{ "id": "ep9151h937", "slug": "immunoaffinity-chromatography", "title": "Immunoaffinity chromatography", "updated": "2026-10-15", "topic_path": [ { "id": "life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/topics/life" }, { "id": "life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology" }, { "id": "life.biology.biochemistry", "label": "Biochemistry and metabolism", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry" }, { "id": "life.biology.biochemistry.practice", "label": "Biochemistry field and methods", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry.practice" }, { "id": "life.biology.biochemistry.practice.methods", "label": "Biochemical methods and techniques", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry.practice.methods" }, { "id": "life.biology.biochemistry.practice.methods.detect", "label": "Detection methods and analytical reactions", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.biochemistry.practice.methods.detect" } ], "geo": [ { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect", "label": "Non-geographic · 1946 to 2000: Detection methods and analytical reactions", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t1946", "label": "Non-geographic · 1946 to 2000", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946" }, { "id": "geo.nongeo.t1946.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life" }, { "id": "geo.nongeo.t1946.life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry", "label": "Biochemistry and metabolism", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice", "label": "Biochemistry field and methods", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice.methods", "label": "Biochemical methods and techniques", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice.methods" }, { "id": "geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect", "label": "Detection methods and analytical reactions", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.biochemistry.practice.methods.detect" } ] } ], "excerpt": "Immunoaffinity chromatography (IAC) is a liquid chromatography method in which immobilized antibodies selectively capture and purify a target biomolecule from a complex mixture, first reported in 1963.", "snippet": "Immunoaffinity chromatography (IAC) is a liquid chromatography method in which immobilized antibodies selectively capture and purify a target biomolecule from a complex mixture, first reported in 1963.", "node": "life.biology.biochemistry.practice.methods.detect", "markdown": "# Immunoaffinity chromatography\n\nImmunoaffinity chromatography (IAC) is a liquid chromatography method in which the stationary phase is an antibody or antibody-related reagent, used to selectively capture and purify a target biomolecule from a complex mixture. It is a subcategory of affinity chromatography and the most common form of bioaffinity chromatography.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) • [2](https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/) • [3](https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1075&context=chemistryhage)\n\n| Key fact | Value |\n|---|---|\n| Stationary phase | Immobilized antibody or antibody-related ligand[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) |\n| Binding conditions | Neutral pH 7.0–7.4; equilibrium constants typically \\( 10^{6} \\)–\\( 10^{12}\\ \\mathrm{M}^{-1} \\)[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) |\n| Common elution | Low pH (1–3), chaotropes (1.5–8 M), competitive elution[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) |\n| Typical purification | Greater than 1000-fold, with ≥90% activity recovery[4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf) • [5](https://experiments.springernature.com/articles/10.1385/1-59259-742-4:103) |\n| Column reuse | 100 or more cycles, depending on impurity load[5](https://experiments.springernature.com/articles/10.1385/1-59259-742-4:103) |\n| Main variants | Immunoextraction, immunodepletion, chromatographic immunoassays, peptide immunoaffinity capture (SISCAPA)[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) • [6](https://www.sciencedirect.com/science/article/pii/S0003267021005407) |\n\n## How it works\n\nThe immobilized antibody binds its antigen under physiological conditions, pH 7.0–7.4, where antibody–antigen equilibrium constants usually fall between \\( 10^{6} \\) and \\( 10^{12}\\ \\mathrm{M}^{-1} \\).[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) Binding arises from electrostatic and hydrophobic interactions, van der Waals forces, and hydrogen bonding between the antigen and the antibody's binding site.[7](https://experiments.springernature.com/articles/10.1007/978-1-59745-324-0_5) Because these constants are so high, the antigen is effectively irreversibly retained while the sample passes through, and isocratic elution is generally not feasible unless a deliberately low-affinity antibody (\\( K_{\\mathrm{a}} < 10^{6}\\ \\mathrm{M}^{-1} \\)) is used.