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Review Article

Separation of antigens and antibodies by immunoaffinity chromatography

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Pages 1038-1044 | Received 20 Aug 2011, Accepted 22 Dec 2011, Published online: 06 Apr 2012

Abstract

Context: Affinity chromatography is an efficient antibody, antigen and protein separation method based on the interaction between specific immobilized ligands and target antibody, antigen, and so on. Populations of available ligands can be used to separate antibodies or their Fab fragments. Similarly, antigens can be isolated by immunoaffinity chromatography (IAC) on immobilized antibodies of low affinity.

Objective: This review describes the advantages, the applications, as well as the drawbacks, of IAC in the separation and purification of antibodies and antigens.

Methods: The present review discussed all types of purification and isolation of antibodies and antigens by IAC, including purification of antibodies using immobilized and synthetic mimic proteins A, G and L; isolation of Fab fragments of antibodies; separation of antibodies against different antigen forms; isolation of antigens by immobilized antibodies and so on. These methods come from over 60 references compiled from all major databases.

Results: Purification of antigens with antibodies should choose low-affinity antibodies to avoid denaturation of most proteins. Concern for cost and safety, prompted research activities focused on novel synthetic ligands with improved properties such as lower cost, avoidance of the risk of contamination associated with natural ligands of human or animal origin to isolate antibodies and antigens.

Conclusion: It is anticipated that the improvements of IAC will have impact not only on large-scale production of antibodies but also on the generation of new affinity-based methods for the increasing number of proteins and antibody derivatives available by protein engineering and the proteomics revolution.

Introduction

Affinity chromatography, a highly selective separation technique, is a type of adsorption chromatography where the target molecule is reversibly adsorbed by a ligand immobilized onto an insoluble support. The ligand is selected with respect to its affinity for a biomolecule, such as the affinity of an antibody to its antigen or that of an antigen to its antibody. Immunoaffinity chromatography (IAC) is a process in which the binding affinity of an antigen to a parent antibody is used as a basis of separation. Firm and specific interactions between antigens and antibodies allow their versatile applications in IAC by using either immobilized antigens or antibodies (CitationSubramanian, 2002; CitationMartins et al., 2007). IAC was discovered in the 1930s. During the last 20–30 years, it has steadily become an invaluable tool in the life sciences with the development of chromatographic materials and immobilization methods.

Antibodies are isolated as an antibody class for immunodiagnostics and biopharmaceutics, and the higher levels of purification are beneficial for the diagnostic methods and for the therapeutic applications. Highly selective methods of antibodies and their Fab fragments purification are important for obtaining catalytic antibodies (CitationDubrovskaya et al., 2003; CitationPagetta et al., 2007), as well as revealing autoimmune catalytic antibodies (CitationNedonchelle et al., 2000). Pure antibodies are also necessary for biochips and related systems, a field which is growing rapidly (CitationKonovalova et al., 2007; CitationWarsinke, 2008). Monoclonal antibodies (MAbs) are useful for the treatment of a wide array of indications including autoimmune diseases, infectious diseases, cardiovascular diseases, transplant rejection and cancer. For instance, IgA, IgE, IgM and IgY increasingly find application in the cure and/or diagnosis of important diseases, especially for the IgG class, which plays most important role in the clinical application (CitationD’Agostino et al., 2008). Meanwhile, extremely purified-pooled polyclonal IgG, intravenous IgG and specific antibodies (hyperimmune IgG) have become the basis for standard therapies in a number of malignancies (CitationVerdoliva et al., 2002). With respect to the importance of purification of antibodies, more and more attention has been paid to IAC, which is an efficient protein separation method based on the interaction between target proteins and specific immobilized antibodies (CitationGrønborg et al., 2002; CitationSteen et al., 2002). In this review, we will describe advantages, applications as well as drawbacks of IAC in the separation and purification of antibodies and antigens.

Isolation of antibodies with affinity chromatography

Antibodies can be isolated from sera of immunized animals (polyclonal antibodies) or from the ascites or culture supernatant (MAbs). To isolate specific polyclonal antibodies from a serum, affinity chromatography on immobilized antigens is often used (CitationMuronetz & Korpela, 2003).

