The Introduction of Antibody Purification
Antibodies with high titre, high specification and high stability are the guarantee of a successful immunology test .The quality of antibodies significantly affects the research results. The requirement of the titre, concentration and purification of antibodies differs in different immunological techniques such as ELISA，IHC，IP，ICC,SDS-PAGE and WB.
In general, immune serum consists of specific antibodies, non-specific antibodies, serum protein and impure proteins. When we have the desired titre of antibodies, the purification of antibodies is determined by the purification methods we select. We will introduce some generally used purification methods and explain in detail their principles as follows.
1. salting-out method
According to the method, saturated ammonium-sulfate solution is added to the immune serum which is to be purified. As antibody is also a kind of protein, the solubility is determined by the protein’s number of hydrophilic groups and the number of charges. After the addition of saturated ammonium-sulfate solution，sulfate ions and ammonium ions compete against antibodies for water molecules. As these two ions have higher hydrophilicity than antibody molecules, the hydrated shell of the antibody molecule is damaged. Meanwhile, the exposed electriferous groups are neutralized by salt ions. Thus, the solubility of antibodies is greatly reduced. Utilizing this method, we can separate antibodies from antiserum.
The procedures are listed as follows:
1. Mix 5ml antiserum with an equal volume of 0.01M PBS in centrifuge tube .Then add 10 ml saturated ammonium-sulfate solution drop by drop and stir it well. Then let it stand for a night at 2-8℃.
2. Centrifuge the substance to be isolated (as in step1) in the centrifuge at 8000r/min for 15 to 20 minutes. Remove the supernatant.
3. Dissolve the centrifuged precipitate (as in step2) in 2ml 0.01M PBS. Slowly add 3 ml saturated ammonium-sulfate solution drop by drop and stir it well. Then let it stand for two hours at 2-8℃.
4. Centrifuge the substance to be isolated (as in step3) in the centrifuge at 8000r/min for 15 to 20 minutes. Remove the supernatant.
5. Dissolve the centrifuged precipitate (as in step 4) in1.65ml 0.01M PBS. Add 3 ml saturated-ammonium sulfate solution drop by drop and stir it well. Then let it stand for two hours at 2-8℃.
6. Centrifuge the substance to be isolated (as in step5) in the centrifuge at 8000r/min for 15 to 20 minutes. Remove the supernatant.
7.Dissolve the centrifuged precipitate (as in step 6)in1ml 0.01M PBS.Transfer it to MD 14000 dialysis bag. Dialyze it with 0.01 M PBS. Repeat the dialyzing process at least four times.The duration of each dialysis must be at least one hour.
2. Octanoic acid- saturated ammonium-sulfate solution precipitate method
While the Octanoic acid remains acid, it can bind with impure proteins in antiserum or in the mouse ascite fluid, Precipitating it at the isoelectric point and leaving IgG class of antibodies in the supernatant. Then utilize the method of saturated ammonium-sulfate precipitation for further purification.
The procedure is listed as follows:
1. Centrifuge the antiserum or mouse ascite fluid in the Centrifuge at 8000r/min for 15 to 20 minutes. Remove the cell fragments or other residual impurities.
2. Mix one part of antiserum or mouse ascite fluid with two parts of 0.06M PH 4.8 acetate buffer solution. Gently add the Octanoic acid or while stirring it constantly at room temperature. The proportion of Octanoic acid to antiserum or the mouse ascite fluid in volume should be 33ul to 1 ml.
3. keep it at the room temperature for 15-30minutes.
4. Keep it stand for a night and allow for sufficient precipitation.
5. Centrifuge it at 8000r/min for 15 to 20 minutes. Remove the precipitate and save the supernatant.
6. Dialyze it with 0.01 M PBS. Repeat the dialyzing process at least four times.The duration of each dialysis must be at least one hour.
7. Apply salting-out method( the precipitation method with saturated ammonium-sulfate solution ) to the purification of supernatant. Please see the procedure in the salting-out method.
