserum, in 4 DEG C of environment, when 3000g, centrifugal 14-16min, takes supernatant, is positioned over-80 DEG C and stores for future use。
7. as claimed in claim 1 a kind of utilize feature diagnosing sequence prepare ox hair detection antibody method, it is characterized in that, the detailed process of step e is: mixed by the proportioning of 1.5g/ (6-7) mL with phosphate buffered solution by protein A filler, evacuation 20-25min after stirring, obtains bubble-free protein A filler liquid；Then the protein A filler liquid of 6-7 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post；By the phosphate buffered solution of 20 parts by volume, albumen post being carried out, cleaning flow velocity is 60mL/h；By phosphate buffered solution, the antiserum of 15 parts by volume is diluted to 20 parts by volume, joins after dilution and albumen post carries out loading, repeat loading once；By the phosphate buffered solution of 30 parts by volume, albumen post being cleaned again, cleaning flow velocity is 60mL/h；With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out eluting；Wash albumen post by phosphate buffered solution after eluting, prepare antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations。
8. a kind of method utilizing feature diagnosing sequence to prepare ox hair detection antibody as claimed in claim 7, it is characterised in that the concentration of phosphate buffered solution used in step e is 0.05mol/L, pH is 7.4。
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Application publication date: 20160622 |