Purification of an antibody from its original source will vary greatly depending upon the properties of the starting material. The advantage to purification is the depth of information available for the antibody, its source and the contaminants that may be present. This knowledge enables the best selections to be made for the purification process, producing the highest yield of antibody in as few as 2 purification steps.
Sample preparation before purification is often required this involves the removal of impurities such as lipoproteins, phenol red, bulk proteins and salts.
Where the antibody has been sourced from will determine the best method for purification.
It is important to chose procedures that are as gentle on the protein as possible, reducing the risk of degradation but also choosing a process that enables efficient extraction.
The use of additives should also be considered as whilst they are essential for the stabilization of the product and can help the extraction process choosing an additive that binds to strongly to the protein can add further steps to the purification process.
Antibody purification from serum samples can include processes such as salt precipitation, this method separates the antibodies through the removal of water from the proteins structure, the higher the salt concentration the less soluble the protein will become as more of the proteins water is removed, this in turn causes the protein to precipitate. The theory is to remove the unwanted proteins first whilst the antibody of interest remains soluble.
Based on the high affinity protein G and protein A have for the Fc region of polyclonal and monoclonal IgG type antibodies. Protein A and G are bacterial proteins from Streptococci and Staphylococcus bacteria respectively, when coupled with Sepharose the purification process is made much easier for the routine purification of antibodies.
Protein G is often the preferred method for purification as it tends to have a higher affinity for IgG compared to protein A it also binds to a broader range of IgG from eukaryotic species and more classes of IgG. the strength of binding is however dependent on the source and subclass of the IgG. These factors all tend to mean protein G produces cleaner purification's and greater yields.
Protein A contains 5 regions that bind to the Fc region of IgG, combining protein A with Sepharose frees these regions enabling them to bind. Protein A is recognized to have an affinity for the Fab region which means it can also be used to for the purification of fab fragments
Protein L from Peptostreptococcus magnus bacteria has also been found beneficial for the purification process, binding with high affinity to an immunoglobulins light chain. As the protein binds only to the light chain it enables greater binding to all antibody classes, IgG, IgM, IgA, IgE and IgD.
Further purification may be required for the removal of specific contaminants that could interfere with the intended application. In some cases the sample can be used directly after affinity purification.
Contaminants can include albumin, transferrin, antibody aggregates, leached protein A, DNA and host or bovine Ig that originate from ascites or cell culture serum. With albumin, transferrin and host/ bovine Ig posing the most difficult for removal. host/bovine Ig are difficult to remove due to the similarity shared with the target antibody. Albumin and transferrin are difficult to remove due to their abundance and similarity to the charge characteristics of many antibodies respectively.
It is important to optimise the assay to improve the performance. there are many parameters that can be altered to meet the requirements including antibody quality, concentration, incubation time and temperature, detection reagent quality, reagent concentration, substrate type and concentration.
Antibody concentration can be optimised by determining the best capture and detection antibody concentrations. One of the best methods to do this is to perform a grid experiment which enable the user to test a large number of antibody pair concentrations at once. The optimal concentration required will vary depending upon the use of monoclonal or poly clonal antibodies. This table briefly shows the recommended starting antibody concentrations for each combination.