Purification of antibodies on immobilized proteins A, G and L

The rapid detection and separation of Staphylococcus aureus and group G Streptococcus were based on the affinity chromatography interactions between Fc fragment of human IgG and protein A/G (located on the cell wall of S. aureus and group G Streptococcus) () (CitationXiao et al., 2007). Immobilized protein A, G and L possessing affinity for type 1–4 of IgGs can be used for the isolation of MAbs from ascites or culture supernatant () (CitationRoque et al., 2005a, 2007; CitationHahn et al., 2006; CitationCarter-Franklin et al., 2007; CitationPavlovic et al., 2007). Protein A is a cell wall protein of S. aureus, which binds selectively to Fc region of IgG but cannot form a complex with human IgG3. Protein A also binds to IgM and IgE (CitationJaoko et al., 2001; CitationCheng et al., 2006). Protein G, a surface IgG-binding protein of Streptococci groups C and G, has a special affinity for the Fc region of IgG. It binds to IgG such as human IgG of all four subclasses and also to mouse monoclonal IgG, as well as to IgM (CitationOmtvedt et al., 2006). It has been reported that the affinity constant is higher for protein G than for protein A for IgG. Protein L from Peptostreptococcus magnus (PpL) interacts with two-thirds of mouse and almost half of the human Ig repertoires. IgG-binding domains from PpL have been shown to bind specifically to κ-light chains (κ1, κ3 and κ4, but not κ2) and λ light chains of antibodies (CitationCossins et al., 2007).

Figure 1.  (a) Schematic representation of an IgG molecule. IgG has a basic four-chain monomeric structure consisting of two identical heavy chains which contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), and two identical light chains. Between the CH1 and CH2 is the hinge region. An IgG molecule can be divided into two parts functionally: Fragment antigen-binding (Fab) fragment, which is the antigen-binding site, and Fragment crystallizable (Fc) fragment, which is the protein A-binding site (CitationYang et al., 2003). (b) The Staphylococcal protein A shown here is comprised with five homologous IgG-binding domains (E, D, A–C), which have high affnity with the side chains of His435 and Tyr436 of Fc1, and a cell-wall attaching structure (XM) (CitationHober et al., 2007).

Figure 1.  (a) Schematic representation of an IgG molecule. IgG has a basic four-chain monomeric structure consisting of two identical heavy chains which contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), and two identical light chains. Between the CH1 and CH2 is the hinge region. An IgG molecule can be divided into two parts functionally: Fragment antigen-binding (Fab) fragment, which is the antigen-binding site, and Fragment crystallizable (Fc) fragment, which is the protein A-binding site (CitationYang et al., 2003). (b) The Staphylococcal protein A shown here is comprised with five homologous IgG-binding domains (E, D, A–C), which have high affnity with the side chains of His435 and Tyr436 of Fc1, and a cell-wall attaching structure (XM) (CitationHober et al., 2007).

Table 1.  Examples of purification of antibodies using synthetic mimic ligands of proteins A and L.

For example, protein A chromatography can separate several IgG1s, IgG2s, antibody fragments and Fc-fusion proteins using eluent with different pH value (CitationGhose et al., 2005). Protein G affinity chromatography can isolate free subunits of Fab (CitationPagetta et al., 2007).

Purification of antibodies using synthetic mimic ligands of proteins A and L

Compared to conventional protein A and L ligands, synthetic mimic ligands (mimic ligands of protein A and L) with pseudobiospecific properties can alternate them for purification of antibodies and surmount some drawbacks such as high cost, low binding capacity, limited life cycles and so on. This kind of ligands includes biological (peptides and engineered protein domains) and completely synthetic (designed dyes and de novo designed) molecules () (CitationPalombo et al., 1998; CitationRoque et al., 2005b).

