CN101158681A - Complementary type affinity chromatography purification method of autoantibody - Google Patents
Complementary type affinity chromatography purification method of autoantibody Download PDFInfo
- Publication number
- CN101158681A CN101158681A CNA2007101902436A CN200710190243A CN101158681A CN 101158681 A CN101158681 A CN 101158681A CN A2007101902436 A CNA2007101902436 A CN A2007101902436A CN 200710190243 A CN200710190243 A CN 200710190243A CN 101158681 A CN101158681 A CN 101158681A
- Authority
- CN
- China
- Prior art keywords
- post
- liquid
- affinity chromatography
- antibody
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a complementary affinity chromatography purified method, which can highly extract a polyclonal antibody of the autologous antigen of purification anti-organization cell from the serum of the people or the serum of the immune animal. For the autologous antigen which has more components in the histiocyte and is not suitable to be expressed by the genetic engineering, and the autologous antigen got from the monoclonal antibody affinity chromatography, the relative autologous antibodies are various, and the autologous antigen is suitable for the purification of the polyclonal antibody of the invention. The high specificity autologou antibody can be used as an antibody standard, and the antibody standard is utilized to produce quantitative detection kits of autologou antibody, and the producing is based on the empirical approaches of various immunologies, and the quantitative detection kits have very great value of diagnosis, treatment, prognosis and curative effect observation of the autoimmune disease. In the practice of purifying anti-sperm antibody, the method is scientific and feasible, and the cost is low, and the immunity anti-sperm polyclonal antibody got from the practice has great purity quotient and specificity.
Description
Technical field
The present invention be a kind of be the technical scheme of the specificity purifying autoantibody of main innovate point with complementary immunoaffinity chromatography, be particularly useful for be unwell to the polyclonal antibody purifying of the autoantigen that obtains with gene engineering expression and monoclonal antibody affinity chromatography, the immunological technique field that belongs to the antigen-antibody preparation and detect in the histocyte because of composition is more.
Background technology
Have autoantigen in a lot of people's body, they are of a great variety, can stimulate body immune system generation immune response, produce antibody and sensitized lymphocyte at autoantigen, cause a lot of autoimmune diseases therefrom.How from the histocyte of body or body fluid, extract these autoantigens of purifying, difficult always technically, subject matter is that the antigentic specificity that obtains is not high, be difficult for rejecting other normal structure compositions of body, generally have very high nonspecific reaction when therefore utilizing these autoantigens to make kit detection specificity autoantibody, promptly false positive is more.The antibody of these autoantigens of purifying difficulty more just specifically from body serum how is especially at the antibody of the numerous autoantigen of those compositions.Owing to be difficult to obtain the standard items of the very high autoantibody of specificity and purity, the detection by quantitative of autoantibody just is difficult to carry out, therefore at present the detection by quantitative kit of domestic and international most autoantibodies all can only sxemiquantitative, with the relative content of relative unit number as autoantibody in the body body fluid, and each producer defines relative unit number separately voluntarily, and is not comparable mutually.
For example, do not breed the 8-15% that disease accounts for China married couple, wherein a lot of Mr. and Mrs are because body to sperm antigen and endometrium antigen etc. immune response has taken place, AsAb that produces and AEA can destroy sperm and endometrium cause infertile, therefore detect these autoantibodies to have or not with content be the important indicator of infertility diagnosis and observation of curative effect.But detecting antibody must have specific antigen.Autoantigen such as purifying sperm antigen, endometrium antigen how? the domestic and foreign literature reported method mainly contains in recent years: physical methods such as ultrasonic-wave crushing, the broken spermatoblast of multigelation or endometrial cell; Chemical extraction method or collagenase digestions such as chemical reagent such as LTS, DTT, NP-40 and Triton X-100; Gene cloning and expression list kind sperm antigen and utilize method of purification of Monoclonal Antibody affinity column etc.
The specificity of the two kinds of method purifying gained antigens in back is the highest, but shortcoming is also very obvious.Technique for gene engineering can only the clonal expression one-component antigen, and the composition of a lot of autoantigens is various, as sperm antigen kind surplus in the of 100 nearly, endometrium antigen also kind is numerous, the AsAb and the AEA that detect in the body with the antigen that only comprises the only a few component of gene cloning and expression very easily cause omission, thereby still far away from practical application; Another shortcoming is that a lot of autoantigens are all unclear from the gene to the protein sequence, can not carry out gene cloning and expression.Utilize the Monoclonal Antibody affinity column to carry out affinity chromatography, autoantigen that equally also can only the purifying one-component, and need the preparation monoclonal antibody.The preparation of monoclonal antibody not only technology is numerous and diverse, many times can't carry out the screening of monoclonal antibody as target antigen with body tissue cell that contains purpose antigen or body fluid, thereby can't obtain monoclonal antibody, also therefore a lot of autoantigens can't come purifying by the affinity chromatography of monoclonal antibody.The autoantigen purity that physics method and chemical method extract is relatively poor, the nonspecific reaction of other compositions in extremely difficult removing and the body serum.