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/)\n\nFor quantitative capture with low nonspecific background, an antibody affinity of at least \\( 10^{8}\\ \\mathrm{M}^{-1} \\) and about two hours of antigen–antibody contact are recommended.[4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf) Binding is reversed by changing the mobile phase so that desorption is favored: a step decrease in pH is the most common approach, and chaotropic salts or competitive agents are alternatives.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) • [8](https://b3p.it.helsinki.fi/download/GE_Protein_Purification_Handbooks/Affinity_Chromatography_Handbook.pdf)\n\n## How it is done\n\n**Coupling the antibody.** The classic support is CNBr-activated Sepharose, a cheap and fast covalent method; a working scale is 1 ml of slurry derivatized with 5 mg of antibody.[4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf) Amine-reactive activations include N,N'-carbonyldiimidazole, cyanogen bromide, N-hydroxysuccinimide, and tresyl or [tosyl chloride](https://www.edgepedia.org/tosyl-chloride).[9](https://pmc.ncbi.nlm.nih.gov/articles/PMC4820777/) Because random coupling through lysines can point the binding site away from the mobile phase, oriented methods are preferred for scarce antibodies, such as oxidized Fc carbohydrate residues reacted with [hydrazide](https://www.edgepedia.org/hydrazide)- or amine-containing supports, free Fab sulfhydryls on thiol-reactive supports, or reversible attachment through Protein A/G followed by cross-linking.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) • [4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf) • [9](https://pmc.ncbi.nlm.nih.gov/articles/PMC4820777/)\n\n**Loading, washing, eluting.** A standard single-step protocol uses 25 mM Tris-HCl pH 8.0 with 300 mM NaCl as binding buffer, a two- to fivefold molar excess of antibody over target, overnight incubation at 4 °C, washing with 10–25 column volumes, and elution with 100 mM glycine pH 2.7 collected in fractions; each milliliter of eluate is neutralized immediately with 200 µl of 1 M Tris-HCl pH 9.0.[10](https://doi.org/10.1016/bs.mie.2014.11.006) Columns are stored as a 50% PBS slurry at 4 °C.[10](https://doi.org/10.1016/bs.mie.2014.11.006)\n\n## Origin\n\nThe earliest affinity-type separation involved binding of α-amylase to insoluble starch.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/) [Immunoprecipitation](https://www.edgepedia.org/immunoprecipitation) appeared in the 1930s as a tool for antibody characterization and purification.[3](https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1075&context=chemistryhage) Haptens were coupled by diazo chemistry to chicken erythrocyte stroma to isolate antibodies.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/)\n\nAntibody bound to modified cellulose as an immunospecific adsorbent of antigens was reported by A.T. Jagendorf, A. Patchornik, and M. Sela in 1963 in Biochimica et Biophysica Acta.[11](https://doi.org/10.1016/0006-3002%2863%2990913-2) Two later advances made modern IAC possible: beaded agarose, and the cyanogen bromide immobilization method described by Porath, Axén, and Ernback in 1967 in Nature.[3](https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1075&context=chemistryhage) • [12](https://doi.org/10.1038/2151491a0)\n\nIn 1968, P. Cuatrecasas, [Meir Wilchek](https://www.edgepedia.org/meir-wilchek), and [Christian B. Anfinsen](https://www.edgepedia.org/christian-b-anfinsen) combined beaded agarose with CNBr coupling to immobilize nuclease inhibitors and purify nucleases in the [Proceedings of the National Academy of Sciences](https://www.edgepedia.org/proceedings-of-the-national-academy-of-sciences); this paper was the first to use the name \"affinity chromatography\".[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/) • [13](https://doi.org/10.1073/pnas.61.2.636) Cuatrecasas and Anfinsen reviewed the method in the 1971 Annual Review of Biochemistry,[14](https://doi.org/10.1146/annurev.bi.40.070171.001355) and Antibodies attached to agarose were used for isolating modified peptides, an early IAC example.[2](https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/)\n\n## Variants\n\n**Immunoextraction** uses the antibody column to remove a specific analyte or group of analytes from a sample before a second analytical method; columns can be run offline or placed online with HPLC, MS, CE, or GC–MS, and large sample volumes can be applied to concentrate the analyte.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) • [9](https://pmc.ncbi.nlm.nih.gov/articles/PMC4820777/) When IAC is combined with silica or monolithic HPLC supports, the format is called high performance immunoaffinity chromatography (HPIAC).