CN105646706A - Preparing method for sheep keratin antibody - Google Patents
Preparing method for sheep keratin antibody Download PDFInfo
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- CN105646706A CN105646706A CN201610078877.1A CN201610078877A CN105646706A CN 105646706 A CN105646706 A CN 105646706A CN 201610078877 A CN201610078877 A CN 201610078877A CN 105646706 A CN105646706 A CN 105646706A
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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Abstract
The invention relates to the technical field of historical relics and archaeology, and discloses a preparing method for a sheep keratin antibody. The preparing method for the sheep keratin antibody comprises the first step of keratin extracting, the second step of antigen preparing, immunizing and blood collecting, the third step of antiserum separating and the fourth step of antibody preparing. The sheep keratin antibody prepared through the method is high in reaction sensitivity on sheep keratin, and an extremely small quantity of antigens is needed.
Description
Technical field
The present invention relates to archaeology of cultural relic technical field, particularly relate to wool product historical relic detection field.
Background technology
Sheep's wool is a kind of natural animal hair fibre. There is collenchyme, present gloss, tough and tensile and flexible. Because its output height, kind are many, multiple woolen knitwear can be produced, it is main woolen industry raw material.
Chinese history is long, and being unearthed from the grave that China is early stage has a large amount of wool products, and these, wool product contained a large amount of archaeological informations in ancient times. Usually to the qualification of wool product mainly to the qualification of wool keratin. Wool keratin is a kind of hardening, not soluble protein, wherein existing containing carboxyl etc. at interior acidic groups, also have amino-contained, imido grpups etc. are at interior basic group, so wool all has certain unstable in acid, alkali, salt, oxygenant, reductive agent, halogen etc. The discriminating of wool product is caused certain difficulty by this.
At present, protein detection technology such as the enzyme linked immunological technology combined based on Ag-Ab is a kind of extensively for the modern technique of historical relic detection, and the wool goat's horn protein antibodies preparation in early stage is a big difficult problem.
Summary of the invention
In order to solve the problems of the technologies described above, the present invention provides the preparation method of a kind of sheep keratin antibody. Sheep keratin antibody prepared by the inventive method is to the reaction sensitivity height of sheep Keratin sulfate, and the amount of antigen of required use is few.
The concrete technical scheme of the present invention is: the preparation method of a kind of sheep keratin antibody, and step is as follows:
A) prepare an aqueous potassium hydrogen sulfate of 5-7wt%, it is 3.5-5 with saturated sodium hydroxide solution by the pH regulator of an aqueous potassium hydrogen sulfate; Then by 1:(40-60) bath raio sheep's wool is added in solution, at 30-35 DEG C water-bath concussion 0.5-1.5h; Then take out wool and it is washed to neutrality; Wool is joined in the mixed solution A of its 50-90 times of quality, filter after reacting 3.5-4.5h at 95-105 DEG C, obtain thick keratin solution, by sheep keratin powder obtained after the dialysis of thick keratin solution, lyophilize.
The inventive method extracts obtained sheep keratin powder, and purity height is effective after being re-dubbed antigen liquid.
B) with physiological saline, above-mentioned obtained sheep keratin powder is mixed with the keratin solution of 0.75-1.25mg/mL, by keratin solution and QuickAntibody-Rabbit5W adjuvant 1:(0.8-1.1 by volume) mix, obtain immunizing antigen liquid, use immunizing antigen liquid that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity; During after initial immunity the 3rd, 5 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization; During after carrying out initial immunity the 7th, 9 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization again; When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood.
The QuickAntibody-Rabbit5W adjuvant that the present invention uses is nontoxic, not containing protein component, does not destroy antigen native configurations, and immunization ways is simple, it is not necessary to emulsification antigen, it is only necessary to simple mixing can be immune, the immunity cycle is short simultaneously, and antigen consumption is few.
C) from the blood of above-mentioned collection, separation obtains antiserum(antisera).
D) albumin A filler is mixed with phosphate buffer soln, after stirring, vacuumize 20-25min, obtain the albumin A filler liquid of bubble-free; Albumin A filler liquid is joined in glass column under air bubble free conditions, obtains albumen post; Albumen post is cleaned; Above-mentioned obtained antiserum(antisera) is diluted, joins after dilution and albumen post carries out loading, repeat loading once; Albumen post is cleaned again, carries out wash-out with glycine solution antagonist, after wash-out, clean albumen post, obtained antibody.