Aspect the purifying of autoantibody, domestic rare report abroad mainly is the monoclonal antibody of preparation at the autoantigen of gene engineering expression.Clone's number of these monoclonal antibodies is limited, is used for scientific research mostly.As the detection by quantitative of other any antibody, the detection by quantitative of AsAb also needs the standard items of AsAb.But human sperm's antigen is nearly hundreds of, and great majority fail to carry out that gene is determined and clonal expression, therefore can't utilize gene recombinant antigens to filter out so numerous monoclonal or polyclonal antibodies.The synantigen composition is not induced the antibody of generation, and its affinity is widely different.Promptly enable to obtain a few genetic recombination sperm antigen, the monoclonal antibody that it is corresponding or the potpourri of several monoclonal antibodies can only combine with the only a few antigenic determinant of sperm, therefore they are when combining with all quite complicated antigen of the composition of bag quilt and determinant on the solid phase carrier, probability is very little, and their affinity also is not enough to represent the affinity of the corresponding antibody of hundreds of kind sperm antigen in the body simultaneously.This affinity difference will directly cause detection by quantitative result's difference.By contrast, from antiserum, extract the artificial active immunity of purifying or the polyclonal antibody that the natural immunity state produces down, because of its at the antigen composition and the diversity and the randomness of determinant, more can represent in the detection by quantitative binding ability of antibody and antigen in the sample to be measured, therefore be more suitable for the antibody standard substance in as the detection by quantitative autoantibody time.
Calendar year 2001, we have reported and have utilized two kinds of polyclonal antibody preparation two kinds of affinity columns (A post and B post), people's sperm membrane antigen is carried out complementary type affinity chromatography purification, obtained the very high sperm membrane antigen of purity, made the qualitative enzyme mark detection kit of AsAb, pass through the on probation of 4 tame units such as Nanjing, Nanjing hospital of andrology, Zhengzhou Bo Sai bio-engineering corporation, and participated in Southeast China University's first scientific and technological achievement exhibition.(Shen is transmitted, Zhang Jianqiong, Jia Limin etc.The method of affinitive layer purification sperm membrane antigen and the detection of sperm antibody, Shanghai Journal of Immunology, 2001; 21:102-104).After 2002, for high degree of specificity ground purifying AsAb from patients serum or immune serum, with further preparation AsAb detection by quantitative kit, we begin to improve the purification scheme of sperm antigen, and further design the purification scheme of sperm antibody on this basis.Finally, we have prepared affinity chromatography A, B, C post purifying sperm antigen, prepare affinity chromatography D, E, F post again, the anti-sperm polyclonal antibody that further from the rabbit anti-serum of anti-people's sperm, extracted purifying, and the quantitative enzyme mark that is used for sperm antibody detects.Immunoblot experiment confirms that the anti-sperm polyclonal antibody of gained does not react with the composition that normal person's whole serum, health adult tissue's cell pyrolysis liquid and bovine serum albumin(BSA) etc. may appear in the detection system, for its antibody standard substance that becomes in the autoantibody quantitative test is laid a good foundation.Still do not have the sperm antibody detection by quantitative kit based on antibody standard substance in the market, having only is the half-quantitative detection kit of relative measurement unit from external import with IU/ml.
Up to now, there are not other people to report the polyclonal antibody that utilizes the present invention program from human serum or immune serum, to extract the anti-autoantigen of purifying.
Summary of the invention
Technical matters: the object of the invention is to provide a kind of complementary type affinity chromatography purification method of autoantibody, the autoantibody of adopting said method purifying is specificity and based on very high purity not only, and component is numerous, is specially adapted to the purifying of the autoantibody that the more autoantigen of composition induces.
Technical scheme: this scheme has been avoided numerous and diverse technology such as gene cloning and expression and Monoclonal Antibody, only need preparation polyclonal antibody, haemocyanin, histiocyte lysate capable and affinity column, easy and simple to handle, expend cheap, laboratory condition is less demanding, and is time saving and energy saving relatively.The polyclone autoantibody of this programme purifying gained can be used as antibody standard substance, be used for making autoantibody detection by quantitative kit, diagnosis, treatment, prognosis and the observation of curative effect of autoimmune disease had important value based on all kinds of immunological experiment methods.
In order to solve the technical barrier of purifying autoantibody from serum, we utilize 6 kinds of affinity columns of preparation such as polyclonal antibody, haemocyanin, histiocyte lysate capable, and having set up with the complementary type affinity chromatography is the purification process of main innovate point.
This method is:
At first prepare autoantigen with complementary type affinity chromatography method purifying:
11) collector's purpose histocyte extracts membrane antigen with NP-40, uses the ultrasonic-wave crushing histocyte again, gets supernatant behind the ultracentrifugation, as rough antigen A liquid,
12) with purpose histocyte immunization experiment animal, get antiserum, slightly carry the γ immunoglobulin (Ig), claim immune serum IgG through salting out method; Get simultaneously not by the pooled serum of this kind animal used as test of immunity, slightly carry the γ immunoglobulin (Ig), claim not immune serum IgG through salting out method; Mix whole serum immunization experiment animal with the normal person, get antiserum, slightly carry the γ immunoglobulin (Ig) through salting out method equally, claim anti-human whole serum IgG,
13) coupling becomes immunoaffinity chromatography A post, B post and C post with anti-human whole serum IgG with above-mentioned immune serum IgG, not immune serum IgG respectively with the Sepharose-4B Ago-Gel,
14) get rough antigen A liquid and cross affinity chromatography A post, collect the antigenic substance that combines with IgG on the A post, concentrating the back is rough antigen B liquid; Get rough antigen B liquid again and cross affinity chromatography B post, collect the antigenic substance that does not combine with IgG on the B post, concentrating the back is rough antigens c liquid; Get rough antigens c liquid at last and cross affinity chromatography C post, collect the antigenic substance that does not combine, concentrate the back and be refining antigen liquid with IgG on the C post; The polyclonal antibody for preparing anti-autoantigen then with complementary type affinity chromatography method purifying:
21) collect the patients serum of the autoantibody positive or with the antiserum of the animal used as test of autoantigen immunity inoculation; With salting out method and DEAE52 cellulose column IgG purification type and IgA type immunoglobulin (Ig), and be called rough IgG type antibody D liquid and rough IgA type antibody D liquid,
22) collection does not contain the healthy people's of purpose autoantigen pooled serum; Collect the people's who does not contain purpose antigen histocyte, and be prepared into histiocyte lysate capable,
23) get married and chromatography D post, E post and F post with above-mentioned refining antigen liquid, human tissue cell's lysate coupling of not containing healthy people's pooled serum of purpose antigen and not containing purpose antigen respectively with the Sepharose-4B Ago-Gel,
24) get rough IgG type or IgA type antibody D liquid is crossed affinity chromatography D post, collect the antibody materials that combines with autoantigen on the D post, concentrating afterwards is rough IgG type or IgA type antibody E liquid; The latter crosses affinity chromatography E post, collects the antibody materials that does not combine with albumen on the E post, and concentrating the back is rough IgG type or IgA type antibody F liquid; F liquid is collected the antibody materials that does not combine with albumen on the F post after affinity chromatography F post, concentrates the back and is refining autoantibody liquid IgG type or IgA type.