Nilsson et al. firstly introduced combinatorial engineering of immunoglobulin-binding protein domains (domain Z, an SpA-analogue domain) and corresponding research has been focused on until now. Lead PpL mimetics (a 169-membered solid-phase ligand library) were synthesized by means of rational design and combinatorial chemistry for the purification of antibodies and small fragments, such as Fab and scFv, and as potential diagnostic or therapeutic agents. The results show that the most promising lead, ligand 8/7, behaves in a similar fashion to PpL in isolating Fab fragments from papain digests of human IgG to a final purity of 97%. Its properties of binding to IgG1 with K and λ isotypes (92% and 100% of loaded protein) and polyclonal IgG from sheep, cow, goat and chicken were also reflected in the efficient isolation of IgGs from crude samples (CitationRoque et al., 2005a) ().

Table 2.  Properties of the Ig-binding bacterial proteins A, G and L.

The construction of peptide libraries also plays an important role in the synthesis of peptidic ligands. Protein A mimetic ligands such as a synthetic peptide (TG19318) or PAM and its inverse derivative (D-PAM), as well as ligand 22/8 were researched (CitationKabir, 2002; CitationVerdoliva et al., 2002; CitationD’Agostino et al., 2008). TG 19318, comprising four identical tripeptide chains linked to a central polylysine core, has been used to isolate polyclonal and MAbs of different classes (IgG, IgM, IgA and IgE) from different sources (serum, ascites and cell supernatants) and species. D-PAM, which is from the PAM peptide by replacing the natural amino acids with the corresponding D isomers, was able to achieve monoclonal IgG isolation from ascetic fluids and cellular supernatants and to obtain purification of polyclonal antibodies from serum. Ligand 22/8, consisting of two organic aromatic amines (3-aminophenol and 4-amino-1-napthol) linked to a scaffold of cyanuric chloride (triazine), displayed wider specificity than protein A, as it isolated IgG from a number of species, the order of adsorption being human > chicken > cow > rabbit > pig > horse > rat > goat > sheep > mouse.

Another approach has used a combination of molecular modeling and synthetic chemistry to design small molecule ligands that can mimic the Fc–protein A interaction. This method originates from the use of dyes as biomimetic ligands for protein binding (CitationGhose et al., 2006). Based on the principle that the dipeptide motif Phe-132:Tyr-133 plays an important role in the interaction of protein A with Fc portion of IgGs, corresponding synthesis of mimetic peptides has been paid attention to. For example, CitationLi et al. (1998) have designed and synthesized several molecules to mimic the Phe-132:Tyr-133 dipeptide by using 1,3,5-trichloro-triazine as the scaffold. Two synthetic, nonpeptidyl protein A mimetic ligands – Mab sorbent A1P and A2P were investigated to be used in the initial purification step for capturing of polyclonal antibodies (CitationGhose et al., 2006). They have small molecules mimicking the amino acids that are responsible for the specific binding interaction in protein A and the results showed that the initially achieved purity was approximately 80%.

Isolation of Fab fragments of antibodies

Advances in affinity chromatography, immobilized proteolytic enzymes (CitationJosic & Clifton, 2007), immobilized protein A (CitationBloom et al., 1989) and protein L (CitationRoque et al., 2005b) for separation of Fc fragments have been described. For example, CitationNing et al. (2003) reported a yield of anti-HBs Fab fragment by affinity chromatography with a purity of 95%.

Fab fragments can be obtained by using nonstoinchiometric polyelectrolyte complexes with attached antigens (CitationDainiak et al., 1998). The main problem for the production of Fab fragments from antibodies is the design of the proteolytic degradation so that splitting of antibodies into Fab and Fc fragments is nearly complete without damaging the Fab moiety. A 1.8-fold purification of antibodies was achieved without a chromatographic step yielding a preparation with more than 95% purity. Rather small damage of the antibodies not only protects binding sites of MAbs from proteolytic damage but also facilitates the proteolysis probably by exposing the antibody molecules in a way convenient for proteolytic attack by the enzyme. The described method is gentler toward antibodies because their binding sites are protected by binding to immobilized antigens.

