Editing Affinity chromatography (section)

===Immunoaffinity===
Another use for the procedure is the affinity purification of antibodies from blood serum. If the serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunized against the antigen concerned) then it can be used for the affinity purification of that antigen. This is also known as Immunoaffinity Chromatography. For example, if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum.

For thoroughness, the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured on the solid support. Elution of the antibodies of interest is most often achieved using a low [[pH]] buffer such as glycine pH 2.8. The eluate is collected into a neutral [[tris]] or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.

Monoclonal antibodies can also be selected to bind proteins with great specificity, where protein is released under fairly gentle conditions. This can become of use for further research in the future.<ref>{{Cite book|last1=Thompson|first1=Nancy E.|last2=Foley|first2=Katherine M.|last3=Stalder|first3=Elizabeth S.|last4=Burgess|first4=Richard R.|chapter=Chapter 28 Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography |pages=475–494|language=en|doi=10.1016/s0076-6879(09)63028-7|pmid=19892188|title=Guide to Protein Purification, 2nd Edition|volume=463|series=Methods in Enzymology|year=2009|isbn=9780123745361}}</ref>

A simplified strategy is often employed to purify antibodies generated against peptide [[antigen]]s. When the peptide antigens are produced synthetically, a terminal [[cysteine]] residue is added at either the N- or C-terminus of the peptide. This cysteine residue contains a [[sulfhydryl]] functional group which allows the peptide to be easily conjugated to a carrier protein (e.g. Keyhole limpet hemocyanin (KLH)). The same cysteine-containing peptide is also immobilized onto an agarose resin through the cysteine residue and is then used to purify the antibody.

Most [[monoclonal antibody|monoclonal antibodies]] have been purified using affinity chromatography based on [[immunoglobulin]]-specific [[Protein A]] or [[Protein G]], derived from bacteria.<ref name="Uhlen 2008">{{cite journal |last1=Uhlén |first1=Mathias |title=Affinity as a Tool in Life Science |journal=BioTechniques |date=April 2008 |volume=44 |issue=5 |pages=649–654 |doi=10.2144/000112803 | pmid=18474040 | doi-access=free }}</ref>

Immunoaffinity chromatography with monoclonal antibodies immobilized on monolithic column has been successfully used to capture extracellular vesicles (e.g., exosomes and exomeres) from human blood plasma by targeting tetraspanins and integrins found on the surface of the EVs.<ref name="Multia et al 2019">{{cite journal |last1=Multia |first1=Evgen |last2=Tear |first2=Crystal Jing Ying |last3=Palviainen |first3=Mari |last4=Siljander |first4=Pia |last5=Riekkola |first5=Marja-Liisa |title=Fast isolation of highly specific population of platelet-derived extracellular vesicles from blood plasma by affinity monolithic column, immobilized with anti-human CD61 antibody |journal=Analytica Chimica Acta |date=December 2019 |volume=1091 |pages=160–168 |doi=10.1016/j.aca.2019.09.022 |pmid=31679569  |bibcode=2019AcAC.1091..160M |hdl=10138/321264 |s2cid=203147714 |hdl-access=free}}</ref><ref name="Multia et al 2020">{{cite journal |last1=Multia |first1=Evgen |last2=Liangsupree |first2=Thanaporn |last3=Jussila |first3=Matti |last4=Ruiz-Jimenez |first4=Jose |last5=Kemell |first5=Marianna |last6=Riekkola |first6=Marja-Liisa |title=Automated On-Line Isolation and Fractionation System for Nanosized Biomacromolecules from Human Plasma |journal=Analytical Chemistry |date=6 October 2020 |volume=92 |issue=19 |pages=13058–13065 |doi=10.1021/acs.analchem.0c01986 |pmid=32893620 |pmc=7586295 |doi-access=free}}</ref>

Immunoaffinity chromatography is also the basis for immunochromatographic test (ICT) strips, which provide a rapid means of diagnosis in patient care. Using ICT, a technician can make a determination at a patient's bedside, without the need for a laboratory.<ref>{{cite book | last=Luppa | first=Peter | title=Point-of-care testing: principles and clinical applications |pages=71–72 | publisher=Springer | location=Berlin, Germany | year=2018 | isbn=9783662544976}}</ref> ICT detection is highly specific to the microbe causing an infection.<ref>{{cite journal |last1=Muller |first1=JD |last2=Wilks |first2=CR |last3=O'Riley |first3=KJ |last4=Condron |first4=RJ |last5=Bull |first5=R. |last6=Mateczun |first6=A. |title=Specificity of an immunochromato-graphic test for anthrax |journal=Australian Veterinary Journal |date=April 2004 |volume=82 |issue=4 |pages=220–222 |doi=10.1111/j.1751-0813.2004.tb12682.x|pmid=15149073 }}</ref>