In step 13) and step 23) when preparing affinity chromatography A, B, C, D, E and F post, replace with the Ago-Gel of non-Sepharose-4B with the Sepharose-4B Ago-Gel.
In step 24) unite when carrying out complementary type affinity chromatography with affinity chromatography D, E and F post, only unite with the D post or with D post and E post.
Not to collect the purpose histocyte when step 11), but collect the people's of containing purpose antigen body fluid,, directly use the people's of containing purpose antigen humoral immunity animal used as test then without NP-40 extraction membrane antigen and ultrasonic-wave crushing histocyte.
Beneficial effect:
1. the invention provides a kind of from human serum or immune serum the technical scheme of specificity purifying autoantibody.
2. the autoantibody of using this programme purifying is specificity and based on very high purity not only.
3. this programme is specially adapted to the polyclone autoantibody that the more autoantigen of purifying composition is induced.Because the more autoantigen of composition is much all unclear from the gene to the protein sequence, can not carry out gene cloning and expression and obtain, can not all obtain by the affinity chromatography of monoclonal antibody.The autoantibody kind that body that these autoantigens are induced produces is numerous, because of antigen is difficult to obtain, also therefore be difficult to the purifying component numerous at autoantibody with a kind of autoantigen.
4. this programme has been avoided numerous and diverse technology such as gene cloning and expression and Monoclonal Antibody, and is easy and simple to handle, expends cheaply, and laboratory condition is less demanding, and is time saving and energy saving relatively.
5. the polyclone autoantibody of using this programme purifying gained can be used as antibody standard substance, be used for making autoantibody detection by quantitative kit based on all kinds of immunological experiment methods, diagnosis, treatment, prognosis and observation of curative effect to autoimmune disease have important value, therefore can create good economic benefits and social benefit.
Embodiment:
The complementary type affinity chromatography purification method of autoantibody of the present invention is:
At first prepare autoantigen with complementary type affinity chromatography method purifying:
11) collector's purpose histocyte extracts membrane antigen with NP-40, uses the ultrasonic-wave crushing histocyte again, gets supernatant behind the ultracentrifugation, as rough antigen A liquid,
12) with purpose histocyte immunization experiment animal, get antiserum, slightly carry the γ immunoglobulin (Ig), claim immune serum IgG through salting out method; Get simultaneously not by the pooled serum of this kind animal used as test of immunity, slightly carry the γ immunoglobulin (Ig), claim not immune serum IgG through salting out method; Mix whole serum immunization experiment animal with the normal person, get antiserum, slightly carry the γ immunoglobulin (Ig) through salting out method equally, claim anti-human whole serum IgG,
13) coupling becomes immunoaffinity chromatography A post, B post and C post with anti-human whole serum IgG with above-mentioned immune serum IgG, not immune serum IgG respectively with the Sepharose-4B Ago-Gel,
14) get rough antigen A liquid and cross affinity chromatography A post, collect the antigenic substance that combines with IgG on the A post, concentrating the back is rough antigen B liquid; Get rough antigen B liquid again and cross affinity chromatography B post, collect the antigenic substance that does not combine with IgG on the B post, concentrating the back is rough antigens c liquid; Get rough antigens c liquid at last and cross affinity chromatography C post, collect the antigenic substance that does not combine, concentrate the back and be refining antigen liquid with IgG on the C post; The polyclonal antibody for preparing anti-autoantigen then with complementary type affinity chromatography method purifying:
21) collect the patients serum of the autoantibody positive or with the antiserum of the animal used as test of autoantigen immunity inoculation; With salting out method and DEAE52 cellulose column IgG purification type and IgA type immunoglobulin (Ig), and be called rough IgG type antibody D liquid and rough IgA type antibody D liquid,
22) collection does not contain the healthy people's of purpose autoantigen pooled serum; Collect the people's who does not contain purpose antigen histocyte, and be prepared into histiocyte lysate capable,
23) get married and chromatography D post, E post and F post with above-mentioned refining antigen liquid, human tissue cell's lysate coupling of not containing healthy people's pooled serum of purpose antigen and not containing purpose antigen respectively with the Sepharose-4B Ago-Gel,
24) get rough IgG type or IgA type antibody D liquid is crossed affinity chromatography D post, collect the antibody materials that combines with autoantigen on the D post, concentrating afterwards is rough IgG type or IgA type antibody E liquid; The latter crosses affinity chromatography E post, collects the antibody materials that does not combine with albumen on the E post, and concentrating the back is rough IgG type or IgA type antibody F liquid; F liquid is collected the antibody materials that does not combine with albumen on the F post after affinity chromatography F post, concentrates the back and is refining autoantibody liquid IgG type or IgA type.