The antibody that the present invention adopts the preparation of sheep Keratin sulfate and obtains has stronger specificity, and this antibody can have obvious immune response with sheep Keratin sulfate, highly sensitive, it is possible to the sheep keratin molecule ruptured in sheep's wool goods ancient times makes detection.
As preferably, rapid A) in described mixed solution A be made up of sodium bisulfite, urea, Sodium dodecylbenzene sulfonate and water; Wherein the concentration of sodium bisulfite is 5wt%, and the concentration of urea is 6M, and the concentration of Sodium dodecylbenzene sulfonate is 1wt%.
In the mixed solution A of the present invention, sodium bisulfite can open rapidly the disulfide linkage in wool sulfoprotein, makes wool scale layer impaired, thus be beneficial to solvent and enter lamina corticalis. The speed that can accelerate to dissolve wool of urea. And Sodium dodecylbenzene sulfonate can reduce solution surface tension, impel solution to penetrate into wool inside, increase the dissolution rate of wool. The sodium ion of sodium bisulfite and Sodium dodecylbenzene sulfonate has strong hydratability simultaneously, wool is produced swelling action so that the material stripping in lamina corticalis. The present invention adopts anionic surfactant sodium dodecylbenzene sulfonate, and cost is low, and micelle-forming concentration is low, and consumption is few.
As preferably, steps A) in described mixed solution A be made up of sodium bisulfite, urea, saponin, Sodium dodecylbenzene sulfonate and water; Wherein the concentration of sodium bisulfite is 5wt%, and the concentration of urea is 6M, and the concentration of saponin is 0.5wt%, and the concentration of Sodium dodecylbenzene sulfonate is 0.8wt%.
Saponin in above-mentioned mixed solution A is natural nonionogenic tenside, after the composite use of Sodium dodecylbenzene sulfonate, the penetrating power of wool is stronger, and the extraction yield of Keratin sulfate is improved effect.
As preferably, steps A) described in the dialysis process of thick Keratin sulfate liquid solution as follows: thick Keratin sulfate liquid solution is loaded in the dialysis tubing that molecular weight cut-off is 14000, it is soaked in the deionized water of 50-100 times of quality by dialysis tubing to dialyse 4-5 days, change deionized water every 7-9h, finally obtain pure keratin solution.
As preferably, rabbit carrying out initial immunity, booster immunization and again in booster immunization process, the injection volume of each injection point is 100 �� L.
As preferably, in step C) in, being separated the method obtaining antiserum(antisera) from blood is: the blood collected is placed in set at room temperature, then solidificating blood is placed in the warm case of 36-38 DEG C and places 25-35min, the refrigerator 11-13h being placed in 4 DEG C again, makes clot fully shrink and obtain the thick antiserum(antisera) that precipitates out completely; Collecting thick antiserum(antisera), in 4 DEG C of environment, when 3000g, centrifugal 14-16min, gets supernatant liquor, is positioned over-80 DEG C of storages for subsequent use.
As preferably, step D) detailed process be:
By albumin A filler with phosphate buffer soln by 1.5g/(6-7) proportioning of mL mixes, vacuumizes 20-25min, obtain the albumin A filler liquid of bubble-free after stirring; Then the albumin A filler liquid of 6-7 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post; Being cleaned by albumen post with the phosphate buffer soln of 20 parts by volume, cleaning flow velocity is 60mL/h; With phosphate buffer soln, the antiserum(antisera) of 15 parts by volume is diluted to 20 parts by volume, joins after dilution and albumen post carries out loading, repeat loading once; Again being cleaned by albumen post with the phosphate buffer soln of 30 parts by volume, cleaning flow velocity is 60mL/h; With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out wash-out; Wash albumen post with phosphate buffer soln after wash-out, obtained antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations.
As preferably, step D) in the concentration of phosphate buffer soln used be 0.05mol/L, pH be 7.4.