Separation of antibodies against different antigen forms

Antibodies capable of interaction with a certain antigen form are necessary for research purposes and practical applications. In the case of MAbs, it is sufficient to select critically the required hybridomas. Although polyclonal antibodies against a protein still prove to be powerful tools to study proteins and their functions, CitationHata and Nakayama (2007) obtained specific antibodies from polyclonal antisera with the new HaloTag-based procedure in just two short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum. This method is quite rapid and simple; potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.

Recently, systematic studies for obtaining antibodies recognizing only nonnative molecular forms of a protein were carried out (CitationO’Nuallain et al., 2007). MAbs of two clones (antibodies of clone 6C5 and clone 6C7) reacting with the nonnative forms of denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), EC 1.2.1.12, were obtained. The investigation of the selected clones by IAC on immobilized oligomeric forms (varying from nonnative monomers to native tetramers) of antigen, including unfolded protein subunits, indicated that both clones of the antibodies effectively bound to active and inactive monomers and dimmers, as well as to the unfolded polypeptide, but not to the native tetrameric protein (CitationGoldberg, 1991; CitationGuijarro et al., 1995).

Polyclonal sera inherently contain a set of different forms of antibodies against an antigen. Different fractions of antibodies are achieved by IAC on immobilized native and denatured forms of antigens. Based on this method, two types of polyclonal antibodies specific to holoenzyme and dGAPDH respectively were separated from the same antiserum of chinchilla rabbits using the standard procedure of affinity chromatography (CitationMuronetz & Korpela, 2003; CitationArutyunova et al., 2004). In general, a great many of antibodies can be separated from polyclonal sera for their homogeneous properties against some antigens.

Isolation of antigens by immobilized antibodies

The use of immobilized antigens for the isolation of antibodies seems to be an optimal method in the field of the affinity chromatography, which is based on the extremely specific interactions. Similarly, because of its high affinity and selectivity, IAC could be in principle of great value in the rapid purification of antigens proteins to high degrees of purity. The antibodies could either be used to purify natural proteins or be used for recombinant proteins purification in conjunction with other methods (CitationBlank et al., 2002). However, there are several main factors limiting the use of antibodies for affinity chromatography: (1) it needs the large investment of time and effort and thus cost to generate MAbs and to produce them at the scale required; (2) high specificities of the antigen–antibody interactions require isolation of specific antibodies for the isolation of antigens from a certain source; (3) immobilization of antibodies on a support often results in a decrease or complete loss of their antigen-binding properties and (4) high-affinity antibody–antigen complexes are difficult to dissociate, often leading to inactivation of the protein product during elution from the immobilized antibody (CitationThompson & Burgess, 2001).

Selection of low-affinity antibodies applicable for antigen purification

IAC has not been generally useful for the purification of proteins in their native state, because the relatively harsh conditions required for the dissociation of antigens from antibodies will cause denaturation of most proteins. However, it has been possible to obtain MAbs that bind to their antigens with low affinity. The antigens may be dissociated from these low-affinity antibodies under relatively mild conditions. Immunoaffinity columns constructed with such low-affinity MAbs have been very effective in the purification of proteins (CitationKellogg & Alberts, 1992).

Therefore, it is preferable to use antibodies of low affinity to purify antigens. Antibodies fragments (mono- and bivalent Fab fragments), as well as mini-antibodies (fragments of the hypervariable sites) usually exhibit a lower affinity to antigens (CitationEngström et al., 2005). The scheme of immunoaffinity isolation of surface antigen of hepatitis B virus was developed. The yield of purified HBs-antigen obtained with MAbs exceeded 90% (CitationNikolaenko et al., 2007). Immunodiagnostically useful Mycobacterium tuberculosis H37Ra protein antigens ES-31, ES-43 and EST-6 were isolated from detergent soluble sonicate (DSS) antigen using monospecific antibodies by affinity chromatography. ES-31, ES-43 and EST-6 antigens purified from both culture filtrate and DSS antigen showed similar seroreactivity with overall sensitivity 85, 80 and 75%, respectively (CitationUpadhye et al., 2007).