In step 13) and step 23) when preparing affinity chromatography A, B, C, D, E and F post with the Sepharose-4B Ago-Gel, the Ago-Gel of available non-Sepharose-4B replaces.
Carry out complementary type affinity chromatography with affinity chromatography A, B, C, D post, the purifying autoantibody; Carry out complementary type affinity chromatography with affinity chromatography A, B, C, D, E post, the purifying autoantibody.
Get the people's of containing purpose antigen body fluid, cell pyrolysis liquid or histocyte suspension, immunization experiment animal such as sheep, horse, rabbit, cavy, rat, mouse, pig etc. obtain immune serum IgG, IgA or IgM; From this kind animal used as test serum, obtain not immune serum IgG, IgA or IgM simultaneously; The same preparation affinity chromatography A, B, C post carry out complementary type affinity chromatography, from people's body fluid, in the cell membrane or surface of cell membrane obtain refining antigen liquid; The same again preparation affinity chromatography D, E and F post carry out complementary type affinity chromatography, obtain the polyclonal antibody of refining anti-people's autoantigen.
Purifying with anti-people's sperm autoantibody is the embodiment that example is set forth the present invention program below:
1. collector's purpose histocyte extracts membrane antigen with NP-40, uses the ultrasonic-wave crushing histocyte again, gets supernatant behind the ultracentrifugation, as rough antigen A liquid;
2. use purpose histocyte immunization experiment animal, get antiserum, slightly carry γ immunoglobulin (Ig) (IgG), claim immune serum IgG through salting out method; Get simultaneously not by the pooled serum of this kind animal used as test of immunity, slightly carry IgG, claim not immune serum IgG through salting out method; With normal person's whole serum immunization experiment animal, get antiserum, slightly carry IgG through salting out method equally, claim anti-human whole serum IgG;
3. get married and chromatography A post, B post and C post with above-mentioned immune serum IgG, not immune serum IgG and AHS IgG coupling respectively with the Sepharose-4B Ago-Gel;
4. get rough antigen A liquid and cross affinity chromatography A post, collect the antigenic substance that combines with IgG on the A post, concentrating the back is rough antigen B liquid; Get rough antigen B liquid again and cross affinity chromatography B post, collect the antigenic substance that does not combine with IgG on the B post, concentrating the back is rough antigens c liquid; Get rough antigens c liquid at last and cross affinity chromatography C post, collect the antigenic substance that does not combine, concentrate the back and be refining antigen liquid with IgG on the C post.
5. collect the patients serum of the autoantibody positive or with the antiserum of the animal used as test of autoantigen immunity inoculation; With salting out method and DEAE52 cellulose column purified blood serum IgG (γ immunoglobulin (Ig)) and serum IgA (alpha immunization globulin), and be called rough IgG type antibody D liquid and rough IgA type antibody D liquid;
6. collect the healthy people's who does not contain the purpose autoantigen pooled serum; Collect the people's who does not contain purpose antigen histocyte, and be prepared into histiocyte lysate capable;
7. get married and chromatography D post, E post and F post with above-mentioned refining antigen liquid, human tissue cell's lysate coupling of not containing healthy people's pooled serum of purpose antigen and not containing purpose antigen respectively with the Sepharose-4B Ago-Gel;
8. get rough IgG type or IgA type antibody D liquid is crossed affinity chromatography D post, collect the antibody materials that combines with autoantigen on the D post, concentrating afterwards is rough IgG type or IgA type antibody E liquid; The latter crosses affinity chromatography E post, collects the antibody materials that does not combine with albumen on the E post, and concentrating the back is rough IgG type or IgA type antibody F liquid; F liquid is collected the antibody materials that does not combine with albumen on the F post after affinity chromatography F post, concentrates the back and is refining autoantibody liquid (IgG or IgA type).Preservation was standby after vacuum freezedrying became pulvis.
The complementary type affinity chromatography purification method of AsAb
1. slightly carrying of people's sperm membrane antigen prepares rough antigen A liquid:
Collect fresh normal semen 100-500ml, use 0.01mol/L, the Tris-HCl damping fluid cyclic washing of pH7.4 6 times is got the sperm precipitation and is added antigen extract (Tris 0.061g, EDTA 0.012g, NaCl0.07g, NP-40 50ul, adding distil water is to 100ml, transfer pH9.0), 4 ℃ of magnetic agitation 30 minutes then add 4 kinds of protease inhibitors (PMSF, 1mmol/L in addition; Leupeptin, 10ug/ml; Aprotinin, 10ug/ml; Iodoacetamide, 1.8mg/ml), the packing test tube is put in the ice cup and is carried out ultrasonic-wave crushing, 300uA, 1min/ time, interval 2min, 12-15 time.Then centrifugal 1200rpm/min, 10min removes the residue fragment.Supernatant is put superspeed refrigerated centrifuge, the centrifugal 50min of 27000g.Supernatant after polyglycol concentrates, PBS solution dialysis 48h above rough antigen A liquid.Behind the BCA method protein quantification, divide device-70 ℃ preservation standby.