3. The purification method of Protein A and Protein G
The principle is illustrated as follows:
Protein A and Protein G can specifically interact with the Fc regions of IgG antibodies. As an aglucon, Protein A and Protein G can be coupled to sepharose. While the antiserum is filtrating through it, the specific IgG can be left by being bound to aglucon. Other impure proteins just pass through it. The binding properties of two proteins differ when they couple with antibodies in different hosts. So we should use Protein A or Protein G or protein A/protein G according to different circumstances.Protein A purification method is recommended in IgG2a,IgG2b,IgG3 of mouse monoclonal antibodies and polyclonal antibodies of rabbit,human and pigs. While the Protein G purification method is recommended in the IgG1 of mouse monoclonal antibodies, monoclonal antibodies of rats, and polyclonal antibodies of mouse, rat and goat.
1. Dissolve 0.5g sepharose4B activated by CNBr in the 2mM HCL solution. Keep it at 4 ℃ for a night and allow for sufficient swelling.
2. Pre-pack the swelled sepharose 4B in the chromatographic column. Wash three times it with 2mM HCL solution which is ten times the volume.Equilibrate the column with 0.1 M NaHCO3 solution.
3. Dissolve 5mg protein A/G in the 0.1 M NaHCO3, then add the equilibrated column(as in step 2). Place it in the shaking table and mix it well at 2-8℃ for a night or at room temperature for 2-4 hours.
4.Wash the column bound with proteins (as in step3) three times with 0.1 M NaHCO3 solution. Pre-collect the supernatant which has been bound. The protein concentration in the supernatant can be determined by the ultraviolet and visible spectrophotometer. Then we will know the binding ratio .
5. Equilibrate the column three times with 0.01M tris base. Add 0.5% BSA to block the unbound sites on the sepharose 4B. Place it in the shaking table and allow for reaction at room temperature for 2-4 hours.
6. equilibrate the column with 0.01M tris base three times.
7. Add the pre-processed 5 ml antiserum which is to be purified and then place it in the shaking table for reaction at room temperature for 2-4 hours.
8. Wash the column at least three times with 0.01M tris base and remove the unbound impure proteins.
9. Elute the antibodies bound in the column with the 9ml 0.1 M PH 2.5 Glycine .
10. Add 1ml 1M NaHCO3 solution in the EP tube to neutralize the antibody which have been eluted (as in step9).
Transfer the antibody solution in the dialysis bag and concentrate the liquid with PEG20000 or saccharose to the volumn of 2ml. Dialyzing it in the 0.01M PBS at least four times. The duration of each dialysis must be at least one hour.
11.Utilize the ultraviolet and visible spectrophotometer to measure the absorbance of the dialyzed antibody at the wavelength of 280nm. The concentration can be obtained after the absorbance is divided by 1.35.
12. Preserve the purified antibodies in the glycerinum with the concentration of 30%-50% at -20 or -80 ℃.It can be preserved for a long time.
Immune affinity purification method
The antibodies are coupled as as a ligand on the sepharose 4b. The specific antibodies specifically interact with antigens on the sepharose 4B. The impure proteins and impure antibodies will not be bound. After a series of steps such as washing and elution, high concentration of target antibodies can be obtained. Although this method is also called affinity purification method as Protein A/G, there are wide difference between the variations of antibodies we get. Generally, non-specific IgG class antibodies can be obtained by Protein A/G ; With Immune affinity purification method, we can mainly get Specific IgG class antibodies. Compared with other methods, this method can help get antibodies with highest specificity and highest concentration. Its test procedure is similar to that of protein A/G purification method. The difference is that we use corresponding target antigens instead of protein A/G.
With the development of the downstream manufacturing techniques in biological science, the purification methods of macro-biomolecules, antibody molecules in particular, are increasing. There are Gel Filtration Chromatography, ion exchange chromatograph, to name a few. The above method is commonly applied to the purification of IgG class antibodies. As for other antibodies such as IgM,IgA，IgD,IgE, the methods must be chosen according to the properties of antibodies.
