Antibody preparation
The preparation of antibodies with high titer, strong specificity and good stability is the basis for the success of immunological experiments. The quality of antibodies directly affects the success or failure of researchers’ research. Different immunological experimental methods (such as ELISA, IHC, IP, ICC, SDS-PAGE, WB, etc.) have different requirements for antibody titer, concentration and purity. We know that the general immune serum contains specific antibodies and non-specific antibodies, serum proteins and various other impurities. The key depends on the purification method chosen. The following is an introduction to the commonly used antibody purification methods and their related principles.
1. Salting out method (saturated ammonium sulfate precipitation method)
The purification method is based on adding saturated ammonium sulfate solution to the immune serum to be purified. Since antibody is also a protein, its solubility in aqueous solution is determined by the number of hydrophilic groups and charges it carries. After adding saturated ammonium sulfate solution to antiserum, sulfate ions and ammonium ions compete with antibodies for water molecules in the solution, because sulfate ions and ammonium ions are more hydrophilic than antibody molecules, so antibodies The hydration film on the surface of the molecule is destroyed, and the exposed charged groups are neutralized by the salt ions in the solution, which greatly reduces its solubility. According to this principle, it is separated from the antiserum.
The specific experimental steps are as follows:
Mix 5ml of antiserum and 0.01M PBS in an equal volume in a centrifuge tube, add 10ml of saturated ammonium sulfate solution to it, shake well while adding dropwise, and then let stand at 2 to 8 degrees Celsius overnight.
Centrifuge the substance to be separated in step a in a centrifuge at 8000 r/min for 15 to 20 minutes, and remove the supernatant.
Dissolve the pellet after centrifugation in step b with 2ml of 0.01M PBS, then slowly add 3ml of saturated ammonium sulfate solution, shake well while adding dropwise, and then place it at 2 to 8 degrees Celsius for 2 hours.
Centrifuge the substance to be separated in step c in a centrifuge at 8000 r/min for 15 to 20 minutes, and remove the supernatant.
Dissolve the precipitate after centrifugation in step d with 1.65ml of 0.01M PBS, then slowly add 3.35ml of saturated ammonium sulfate solution, shake well while adding dropwise, and then place it at 2 to 8 degrees Celsius for 2 hours.
Centrifuge the material to be separated in step e in a centrifuge at 8000 r/min for 15 to 20 minutes, and remove the supernatant.
Dissolve the pellet after centrifugation in step f with 1 ml of 0.01M PBS, transfer it to an MD 14000 dialysis bag, and conduct dialysis with 0.01 M PBS for more than 4 times, each time for at least 1 hour.
2. n-octanoic acid-saturated ammonium sulfate precipitation method
The principle of the purification method is: n-octanoic acid can bind to the impurity protein in the antiserum or mouse ascites under the condition of partial acid, and precipitate it near the isoelectric point, IgG class antibody exists in the supernatant, and then It can be further purified by the precipitation method with saturated ammonium sulfate.
The specific experimental steps are as follows:
Centrifuge the antiserum or mouse ascites in a centrifuge for 15 to 20 minutes at 8000 r/min to remove impurities such as cell debris or other precipitates.
Mix 1 volume of antiserum or ascites with 2 volumes of 0.06M acetate buffer at pH 4.8, and slowly add n-octanoic acid while stirring at room temperature. The amount of n-octanoic acid added is 33ul/ml of antiserum or ascites.
Mix at room temperature for 15 to 30 minutes
Let stand overnight at 2 to 8 degrees Celsius to fully settle.
Centrifuge at 8000 r/min for 15 to 20 minutes, discard the precipitate and take the supernatant.
The supernatant was transferred to an MD 14000 dialysis bag, and dialyzed against 0.01 M PBS for more than 4 times, each time for at least 1 hour.
The supernatant after dialysis in step f is purified by salting out method (saturated ammonium sulfate precipitation method). The specific experiment is described above, and will not be repeated here.