2. prepare immune serum IgG, not immune serum IgG and anti-human whole serum IgG:
Collect many people's fresh normal semen 100ml, with physiological saline washing 6 times, precipitation is resuspended with physiological saline, counting sperm, 2 of conventional subcutaneous multi-point injection experimental rabbits, immunity inoculation 3-4 time/, each 2 weeks at interval, 1 * 10
6Individual sperm/time.Whole rabbit anteserums were got on the 7th day in last immunity back, and it is standby to be sub-packed in-20 ℃ of preservations; Get an experimental rabbit without immunity inoculation simultaneously, get its whole serum, the same preservation is standby; Collect several bachelorettes' whole serum number milliliter in addition, be mixed into behind the Water-In-Oil sample 2 of conventional subcutaneous multi-point injection experimental rabbits with the Fu Shi Freund's complete adjuvant, 3-5 time/only, 1-2ml/ time/only, each 2 weeks of interval.From immunity for the second time, mix whole serum and freund 's incomplete adjuvant mixing.Whole rabbit anteserums were got on the 7th day in last immunity back, and it is standby to be sub-packed in-20 ℃ of preservations.
Get the rabbit anteserum of above-mentioned personnel selection sperm immunization, not by the rabbit anteserum of immunity and with the rabbit anteserum of human whole serum's immunity, extract total IgG type immunoglobulin (Ig)s through 33% ammonium sulfate salting-out process 4 ℃ of routines respectively, precipitation is dissolved the back in 4 ℃ of dialysed overnight with the PBS damping fluid, ℃ preservation of BCA method protein quantification postposition-20 is standby, is called immune serum IgG, not immune serum IgG and anti-human whole serum IgG.
3. prepare affinity chromatography A post, B post and C post:
Getting above-mentioned immune serum IgG, not immune serum IgG and anti-human whole serum IgG gets married and chromatography A post, B post and C post with the Sepharose-4B coupling of cyanogen bromide-activated respectively.Concrete grammar is as follows: the dried glue of Sepharose-4B of getting the 2-5g cyanogen bromide-activated is in beaker, dissolve with 1mM HCl solution, move to after waiting to expand in the funnel that suction filtration uses, take out with 1mM HCl solution and to wash 15min, then take out that to be washed till liquid level concordant with the agar gel face, make gel recovery gloss activity with the HCl solution of 100-200ml.Get above-mentioned serum IgG liquid, melt the aggregation that back 4 ℃ of ultracentrifugations are removed Ig, behind BCA method protein quantification, use 0.1mol/LNaHCO
3/ 0.5mol/L NaCl solution dilution IgG liquid is to 5mg/L.Isopyknic processing back gel and isopyknic IgG liquid are mixed in the beaker, put room temperature and constantly stir 1-2h.Use 5 times again to the 0.1mol/L of gel volume NaHCO
3/ 0.5mol/L NaCl solution is taken out and washed gel, and is concordant with the gel face to liquid level, then gel is dissolved in the 50mmol/L glycine solution of pH8.0, puts 4 ℃ of 16h, with remaining reactive group on the sealing gel.Dress is taken out Shen with 5 times to the 0.1M of gel volume AcetateBuffer (pH4.0)/0.5M NaCl behind the post in glass chromatography pipe, uses 5 times to take out and wash to the 0.1M of gel volume Tris-HClBuffer (pH8.0)/0.5M NaCl again, and repeats two circulations.At last that coupling is good affinity column is stored in the TSA solution, puts under 4 ℃ of conditions and can preserve 1-2.
4. complementary type affinity chromatography purification sperm membrane antigen:
The method of conventional affinity chromatography see " fine works molecular biology experiment guide " (Yan Ziying, Wang Hailin are translated, Beijing: Science Press, 1998:382).The key step of this complementary type affinity chromatography is:
1. get rough antigen A liquid and cross affinity chromatography A post, collect the antigenic substance that combines with IgG on the A post: get and the isopyknic rough antigen A liquid of A post gel (1-10mg/ml), room temperature effect 3h behind the upper prop, then wash the A post successively with following solution, flow velocity 1ml/min, till 0D280 absorbance＜0.02 of flowing out component: the lavation buffer solution of 5 times of bed volumes, pH8.0
The Tris damping fluid of 5 times of bed volumes, pH8.0
The Tris damping fluid of 5 times of bed volumes, pH9.0
Then with the triethanolamine solution wash-out A post of 5 times of bed volumes, till OD280 absorbance＜0.02 of flowing out component, flow velocity 1ml/min, collect eluent simultaneously, and use 1mol/L Tris-HCl (Ph6.7) damping fluid to neutralize immediately, in order to avoid the protein that Shen takes off sex change under the alkali condition, that is: the eluent with each bed volume is collected in the test tube of 1mol/L Tris-HCl (Ph6.7) damping fluid that contains 0.2 volume, and polyglycol concentrates about the adjust pH to 7.4 of back and is rough antigen B liquid.
2. get rough antigen B liquid and cross affinity chromatography B post, collect the antigenic substance that does not combine with IgG on the B post:
With B post on the rough antigen B liquid, room temperature effect 1h, with lavation buffer solution (pH8.0) the washing B post of 5 times of bed volumes, flow velocity 1ml/min is till OD280 absorbance＜0.02 of flowing out component; Use Tris damping fluid (pH8.0) the wash-out B post of 5 times of bed volumes at last, and collect eluent simultaneously, after polyglycol concentrates, be rough antigens c liquid.