[15](https://pmc.ncbi.nlm.nih.gov/articles/PMC3346860/)\n\n**Immunodepletion** is the inverse operation: an antibody column removes high- and mid-abundance proteins so that low-abundance components pass through for analysis.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) Multi-component immunoaffinity subtraction chromatography (IASC), reported by Rembert Pieper and colleagues in 2003 in PROTEOMICS, pooled antibody-coupled matrices to remove up to ten high-abundance plasma proteins in one step.[16](https://doi.org/10.1002/pmic.200390057) Commercial depletion columns based on this principle followed, including Agilent's MARS, Beckman Coulter IgY12, and Sigma-Aldrich IgY14 columns.[17](https://pmc.ncbi.nlm.nih.gov/articles/PMC3261331/) • [18](https://www.osti.gov/pages/servlets/purl/1251560)\n\n**Chromatographic immunoassays** use the antibody column itself as the recognition element in an LC format.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) **Peptide immunoaffinity capture** operates after proteolysis: SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies) uses antibodies against proteotypic peptides with stable isotope-labeled standards for quantification, and multiplex immuno-MRM assays now determine more than 60 targets.[6](https://www.sciencedirect.com/science/article/pii/S0003267021005407)\n\n## Applications\n\nIAC is used for selective purification of proteins and for sample clean-up before analysis of foods for mycotoxins, veterinary drug residues, pesticides, and environmental contaminants, and it has been coupled with HPLC, GC, MS, and CE.[4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf) In bioanalysis, antibodies on beads captured low-level protein doping agents from urine before LC-MS as early as the 1990s, detecting human chorionic gonadotropin at 25 mIU/mL (112.5 fmol/mL).[6](https://www.sciencedirect.com/science/article/pii/S0003267021005407) In high-performance formats, affinity extraction can remove targets in less than a few seconds, a feature used to measure free drug and hormone fractions in serum.[15](https://pmc.ncbi.nlm.nih.gov/articles/PMC3346860/) In bioprocessing, affinity capture is a standard first step for antibody therapeutics; as of 2007, 16 of 17 approved therapeutic monoclonal antibodies used Protein A affinity chromatography as the first capture step.[19](https://scialert.net/fulltext/?doi=jas.2012.1136.1141)\n\n## Limitations and alternatives\n\n**Random coupling wastes antibody.** With random covalent coupling, statistically two of three attachment events destroy antigen-binding capacity, and only one in three antibodies is optimally immobilized; oriented methods address this but Protein A/G-based beads are expensive, bind extraneous antibody, and their specificities are isotype-dependent.[4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf)\n\n**Elution stresses the product and the column.** Low pH 2.0–2.5 is the elution method of choice because it disrupts ionic and hydrogen bonds, but denaturing agents (8 M urea, 6 M guanidinium hydrochloride) and chaotropic salts (3 M sodium thiocyanate, magnesium chloride) can damage labile proteins and shorten column lifetime; chaotropes should be avoided when possible because they are likely to denature the eluted protein.[4](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf) • [8](https://b3p.it.helsinki.fi/download/GE_Protein_Purification_Handbooks/Affinity_Chromatography_Handbook.pdf) Mild alternatives include basic pH 9 elution, competitive small molecules, 1 M NaCl, or high propylene glycol concentrations.[10](https://doi.org/10.1016/bs.mie.2014.11.006) Alkaline elution cannot be used with silica or glass HPIAC supports, which are unstable above pH 8.0.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/)\n\n**Capacity decay and nonspecific loss.** Organic modifiers such as methanol decrease column capacity through denaturation whose kinetic reversal can take a few days.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/) Reducing agents cannot be used at any step because they reduce the antibody's disulfide bonds and diminish activity.[10](https://doi.org/10.1016/bs.mie.2014.11.006) In immunodepletion, significant nonspecific or specific loss of low-abundance proteins is a key caveat, especially for the SuperMix system with its large number of different antibodies.[18](https://www.osti.gov/pages/servlets/purl/1251560)\n\n**Alternatives.** Protein A and Protein G bind the Fc region of IgG, and Protein L binds the variable domains of certain kappa light-chain subtypes, such as human Vk1, Vk3, and Vk4, with binding depending on the antibody species and sequence, giving complementary antibody-capture media that do not recognize a specific antigen.