In general, immune serum consists of specific antibodies, non-specific antibodies, serum protein and impure proteins. When we have the desired titre of antibodies, the purification of antibodies is determined by the purification methods we select. We will introduce some generally used purification methods and explain in detail their principles as follows.
1. salting-out method
According to the method, saturated ammonium-sulfate solution is added to the immune serum which is to be purified. As antibody is also a kind of protein, the solubility is determined by the protein’s number of hydrophilic groups and the number of charges. After the addition of saturated ammonium-sulfate solution，sulfate ions and ammonium ions compete against antibodies for water molecules. As these two ions have higher hydrophilicity than antibody molecules, the hydrated shell of the antibody molecule is damaged. Meanwhile, the exposed electriferous groups are neutralized by salt ions. Thus, the solubility of antibodies is greatly reduced. Utilizing this method, we can separate antibodies from antiserum.
The procedures are listed as follows:
1. Mix 5ml antiserum with an equal volume of 0.01M PBS in centrifuge tube .Then add 10 ml saturated ammonium-sulfate solution drop by drop and stir it well. Then let it stand for a night at 2-8℃.
2. Centrifuge the substance to be isolated (as in step1) in the centrifuge at 8000r/min for 15 to 20 minutes. Remove the supernatant.
3. Dissolve the centrifuged precipitate (as in step2) in 2ml 0.01M PBS. Slowly add 3 ml saturated ammonium-sulfate solution drop by drop and stir it well. Then let it stand for two hours at 2-8℃.
4. Centrifuge the substance to be isolated (as in step3) in the centrifuge at 8000r/min for 15 to 20 minutes. Remove the supernatant.
5. Dissolve the centrifuged precipitate (as in step 4) in1.65ml 0.01M PBS. Add 3 ml saturated-ammonium sulfate solution drop by drop and stir it well. Then let it stand for two hours at 2-8℃.
6. Centrifuge the substance to be isolated (as in step5) in the centrifuge at 8000r/min for 15 to 20 minutes. Remove the supernatant.
7.Dissolve the centrifuged precipitate (as in step 6)in1ml 0.01M PBS.Transfer it to MD 14000 dialysis bag. Dialyze it with 0.01 M PBS. Repeat the dialyzing process at least four times.The duration of each dialysis must be at least one hour.
2. Octanoic acid- saturated ammonium-sulfate solution precipitate method
While the Octanoic acid remains acid, it can bind with impure proteins in antiserum or in the mouse ascite fluid, Precipitating it at the isoelectric point and leaving IgG class of antibodies in the supernatant. Then utilize the method of saturated ammonium-sulfate precipitation for further purification.
The procedure is listed as follows:
1. Centrifuge the antiserum or mouse ascite fluid in the Centrifuge at 8000r/min for 15 to 20 minutes. Remove the cell fragments or other residual impurities.
2. Mix one part of antiserum or mouse ascite fluid with two parts of 0.06M PH 4.8 acetate buffer solution. Gently add the Octanoic acid or while stirring it constantly at room temperature. The proportion of Octanoic acid to antiserum or the mouse ascite fluid in volume should be 33ul to 1 ml.
3. keep it at the room temperature for 15-30minutes.
4. Keep it stand for a night and allow for sufficient precipitation.
5. Centrifuge it at 8000r/min for 15 to 20 minutes. Remove the precipitate and save the supernatant.
6. Dialyze it with 0.01 M PBS. Repeat the dialyzing process at least four times.The duration of each dialysis must be at least one hour.
7. Apply salting-out method( the precipitation method with saturated ammonium-sulfate solution ) to the purification of supernatant. Please see the procedure in the salting-out method.