3. get rough antigens c liquid at last and cross affinity chromatography C post, collect the antigenic substance that does not combine: with C post on the rough antigens c liquid with IgG on the C post, room temperature effect 1h, lavation buffer solution (pH8.0) washing C post with 5 times of bed volumes, flow velocity 1ml/min is till OD280 absorbance＜0.02 of flowing out component; Use Tris damping fluid (pH8.0) the wash-out C post of 5 times of bed volumes at last, and collect eluent simultaneously,, be refining antigen liquid more than the dialysis 24h through polyglycol simmer down to 1-5ml.BCA method protein quantification, the SDS-PAGE electrophoresis is identified purity and composition, divides device-70 ℃ preservation standby.
TSA preserves damping fluid: 0.002mol/L Tris-Cl; 0.14mol/L NaCl; 0.025% (W/V) NaN
31mmol/L EDTA; The 20ug/ml gentamicin, pH8.0, put 4 ℃ ice-cold
Lavation buffer solution: 0.01mol/L Tris-Cl; 0.14mol/L NaCl; 0.025% (W/V) NaN
30.5% (V/V) Triton X-100; 0.5% (V/V) NaTDC, pH8.0, put 4 ℃ ice-cold
Tris damping fluid: 50mmol/L Tris-Cl; 0.1% (V/V) Triton X-100; 0.5mol/L NaCl, pH8.0 or pH9.0, put 4 ℃ ice-cold
Triethanolamine damping fluid: 50mmol/L triethanolamine; 0.1% (V/V) Triton X-100; 0.15mol/LNaCl pH1 1.5, put 4 ℃ ice-cold
5. from the AsAb positive serum, extract total IgG and total IgA:
Collect the patients serum of the AsAb positive or the rabbit anteserum of personnel selection sperm immunization inoculation; In conjunction with DEAE52 cellulose column purifying total IgG type and IgA type immunoglobulin (Ig), and be called rough IgG type antibody D liquid and rough IgA type antibody D liquid with salting out method.
Total IgG extracts: get AsAb IgG type positive patient pooled serum or rabbit anti-serum, slightly carry IgG type immunoglobulin (Ig) through conventional 33% ammonium sulfate salting-out process, concrete steps see that (Yan Ziying, Wang Hailin are translated " fine works molecular biology experiment guide ", Beijing: Science Press, 1998:436).Cross DEAE52 cellulose chromatography post behind the dialysis desalination in batches, with 0.01M pH7.4 PBS buffer solution elution, collect eluent, the total IgG liquid through polyglycol simmer down to 2-4mg/ml is purifying is called rough IgG type antibody D liquid.Divide device-70 ℃ preservation standby.All are saltoutd, dialysis, chromatography and centrifugation step are all carried out at 4 ℃.
Total IgA extracts: get AsAb IgA type positive patient pooled serum or rabbit anti-serum, add equivalent 0.1M ZnSO
4Liquid, adjust pH to 7.0, centrifugal going precipitated after stirring 1h, add equivalent saturated ammonium sulfate liquid in the supernatant, spend the night in 4 ℃ behind the mixing, centrifugal going behind the supernatant in the molten 10%EDTA-Na of the being deposited in solution, cross DEAE52 cellulose chromatography post behind the dialysis desalination more in batches, elder generation is with 0.01M pH7.4 PBS wash-out and abandon eluent, uses 0.1M pH6.4 PBS wash-out again instead, and collected protein peak is IgA.Polyglycol concentrates the back after Sephadex G200 post, and through 0.01M pH7.4 PBS wash-out, first protein peak is total IgA liquid of purifying, is called rough IgA type antibody D liquid.Divide device-70 ℃ preservation standby.All are saltoutd, dialysis, chromatography and centrifugation step are all carried out at 4 ℃.
6. prepare affinity chromatography D post, E post and F post:
Collect bachelorette's pooled serum (not containing the human sperm antigen); Collect the histocyte (not containing the human sperm antigen) of healthy women, and routine is made histiocyte lysate capable.Getting the Sepharose-4B Ago-Gel of cyanogen bromide-activated gets married and chromatography D post, E post and F post with couplings such as refining sperm antigen liquid, bachelorette's pooled serum and healthy women histiocyte lysate capables respectively.Coupling step when concrete coupling step prepares with affinity chromatography A, B, C post is identical, as long as change by conjugate.
7. the anti-people's sperm of complementary type affinity chromatography purification polyclonal antibody:
The method of conventional affinity chromatography see " fine works molecular biology experiment guide " (Yan Ziying, Wang Hailin are translated, Beijing: Science Press, 1998:382).The key step of this complementary type affinity chromatography is:
1. get rough IgG type (IgA type) antibody D liquid and cross affinity chromatography D post, collect the antibody materials that combines with sperm antigen on the D post: get and D post gel isopyknic antibody D liquid (1-10mg/ml), room temperature effect 3h behind the upper prop, then wash the D post successively with following solution, flow velocity 1ml/min, till OD280 absorbance＜0.02 of flowing out component:
The lavation buffer solution of 5 times of bed volumes, pH8.0
The Tris damping fluid of 5 times of bed volumes, pH8.0
The Tris damping fluid of 5 times of bed volumes, pH9.0
Take off the D post with the triethanolamine solution Shen of 5 times of bed volumes then, till OD280 absorbance＜0.02 of flowing out component, flow velocity 1ml/min, collect eluent simultaneously, and use 1mol/L Tris-HCl (pH6.7) damping fluid to neutralize immediately, in order to avoid the protein that elutes sex change under the alkali condition, that is: the eluent with each bed volume is collected in the test tube of 1mol/L Tris-HCl (pH6.7) damping fluid that contains 0.2 volume, and polyglycol concentrates about the adjust pH to 7.4 of back and is rough IgG type (IgA type) antibody E liquid.