It is compared with the prior art, the invention has the beneficial effects as follows: sheep keratin antibody prepared by the inventive method is to the reaction sensitivity height of sheep Keratin sulfate, and the amount of antigen of required use is few.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
A preparation method for sheep keratin antibody, step is as follows:
A) prepare an aqueous potassium hydrogen sulfate of 6wt%, it is 4 with saturated sodium hydroxide solution by the pH regulator of an aqueous potassium hydrogen sulfate. Then by the bath raio of 1:50, sheep's wool is added in solution, water-bath concussion 1h at 32 DEG C. Then take out wool and with distilled water washing filtering until water lotion pH value is 7. Being joined by wool in the mixed solution A of its 70 times of quality, the concentration of described mixed solution A sulfite hydrogen sodium is 5wt%, and the concentration of urea is 6M, and the concentration of Sodium dodecylbenzene sulfonate is 1wt%. Filter after reacting 4h at 100 DEG C, obtain thick keratin solution, thick keratin solution is loaded in the dialysis tubing that molecular weight cut-off is 14000, dialysis tubing is soaked in the deionized water of 50-100 times of quality and dialyses 4.5 days, deionized water is changed every 8h, obtain pure keratin solution, obtained sheep keratin powder after lyophilize.
B) with physiological saline, above-mentioned obtained sheep keratin powder is mixed with the keratin solution of 1mg/mL, by keratin solution and QuickAntibody-Rabbit5W adjuvant by volume 1:0.95 mix, obtain immunizing antigen liquid, use immunizing antigen liquid that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity; During after initial immunity the 3rd, 5 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization; During after carrying out initial immunity the 7th, 9 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization again. Rabbit is carrying out initial immunity, booster immunization and again in booster immunization process, and the injection volume of each injection point is 100 �� L. When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood.
C) blood collected is placed in set at room temperature, then solidificating blood is placed in the warm case of 37 DEG C and places 30min, then be placed in the refrigerator 12h of 4 DEG C, make clot fully shrink and obtain the thick antiserum(antisera) that precipitates out completely; Collecting thick antiserum(antisera), in 4 DEG C of environment, the centrifugal 15min when 3000g, gets supernatant liquor and is antiserum(antisera), is positioned over-80 DEG C of storages for subsequent use.
D) albumin A filler is mixed by the proportioning of 1.5g/6.5mL with phosphate buffer soln, after stirring, vacuumize 22min, obtain the albumin A filler liquid of bubble-free; Then the albumin A filler liquid of 6.5 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post; Being that albumen post is cleaned by 0.05mol/L phosphate buffer soln by the concentration of 20 parts by volume, cleaning flow velocity is 60mL/h; It is that the antiserum(antisera) of 15 parts by volume is diluted to 20 parts by volume by the phosphate buffer soln of 0.05mol/L by concentration, joins after dilution and albumen post carries out loading, repeat loading once; Being that albumen post is cleaned by the phosphate buffer soln of 0.05mol/L again by the concentration of 30 parts by volume, cleaning flow velocity is 60mL/h; With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out wash-out; With the phosphate buffer soln of 0.05mol/L washing albumen post after wash-out, obtained antibody, is loaded in centrifuge tube by antibody and stand-by in 4 DEG C of preservations. The concentration of above-mentioned phosphate buffer soln used is 0.05mol/L, pH is 7.4pH.
E) specific detection: get steps A) obtained sheep Keratin sulfate mass concentration be 1% the bovine serum albumin solution concentration that is mixed with 1 �� g/mL be coated in enzyme plate 12h, wash three times with the phosphate buffer soln of pH7.4. Every hole adds 200 �� L, and mass concentration is bovine serum albumin solution closed 2h at 37 DEG C of 1%; 3 times are washed with the phosphate buffer soln of pH7.4, then it is the bovine serum albumin solution dilution 1:240000 of 1% respectively by mass concentration by sheep keratin antibody, 1:60000,1:20000,1:10000,1:5000, doubly, every hole adds 100 �� L sheep keratin antibodies to 1:1000,1:200, hatch 1h at 37 DEG C, wash 5 times with the phosphate buffer soln of pH7.4; The horseradish peroxidase mark two that every hole adds 100 �� L resists, and hatches 1h at 37 DEG C, described two anti-employing mass concentrations be 1% bovine serum albumin dilute 5000 times; Last every hole add the mass concentration of 100 �� L be 1% tetramethyl biphenyl amine aqueous solution in the colour developing of dark place, after 10min, add the H of 100 �� L2mol/L2SO4Termination reaction; OD is measured by microplate reader450nm, according to OD450nmValue, judges situations such as the associativities of antigen-antibody; Just can determine when OD value reaches positive criteria in sample containing sheep's wool; If containing sheep's wool in sample, then OD will lower than positive criteria, according to this standard, it can be determined that the existence of sheep Keratin sulfate.