3.Protein A and protein G purification method
Basic principle: protein A and protein G can specifically bind to the Fc segment of antibody IgG molecules. Protein A and protein G can be coupled to agarose gel as ligands. When antiserum flows through it, specific IgG binds to the ligands, and other impurities flow through. Since the two proteins have different binding abilities to antibodies from different hosts when purifying antibodies, we need to deal with specific situations and choose protein A or protein G, or a combination of protein A/protein G respectively. It is generally recommended that mouse monoclonal antibodies IgG2a, IgG2b, IgG3, rabbit, human, and pig polyclonal antibodies are purified with protein A, while mouse monoclonal antibodies IgG1, rat monoclonal antibodies, mouse, rat, and goat polyclonal antibodies are Choose to purify with protein G.
The specific experimental steps are as follows:
Weigh 0.5 g of CNBr-activated sepharose 4B, dissolve it in 2 mM HCL solution, and keep it at 4 degrees Celsius overnight to fully swell.
The swollen sepharose 4B was pre-packed in the chromatography column, washed three times with 10 times the volume of 2mM HCL solution, and the column was equilibrated with 0.1 M NaHCO3 solution.
Dissolve 5mg protein A/G in 0.1 M NaHCO3 solution, add to the equilibrated column in step b, and place on a shaker to gently mix at 2 to 8 degrees Celsius overnight or at room temperature for 2 to 4 hours
The protein-bound column in c was washed three times with 0.1 M NaHCO3 solution. The bound supernatant was collected in advance, and the protein concentration in the bound supernatant was measured with a UV-Vis spectrophotometer to determine the binding rate.
The column was equilibrated three times with 0.01M tris base, 0.5% BSA was added to block unbound sites on sepharose 4B, and the reaction was placed on a shaker at room temperature for 2 to 4 hours.
The column was equilibrated 3 times with 0.01M tris base.
Add 5 ml of the pretreated antiserum to be purified, and place on a shaker to react at room temperature for 2 to 4 hours.
Wash the column three more times with 0.01M tris base to remove unbound contaminants
The antibody bound to the column was eluted with 9 ml of 0.1 M glycine pH 2.5
Add 1 ml of 1M NaHCO3 solution to the EP tube to neutralize the antibody eluted in step i
The antibody solution in step j is put into a dialysis bag, concentrated to 2 ml with PEG20000 or sucrose, and then dialyzed in 0.01M PBS for more than 4 times, and the interval between each liquid change is more than 1 hour.
Measure the absorbance value of the dialyzed antibody at a wavelength of 280 nm with a UV-Vis spectrophotometer, and divide the absorbance value by 1.35 to obtain the concentration of the measured antibody.
The purified antibody can be stored for a long time by adding 30%-50% glycerol at -20 degrees Celsius or -80 degrees Celsius.
4. Immunoaffinity purification
Basic principle: The antigen is coupled to sepharose 4B as a ligand, and specific antibodies in biological fluids such as serum are specifically bound to the antigen on sepharose 4B, while other heteroproteins and heteroantibodies are not bound. A series of experimental steps such as elution and elution can obtain high-purity target antibodies. Although this method and Protein A/G are both called affinity purification methods, the final antibodies obtained by purification are quite different. The purified antibodies are mainly specific IgG antibodies. Compared with other purification methods, the antibodies purified by this method have the strongest specificity and the highest purity. The experimental steps of this purification method are basically the same as those of the protein A/G purification method, and the ligand protein A/G of the third experimental method can be replaced with the corresponding target antigen.
With the continuous development of biological downstream processing technology, there are more and more separation and purification methods for various biological macromolecules, especially antibody macromolecules, such as molecular sieve chromatography (gel chromatography or gel filtration), ion exchange chromatography, etc. etc. were also used in experiments. The above-mentioned purification methods are only general IgG antibodies. The purification methods of other IgM, IgA, IgD, IgE and other antibodies should be selected according to their own characteristics according to their own characteristics.