2. get rough IgG type (IgA type) antibody E liquid and cross affinity chromatography E post, collect the antibody materials that does not combine: with E post on the antibody E liquid with human albumin on the E post, room temperature effect 1h, lavation buffer solution (pH8.0) washing E post with 5 times of bed volumes, flow velocity 1ml/min is till OD280 absorbance＜0.02 of flowing out component; Use Tris damping fluid (pH8.0) Shen of 5 times of bed volumes to take off the E post at last, and collect eluent simultaneously, after polyglycol concentrates, be rough IgG type (IgA type) antibody F liquid.
3. get rough IgG type (IgA type) antibody F liquid at last and cross affinity chromatography F post, collect not with the F post on the protein bound antibody materials of human tissue cell: with F post on the antibody F liquid, room temperature effect 1h, lavation buffer solution (pH8.0) washing F post with 5 times of bed volumes, flow velocity 1ml/min is till OD280 absorbance＜0.02 of flowing out component; Use Tris damping fluid (pH8.0) the wash-out F post of 5 times of bed volumes at last, and collect eluent simultaneously,, be refining autoantibody liquid (IgG or IgA type) more than the dialysis 24h through polyglycol simmer down to 1-5ml.BCA method protein quantification, the SDS-PAGE electrophoresis is identified purity and composition, preservation was standby after vacuum freezedrying became pulvis.Divide device-70 ℃ preservation standby.
8. the evaluation and the preservation of refining autoantibody:
Get refining anti-people's sperm autoantibody liquid (IgG or IgA type), the BCA method is carried out protein quantification; The 10%SDS-PAGE electrophoresis is identified the purity of its antibody; Then do immunoblot experiment, detect the reactivity of the composition that refining antibody and sperm antigen, bachelorette's whole serum, human tissue cell's lysate and bovine serum albumin(BSA) etc. may occur in the sperm antibody detection system, identify the specificity of purified autoantibody; Preservation was standby after last vacuum freezedrying became pulvis.
Claims (4)
1. the complementary type affinity chromatography purification method of an autoantibody is characterized in that this method is: at first prepare autoantigen with complementary type affinity chromatography method purifying:
11) collector's purpose histocyte extracts membrane antigen with NP-40, uses the ultrasonic-wave crushing histocyte again, gets supernatant behind the ultracentrifugation, as rough antigen A liquid,
12) with purpose histocyte immunization experiment animal, get antiserum, slightly carry the γ immunoglobulin (Ig), claim immune serum IgG through salting out method; Get simultaneously not by the pooled serum of this kind animal used as test of immunity, slightly carry the γ immunoglobulin (Ig), claim not immune serum IgG through salting out method; Mix whole serum immunization experiment animal with the normal person, get antiserum, slightly carry the γ immunoglobulin (Ig) through salting out method equally, claim anti-human whole serum IgG,
13) coupling becomes immunoaffinity chromatography A post, B post and C post with anti-human whole serum IgG with above-mentioned immune serum IgG, not immune serum IgG respectively with the Sepharose-4B Ago-Gel,
14) get rough antigen A liquid and cross affinity chromatography A post, collect the antigenic substance that combines with IgG on the A post, concentrating the back is rough antigen B liquid; Get rough antigen B liquid again and cross affinity chromatography B post, collect the antigenic substance that does not combine with IgG on the B post, concentrating the back is rough antigens c liquid; Get rough antigens c liquid at last and cross affinity chromatography C post, collect the antigenic substance that does not combine, concentrate the back and be refining antigen liquid with IgG on the C post;
The polyclonal antibody for preparing anti-autoantigen then with complementary type affinity chromatography method purifying:
21) collect the patients serum of the autoantibody positive or with the antiserum of the animal used as test of autoantigen immunity inoculation; With salting out method and DEAE52 cellulose column IgG purification type and IgA type immunoglobulin (Ig), and be called rough IgG type antibody D liquid and rough IgA type antibody D liquid,
22) collection does not contain the healthy people's of purpose autoantigen pooled serum; Collect the people's who does not contain purpose antigen histocyte, and be prepared into histiocyte lysate capable,
23) get married and chromatography D post, E post and F post with above-mentioned refining antigen liquid, human tissue cell's lysate coupling of not containing healthy people's pooled serum of purpose antigen and not containing purpose antigen respectively with the Sepharose-4B Ago-Gel,
24) get rough IgG type or IgA type antibody D liquid is crossed affinity chromatography D post, collect the antibody materials that combines with autoantigen on the D post, concentrating afterwards is rough IgG type or IgA type antibody E liquid; The latter crosses affinity chromatography E post, collects the antibody materials that does not combine with albumen on the E post, and concentrating the back is rough IgG type or IgA type antibody F liquid; F liquid is collected the antibody materials that does not combine with albumen on the F post after affinity chromatography F post, concentrates the back and is refining autoantibody liquid IgG type or IgA type.