Result display except blank be colourless except, sample hole is yellow. The result of the absorbancy measuring OD450nm place with enzyme plate is: the OD obtained after the antibody after purifying carries out 240000,60000,20000,10000,5000,1000,200 times of dilutions450nmValue is respectively 0.287,0.496,0.959,1.414,1.545,1.863,2.169, and the OD value of blank is 0.045. According to platform OD450nmValue reaches 0.6 for positive requirement, even if by the antibody dilution to 20000 times of preparation, the positive signal of sheep Keratin sulfate still being detected, develop the color clear, result is clear and definite. From above result it can be shown that, the antibody prepared by the inventive method is extremely high to the reaction sensitivity at sheep angle, and required amount of antigen is few, and easy and simple to handle, drops into little, should large-scale promotion utilize.
Embodiment 2
A preparation method for sheep keratin antibody, step is as follows:
A) prepare an aqueous potassium hydrogen sulfate of 6wt%, it is 4.2 with saturated sodium hydroxide solution by the pH regulator of an aqueous potassium hydrogen sulfate. Then by the bath raio of 1:50, sheep's wool is added in solution, water-bath concussion 1h at 33 DEG C. Then take out wool and with distilled water washing filtering until water lotion pH value is 7. Being joined by wool in the mixed solution A of its 70 times of quality, the concentration of described mixed solution A sulfite hydrogen sodium is 5wt%, and the concentration of urea is 6M, and the concentration of saponin is 0.5wt%, and the concentration of Sodium dodecylbenzene sulfonate is 0.8wt%. Filter after reacting 4h at 100 DEG C, obtain thick keratin solution, thick keratin solution is loaded in the dialysis tubing that molecular weight cut-off is 14000, dialysis tubing is soaked in the deionized water of 50-100 times of quality and dialyses 4.5 days, deionized water is changed every 8h, obtain pure keratin solution, obtained sheep keratin powder after lyophilize.
B) with physiological saline, above-mentioned obtained sheep keratin powder is mixed with the keratin solution of 1mg/mL, by keratin solution and QuickAntibody-Rabbit5W adjuvant by volume 1:1 mix, obtain immunizing antigen liquid, use immunizing antigen liquid that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity; During after initial immunity the 3rd, 5 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization; During after carrying out initial immunity the 7th, 9 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization again. Rabbit is carrying out initial immunity, booster immunization and again in booster immunization process, and the injection volume of each injection point is 100 �� L. When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood.
C) blood collected is placed in set at room temperature, then solidificating blood is placed in the warm case of 37 DEG C and places 30min, then be placed in the refrigerator 12h of 4 DEG C, make clot fully shrink and obtain the thick antiserum(antisera) that precipitates out completely; Collecting thick antiserum(antisera), in 4 DEG C of environment, the centrifugal 15min when 3000g, gets supernatant liquor and is antiserum(antisera), is positioned over-80 DEG C of storages for subsequent use.
D) albumin A filler is mixed by the proportioning of 1.5g/6.5mL with phosphate buffer soln, after stirring, vacuumize 23min, obtain the albumin A filler liquid of bubble-free; Then the albumin A filler liquid of 6.5 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post; Being that albumen post is cleaned by 0.05mol/L phosphate buffer soln by the concentration of 20 parts by volume, cleaning flow velocity is 60mL/h; It is that the antiserum(antisera) of 15 parts by volume is diluted to 20 parts by volume by the phosphate buffer soln of 0.05mol/L by concentration, joins after dilution and albumen post carries out loading, repeat loading once; Being that albumen post is cleaned by the phosphate buffer soln of 0.05mol/L again by the concentration of 30 parts by volume, cleaning flow velocity is 60mL/h; With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out wash-out; With the phosphate buffer soln of 0.05mol/L washing albumen post after wash-out, obtained antibody, is loaded in centrifuge tube by antibody and stand-by in 4 DEG C of preservations. The concentration of above-mentioned phosphate buffer soln used is 0.05mol/L, pH is 7.4.