3. The purification method of Protein A and Protein G
The principle is illustrated as follows:
Protein A and Protein G can specifically interact with the Fc regions of IgG antibodies. As an aglucon, Protein A and Protein G can be coupled to sepharose. While the antiserum is filtrating through it, the specific IgG can be left by being bound to aglucon. Other impure proteins just pass through it. The binding properties of two proteins differ when they couple with antibodies in different hosts. So we should use Protein A or Protein G or protein A/protein G according to different circumstances.Protein A purification method is recommended in IgG2a,IgG2b,IgG3 of mouse monoclonal antibodies and polyclonal antibodies of rabbit,human and pigs. While the Protein G purification method is recommended in the IgG1 of mouse monoclonal antibodies, monoclonal antibodies of rats, and polyclonal antibodies of mouse, rat and goat.
1. Dissolve 0.5g sepharose4B activated by CNBr in the 2mM HCL solution. Keep it at 4 ℃ for a night and allow for sufficient swelling.
2. Pre-pack the swelled sepharose 4B in the chromatographic column. Wash three times it with 2mM HCL solution which is ten times the volume.Equilibrate the column with 0.1 M NaHCO3 solution.
3. Dissolve 5mg protein A/G in the 0.1 M NaHCO3, then add the equilibrated column(as in step 2). Place it in the shaking table and mix it well at 2-8℃ for a night or at room temperature for 2-4 hours.
4.Wash the column bound with proteins (as in step3) three times with 0.1 M NaHCO3 solution. Pre-collect the supernatant which has been bound. The protein concentration in the supernatant can be determined by the ultraviolet and visible spectrophotometer. Then we will know the binding ratio .
5. Equilibrate the column three times with 0.01M tris base. Add 0.5% BSA to block the unbound sites on the sepharose 4B. Place it in the shaking table and allow for reaction at room temperature for 2-4 hours.
6. equilibrate the column with 0.01M tris base three times.
7. Add the pre-processed 5 ml antiserum which is to be purified and then place it in the shaking table for reaction at room temperature for 2-4 hours.
8. Wash the column at least three times with 0.01M tris base and remove the unbound impure proteins.
9. Elute the antibodies bound in the column with the 9ml 0.1 M PH 2.5 Glycine .
10. Add 1ml 1M NaHCO3 solution in the EP tube to neutralize the antibody which have been eluted (as in step9).
Transfer the antibody solution in the dialysis bag and concentrate the liquid with PEG20000 or saccharose to the volumn of 2ml. Dialyzing it in the 0.01M PBS at least four times. The duration of each dialysis must be at least one hour.
11.Utilize the ultraviolet and visible spectrophotometer to measure the absorbance of the dialyzed antibody at the wavelength of 280nm. The concentration can be obtained after the absorbance is divided by 1.35.
12. Preserve the purified antibodies in the glycerinum with the concentration of 30%-50% at -20 or -80 ℃.It can be preserved for a long time.
Immune affinity purification method
The antibodies are coupled as as a ligand on the sepharose 4b. The specific antibodies specifically interact with antigens on the sepharose 4B. The impure proteins and impure antibodies will not be bound. After a series of steps such as washing and elution, high concentration of target antibodies can be obtained. Although this method is also called affinity purification method as Protein A/G, there are wide difference between the variations of antibodies we get. Generally, non-specific IgG class antibodies can be obtained by Protein A/G ; With Immune affinity purification method, we can mainly get Specific IgG class antibodies. Compared with other methods, this method can help get antibodies with highest specificity and highest concentration. Its test procedure is similar to that of protein A/G purification method. The difference is that we use corresponding target antigens instead of protein A/G.
With the development of the downstream manufacturing techniques in biological science, the purification methods of macro-biomolecules, antibody molecules in particular, are increasing. There are Gel Filtration Chromatography, ion exchange chromatograph, to name a few. The above method is commonly applied to the purification of IgG class antibodies. As for other antibodies such as IgM,IgA，IgD,IgE, the methods must be chosen according to the properties of antibodies.