2. the complementary type affinity chromatography purification method of autoantibody according to claim 1, it is characterized in that: in step 13) and step 23) when preparing affinity chromatography A, B, C, D, E and F post, replace with the Ago-Gel of non-Sepharose-4B with the Sepharose-4B Ago-Gel.
3. the complementary type affinity chromatography purification method of autoantibody according to claim 1 is characterized in that: in step 24) unite when carrying out complementary type affinity chromatography with affinity chromatography D, E and F post, only unite with the D post or with D post and E post.
4. the complementary type affinity chromatography purification method of autoantibody according to claim 1, it is characterized in that: be not to collect the purpose histocyte when step 11), but collect the people's of containing purpose antigen body fluid, then without NP-40 extraction membrane antigen and ultrasonic-wave crushing histocyte, directly use the people's of containing purpose antigen humoral immunity animal used as test.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101902436A CN101158681A (en) | 2007-11-23 | 2007-11-23 | Complementary type affinity chromatography purification method of autoantibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101902436A CN101158681A (en) | 2007-11-23 | 2007-11-23 | Complementary type affinity chromatography purification method of autoantibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101158681A true CN101158681A (en) | 2008-04-09 |
Family
ID=39306840
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101902436A Pending CN101158681A (en) | 2007-11-23 | 2007-11-23 | Complementary type affinity chromatography purification method of autoantibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101158681A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102626422A (en) * | 2012-04-17 | 2012-08-08 | 周建伟 | Human sperm specific transfer factor oral preparation and preparation process thereof |
| CN104977419A (en) * | 2014-04-01 | 2015-10-14 | 苏州浩欧博生物医药有限公司 | Purifying preparation method of autoimmuno antigen positive serum |
| CN105675890A (en) * | 2015-12-31 | 2016-06-15 | 湖南农业大学 | Apparatus for detecting pregnancy of laboratory mice, and method thereof |
-
2007
- 2007-11-23 CN CNA2007101902436A patent/CN101158681A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102626422A (en) * | 2012-04-17 | 2012-08-08 | 周建伟 | Human sperm specific transfer factor oral preparation and preparation process thereof |
| CN102626422B (en) * | 2012-04-17 | 2014-03-12 | 周建伟 | Human sperm specific transfer factor oral preparation and preparation process thereof |
| CN104977419A (en) * | 2014-04-01 | 2015-10-14 | 苏州浩欧博生物医药有限公司 | Purifying preparation method of autoimmuno antigen positive serum |
| CN105675890A (en) * | 2015-12-31 | 2016-06-15 | 湖南农业大学 | Apparatus for detecting pregnancy of laboratory mice, and method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Unanue et al. | The immunogenicity of antigen bound to the plasma membrane of macrophages | |
| Alspaugh et al. | Serum antibody in rheumatoid arthritis reactive with a cell‐associated antigen. Demonstration by precipitation and immunofluorescence | |
| CN102633864B (en) | GPC3 antigen polypeptide, anti-GPC3 polyclonal antibody and application thereof | |
| CN107266569B (en) | Preparation method of tussah silk fibroin monoclonal antibody | |
| CN101831407B (en) | Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof | |
| CN103820394B (en) | Monoclonal antibodies against morphine, produce the cell strain of this antibody, morphine detection kit and preparation method thereof | |
| CN102776153A (en) | Mouse anti-human neutrophil gelatinase-associated lipocalin (NGAL) monoclonal antibodies and hybridoma cell strains and application thereof | |
| CN105112398A (en) | Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody | |
| CN110261616A (en) | A kind of hepatitis c virus detection reagent box | |
| CN102830229A (en) | Detection reagent of treponema pallidum IgM (Immunoglobulin M) antigen colloidal gold method and preparation method thereof | |
| CN109387627A (en) | A kind of Reagent Protocol of screening for cancer and early diagnosis based on placenta sample chondroitin sulfate A (CSA) | |
| CN101158681A (en) | Complementary type affinity chromatography purification method of autoantibody | |
| CN106220730A (en) | A kind of preparation method of Aspergillus fumigatus galactomannan polyclonal antibody | |
| CN105567724A (en) | Gastrin-releasing peptide precursor protein expression vector construction method, monoclonal antibody preparation method and kit preparation method | |
| CN104744580B (en) | A kind of TgVP1 extracellular regions antigen polypeptide, the polyclonal antibody of anti-TgVP1 and its application | |
| CN105646703A (en) | Polyclonal antibody of GXM (glucuronoxylomannan) of Cryptococcus neoformans and preparation method of polyclonal antibody | |
| CN104977419A (en) | Purifying preparation method of autoimmuno antigen positive serum | |
| CN105732810B (en) | A kind of Procalcitonin monoclonal antibody and its application | |
| CN102690788B (en) | Zearalenone anti-idiotypic antibody, preparation method thereof, and application thereof | |
| CN106399294B (en) | A kind of preparation of the monoclonal antibody 7H8 of anti-human Procalcitonin protein N terminal epitope | |
| CN104610441B (en) | For preparing artificial semiantigen, preparation method and the monoclonal antibody of acquisition of glypican-3 (GPC3) monoclonal antibody | |
| CN101735317A (en) | Cathepsin D antigen polypeptide and application thereof as well as detecting kit containing polypeptide | |
| CN102336814A (en) | Polypeptide sequence for preparing anti-liver-neoplasm marker CK-19 antibody, polyclonal antibody and application thereof | |
| CN102221615A (en) | Double-antibody sandwich ELISA method based on Angiogenin detection | |
| CN102329378B (en) | HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080409 |