Embodiment 3
A preparation method for sheep keratin antibody, step is as follows:
A) prepare an aqueous potassium hydrogen sulfate of 5wt%, it is 3.5 with saturated sodium hydroxide solution by the pH regulator of an aqueous potassium hydrogen sulfate. Then by the bath raio of 1:40, sheep's wool is added in solution, water-bath concussion 1.5h at 30 DEG C. Then take out wool and with distilled water washing filtering until water lotion pH value is 7. Being joined by wool in the mixed solution A of its 50 times of quality, the concentration of described mixed solution A sulfite hydrogen sodium is 5wt%, and the concentration of urea is 6M, and the concentration of saponin is 0.5wt%, and the concentration of Sodium dodecylbenzene sulfonate is 0.8wt%. Filter after reacting 4.5h at 95 DEG C, obtain thick keratin solution, thick keratin solution is loaded in the dialysis tubing that molecular weight cut-off is 14000, dialysis tubing is soaked in the deionized water of 50 times of quality and dialyses 5 days, deionized water is changed every 7h, obtain pure keratin solution, obtained sheep keratin powder after lyophilize.
B) with physiological saline, above-mentioned obtained sheep keratin powder is mixed with the keratin solution of 0.75mg/mL, by keratin solution and QuickAntibody-Rabbit5W adjuvant by volume 1:0.8 mix, obtain immunizing antigen liquid, use immunizing antigen liquid that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity; During after initial immunity the 3rd, 5 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization; During after carrying out initial immunity the 7th, 9 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization again. Rabbit is carrying out initial immunity, booster immunization and again in booster immunization process, and the injection volume of each injection point is 100 �� L. When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood.
C) blood collected is placed in set at room temperature, then solidificating blood is placed in the warm case of 36 DEG C and places 35min, then be placed in the refrigerator 11h of 4 DEG C, make clot fully shrink and obtain the thick antiserum(antisera) that precipitates out completely; Collecting thick antiserum(antisera), in 4 DEG C of environment, the centrifugal 14min when 3000g, gets supernatant liquor and is antiserum(antisera), is positioned over-80 DEG C of storages for subsequent use.
D) albumin A filler is mixed by the proportioning of 1.5g/6mL with phosphate buffer soln, after stirring, vacuumize 20min, obtain the albumin A filler liquid of bubble-free; Then the albumin A filler liquid of 6 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post; Being that albumen post is cleaned by 0.05mol/L phosphate buffer soln by the concentration of 20 parts by volume, cleaning flow velocity is 60mL/h; It is that the antiserum(antisera) of 15 parts by volume is diluted to 20 parts by volume by the phosphate buffer soln of 0.05mol/L by concentration, joins after dilution and albumen post carries out loading, repeat loading once; Being that albumen post is cleaned by the phosphate buffer soln of 0.05mol/L again by the concentration of 30 parts by volume, cleaning flow velocity is 60mL/h; With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out wash-out; With the phosphate buffer soln of 0.05mol/L washing albumen post after wash-out, obtained antibody, is loaded in centrifuge tube by antibody and stand-by in 4 DEG C of preservations. The concentration of above-mentioned phosphate buffer soln used is 0.05mol/L, pH is 7.4.
Embodiment 4
A preparation method for sheep keratin antibody, step is as follows:
A) prepare an aqueous potassium hydrogen sulfate of 7wt%, it is 5 with saturated sodium hydroxide solution by the pH regulator of an aqueous potassium hydrogen sulfate. Then by the bath raio of 1:60, sheep's wool is added in solution, water-bath concussion 0.5h at 35 DEG C. Then take out wool and with distilled water washing filtering until water lotion pH value is 7. Being joined by wool in the mixed solution A of its 90 times of quality, the concentration of described mixed solution A sulfite hydrogen sodium is 5wt%, and the concentration of urea is 6M, and the concentration of saponin is 0.5wt%, and the concentration of Sodium dodecylbenzene sulfonate is 0.8wt%. Filter after reacting 3.5h at 105 DEG C, obtain thick keratin solution, thick keratin solution is loaded in the dialysis tubing that molecular weight cut-off is 14000, dialysis tubing is soaked in the deionized water of 100 times of quality and dialyses 4 days, deionized water is changed every 9h, obtain pure keratin solution, obtained sheep keratin powder after lyophilize.