[20](https://research.fredhutch.org/content/dam/research/hahn/methods/Affinity_Chromatography_Handbook-Antibodies.pdf) MEP HyperCel mixed-mode resin binds antibodies at physiological pH and elutes at mildly acidic pH as a gentler alternative to Protein A chromatography, which requires strong acid elution that can denature antibodies and cause loss of potency.[21](https://mdpi-res.com/d_attachment/antibodies/antibodies-12-00015/article_deploy/antibodies-12-00015-v2.pdf?version=1677068652)\n\n## References\n\n1. [Immunoaffinity chromatography: an introduction to applications and recent developments (Hage et al., Bioanalysis 2010)](https://pmc.ncbi.nlm.nih.gov/articles/PMC2903764/)\n2. [Affinity Chromatography: A Review of Trends and Developments over the Past 50 Years](https://pmc.ncbi.nlm.nih.gov/articles/PMC7584770/)\n3. [Affinity Chromatography: A Historical Perspective (Hage & Matsuda, Methods in Molecular Biology, 2015)](https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1075&context=chemistryhage)\n4. [Immunoaffinity Chromatography: A Review (IntechOpen)](https://cdn.intechopen.com/pdfs/33050/InTech-Immunoaffinity_chromatography_a_review.pdf)\n5. [Immunoaffinity Chromatography of Proteins (Gallant, Methods in Molecular Biology)](https://experiments.springernature.com/articles/10.1385/1-59259-742-4:103)\n6. [Affinity capture in bottom-up protein analysis – proteolytic peptide capture using antibodies and MIPs (Analytica Chimica Acta)](https://www.sciencedirect.com/science/article/pii/S0003267021005407)\n7. [Antibody Purification: Affinity Chromatography – Protein A and Protein G Sepharose (Methods in Molecular Biology)](https://experiments.springernature.com/articles/10.1007/978-1-59745-324-0_5)\n8. [Affinity Chromatography Principles and Methods Handbook (GE Healthcare/Cytiva)](https://b3p.it.helsinki.fi/download/GE_Protein_Purification_Handbooks/Affinity_Chromatography_Handbook.pdf)\n9. [Chromatographic immunoassays: strategies and recent developments in the analysis of drugs and biological agents](https://pmc.ncbi.nlm.nih.gov/articles/PMC4820777/)\n10. [Immunoaffinity Purification of Proteins (Kavran & Leahy, Methods in Enzymology, 2015)](https://doi.org/10.1016/bs.mie.2014.11.006)\n11. [Use of antibody bound to modified cellulose as an immunospecific adsorbent of antigens (Biochimica et Biophysica Acta, 1963)](https://doi.org/10.1016/0006-3002%2863%2990913-2)\n12. [JERKER PORATH, ROLF AXÉN, SVERKER ERNBACK (1967). Chemical Coupling of Proteins to Agarose. Nature.](https://doi.org/10.1038/2151491a0)\n13. [P Cuatrecasas, M Wilchek, C B Anfinsen (1968). Selective enzyme purification by affinity chromatography.. Proceedings of the National Academy of Sciences.](https://doi.org/10.1073/pnas.61.2.636)\n14. [P Cuatrecasas, C B Anfinsen (1971). Affinity Chromatography. Annual Review of Biochemistry.](https://doi.org/10.1146/annurev.bi.40.070171.001355)\n15. [Pharmaceutical and biomedical applications of affinity chromatography (Hage et al., 2012)](https://pmc.ncbi.nlm.nih.gov/articles/PMC3346860/)\n16. [Rembert Pieper and colleagues (2003). Multi‐component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome. PROTEOMICS.](https://doi.org/10.1002/pmic.200390057)\n17. [IgY14 and SuperMix immunoaffinity separations coupled with LC-MS for human plasma proteomics biomarker discovery](https://pmc.ncbi.nlm.nih.gov/articles/PMC3261331/)\n18. [Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids](https://www.osti.gov/pages/servlets/purl/1251560)\n19. [Comparison of Binding Capacity and Affinity of Monoclonal Antibody towards Different Affinity Resins (Journal of Applied Sciences)](https://scialert.net/fulltext/?doi=jas.2012.1136.1141)\n20. [Affinity Chromatography Handbook, Vol. 1: Antibodies (Cytiva)](https://research.fredhutch.org/content/dam/research/hahn/methods/Affinity_Chromatography_Handbook-Antibodies.pdf)\n21. [Non-Affinity Purification of Antibodies (Antibodies, 2023)](https://mdpi-res.com/d_attachment/antibodies/antibodies-12-00015/article_deploy/antibodies-12-00015-v2.pdf?version=1677068652)\n\n---\n*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Biochemistry field and methods › Biochemical methods and techniques › Detection methods and analytical reactions*\n\n*Initially written Sep 29, 2026 · Edited: Oct 15, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/immunoaffinity-chromatography", "markdown_url": "https://www.edgepedia.org/immunoaffinity-chromatography.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. AI training is open to everyone. 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Edgepedia Community License 1.0.", "speakable": "Immunoaffinity chromatography is a liquid chromatography method in which immobilized antibodies selectively capture and purify a target biomolecule from a complex mixture, first reported in 1963." }