B) with physiological saline, above-mentioned obtained sheep keratin powder is mixed with the keratin solution of 1.25mg/mL, by keratin solution and QuickAntibody-Rabbit5W adjuvant by volume 1:1.1 mix, obtain immunizing antigen liquid, use immunizing antigen liquid that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity; During after initial immunity the 3rd, 5 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization; During after carrying out initial immunity the 7th, 9 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization again. Rabbit is carrying out initial immunity, booster immunization and again in booster immunization process, and the injection volume of each injection point is 100 �� L. When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood.
C) blood collected is placed in set at room temperature, then solidificating blood is placed in the warm case of 38 DEG C and places 25min, then be placed in the refrigerator 13h of 4 DEG C, make clot fully shrink and obtain the thick antiserum(antisera) that precipitates out completely; Collecting thick antiserum(antisera), in 4 DEG C of environment, the centrifugal 16min when 3000g, gets supernatant liquor and is antiserum(antisera), is positioned over-80 DEG C of storages for subsequent use.
D) albumin A filler is mixed by the proportioning of 1.5g/7mL with phosphate buffer soln, after stirring, vacuumize 25min, obtain the albumin A filler liquid of bubble-free; Then the albumin A filler liquid of 7 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post; Being that albumen post is cleaned by 0.05mol/L phosphate buffer soln by the concentration of 20 parts by volume, cleaning flow velocity is 60mL/h; It is that the antiserum(antisera) of 15 parts by volume is diluted to 20 parts by volume by the phosphate buffer soln of 0.05mol/L by concentration, joins after dilution and albumen post carries out loading, repeat loading once; Being that albumen post is cleaned by the phosphate buffer soln of 0.05mol/L again by the concentration of 30 parts by volume, cleaning flow velocity is 60mL/h; With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out wash-out; With the phosphate buffer soln of 0.05mol/L washing albumen post after wash-out, obtained antibody, is loaded in centrifuge tube by antibody and stand-by in 4 DEG C of preservations. The concentration of above-mentioned phosphate buffer soln above-mentioned used used is 0.05mol/L, pH is 7.4.
Raw materials used, equipment in the present invention, unless otherwise noted, is conventional raw material, the equipment of this area; Method therefor in the present invention, unless otherwise noted, is the ordinary method of this area.
The above; it it is only the better embodiment of the present invention; not the present invention being imposed any restrictions, every any simple modification, change and equivalent transformation above embodiment done according to the technology of the present invention essence, all still belongs to the protection domain of technical solution of the present invention.
Claims (8)
1. the preparation method of a sheep keratin antibody, it is characterised in that step is as follows:
A) prepare an aqueous potassium hydrogen sulfate of 5-7wt%, it is 3.5-5 with saturated sodium hydroxide solution by the pH regulator of an aqueous potassium hydrogen sulfate; Then by 1:(40-60) bath raio sheep's wool is added in solution, at 30-35 DEG C water-bath concussion 0.5-1.5h; Then take out wool and it is washed to neutrality; Wool is joined in the mixed solution A of its 50-90 times of quality, filter after reacting 3.5-4.5h at 95-105 DEG C, obtain thick keratin solution, by sheep keratin powder obtained after the dialysis of thick keratin solution, lyophilize;
B) with physiological saline, above-mentioned obtained sheep keratin powder is mixed with the keratin solution of 0.75-1.25mg/mL, by keratin solution and QuickAntibody-Rabbit5W adjuvant 1:(0.8-1.1 by volume) mix, obtain immunizing antigen liquid, use immunizing antigen liquid that rabbit is carried out subcutaneous multi-point injection, carry out initial immunity; During after initial immunity the 3rd, 5 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization; During after carrying out initial immunity the 7th, 9 week, it may also be useful to rabbit is carried out subcutaneous multi-point injection by immunizing antigen liquid, carries out booster immunization again; When the antibody titer in rabbit blood sample reaches 1:20000, collect rabbit blood;
C) from the blood of above-mentioned collection, separation obtains antiserum(antisera);
D) albumin A filler is mixed with phosphate buffer soln, after stirring, vacuumize 20-25min, obtain the albumin A filler liquid of bubble-free; Albumin A filler liquid is joined in glass column under air bubble free conditions, obtains albumen post; Albumen post is cleaned; Above-mentioned obtained antiserum(antisera) is diluted, joins after dilution and albumen post carries out loading, repeat loading once; Albumen post is cleaned again, carries out wash-out with glycine solution antagonist, after wash-out, clean albumen post, obtained antibody.
2. the preparation method of a kind of sheep keratin antibody as claimed in claim 1, it is characterised in that, steps A) in described mixed solution A be made up of sodium bisulfite, urea, Sodium dodecylbenzene sulfonate and water; Wherein the concentration of sodium bisulfite is 5wt%, and the concentration of urea is 6M, and the concentration of Sodium dodecylbenzene sulfonate is 1wt%.
3. the preparation method of a kind of sheep keratin antibody as claimed in claim 1, it is characterised in that, steps A) in described mixed solution A be made up of sodium bisulfite, urea, saponin, Sodium dodecylbenzene sulfonate and water; Wherein the concentration of sodium bisulfite is 5wt%, and the concentration of urea is 6M, and the concentration of saponin is 0.5wt%, and the concentration of Sodium dodecylbenzene sulfonate is 0.8wt%.
4. the preparation method of a kind of sheep keratin antibody as described in claim 1 or 2 or 3, it is characterized in that, steps A) described in the dialysis process of thick Keratin sulfate liquid solution as follows: thick Keratin sulfate liquid solution is loaded in the dialysis tubing that molecular weight cut-off is 14000, it is soaked in the deionized water of 50-100 times of quality by dialysis tubing to dialyse 4-5 days, change deionized water every 7-9h, finally obtain pure keratin solution.
5. the preparation method of a kind of sheep keratin antibody as claimed in claim 1, it is characterised in that, rabbit is being carried out initial immunity, booster immunization and again in booster immunization process, the injection volume of each injection point is 100 �� L.
6. the preparation method of a kind of sheep keratin antibody as claimed in claim 1, it is characterized in that, in step C) in, being separated the method obtaining antiserum(antisera) from blood is: the blood collected is placed in set at room temperature, then solidificating blood is placed in the warm case of 36-38 DEG C and places 25-35min, the refrigerator 11-13h being placed in 4 DEG C again, makes clot fully shrink and obtain the thick antiserum(antisera) that precipitates out completely; Collecting thick antiserum(antisera), in 4 DEG C of environment, when 3000g, centrifugal 14-16min, gets supernatant liquor, is positioned over-80 DEG C of storages for subsequent use.
7. the preparation method of a kind of sheep keratin antibody as described in claim 1 or 6, it is characterised in that, step D) detailed process be:
By albumin A filler with phosphate buffer soln by 1.5g/(6-7) proportioning of mL mixes, vacuumizes 20-25min, obtain the albumin A filler liquid of bubble-free after stirring; Then the albumin A filler liquid of 6-7 parts by volume is joined in glass column under air bubble free conditions, obtain albumen post; Being cleaned by albumen post with the phosphate buffer soln of 20 parts by volume, cleaning flow velocity is 60mL/h; With phosphate buffer soln, the antiserum(antisera) of 15 parts by volume is diluted to 20 parts by volume, joins after dilution and albumen post carries out loading, repeat loading once; Again being cleaned by albumen post with the phosphate buffer soln of 30 parts by volume, cleaning flow velocity is 60mL/h; With concentration to be 0.2mol/L, pH be 3.0 glycine solution antagonist carry out wash-out; Wash albumen post with phosphate buffer soln after wash-out, obtained antibody, antibody is loaded in centrifuge tube and stand-by in 4 DEG C of preservations.
8. the preparation method of a kind of sheep keratin antibody as claimed in claim 7, it is characterised in that, step D) in the concentration of phosphate buffer soln used be 0.05mol/L, pH be 7.4.
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| CN113004387A (en) * | 2019-12-19 | 2021-06-22 | 中国医学科学院药物研究所 | Sheep keratin, preparation method thereof, pharmaceutical composition thereof and application |
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