Isolation of proteins and other compounds with antibodies

Immobilized antibodies are used for purification or separation of various substrates, containing enzymes, peptides (CitationLiu et al., 2007), antibodies and cells (CitationPappas & Wang, 2007). Compared to standard purification methods, affinity chromatography offers high selectivity, hence high resolution, and usually high capacity for proteins of interest. Purification can be in the order of several thousand-fold. Purification that would otherwise be time-consuming, lower purity, difficult or even impossible using other techniques can be easily achieved with affinity chromatography. For example, β-lactoglobulin, prolactin and human protein C were isolated with antibodies immobilized by different methods. Alkaline phosphatase was purified with a 95% yield and pyruvate kinase was purified 2400-fold from an erythrocyte lysate with an 83% yield (CitationKuckelkorn & Jacobasch, 1990) in one step from a crude extract. Successful isolation of recombinant α-amylase (CitationBibi, 1989) and β-lactamase has also been described. Besides, the large variety of antibodies or other ligands immobilized on solid supports have been used to solve several problems like the selection of phosphorylated, glycosylated proteins. Antibodies specific for phosphorylated tyrosine residues were used for the selection of phosphorylated proteins (CitationPandey et al., 2000; CitationSteen et al., 2002).

For proteomic projects and genomics projects, protein separation from plasma, serum, CSF, urine and other body fluid or cellular sources is an important step (CitationGuijarro et al., 1995; CitationKitao & Takata, 2006). Among various separation technologies, IAC is one of the most effective methods when the selected antibodies have the following properties: strong avidity, high specificity, low nonspecific binding or accumulative production. For instance, plasma proteins were isolated by chicken IgY antibodies (CitationHuang & Fang, 2008). Annexin V was purified from human placenta on column with appropriate monospecific antibodies. The yield of the protein is about 5 mg per 100 g of wet tissue through a two-stage procedure (CitationMikaelyan et al., 2008). With the development of purification methods for recombinant green fluorescent protein (rGFP) or recombinant proteins fused with GFP tag, the purity of rGFP (more than 97% homogeneity) using this method, which is based on high specificity and affinity of MAb against GFP, is superior to other methods (ZCitationHuang et al., 2008). Additionally, IAC with antibody fragments, which can be obtained based on the protein fragment complementation assay and the availability of antibody libraries, would be particularly attractive for the parallel purification of proteins for proteomics projects. For instance, generic, parallel and scalable protein purification was achieved with the method of the directed immobilization of recombinant antibody fragments as ligands (CitationBlank et al., 2002).

IAC is also the important and effective technology which can remove nonnative and denatured protein forms. For the increasing usage of recombinant proteins, it is especially necessary to separate functional proteins from their nonnative forms. Nonnative proteins can also be removed with antibodies immobilized on soluble polyelectrolytes (CitationMuronetz & Korpela, 2003). Based on this principle, the synthesis of the conjugate of poly(methacrylic acid) with MAbs was used to remove dGAPDH (CitationMuronetz et al., 2000).

The separation of proteins with the technology of IAC is also exemplified by various investigations. CitationTaylor et al. (2003) have reported two kinds of methods for immunoaffinity purification of human neutrophil flavocytochrome b (Cyt b) successively to analyze its structure and catalytic mechanism. One is that Cyt b was purified through using the p22phox-specific mAb 44.1, with gentle elution of Cyt b carried out by the addition of epitope-mimicking peptide. Although this method has some advantages such as good recoveries and higher purification of Cyt b, the limit is that purification by mAb 44.1 is only achieved using the low critical micelle concentration detergent dodecylmaltoside (CitationTaylor et al., 2003). Therefore, another improving method was investigated. The epitope-mapped mAb CS9 (p22phox subunit of Cyt b) was coupled to Sepharose beads to be used as an affinity matrix for single-step immunoaffinity purification of Cyt b from both human neutrophil and PLB-985 membrane fractions. The high efficiency of this method overcame the drawback of the previous one mentioned above (CitationLord et al., 2008). IAC using single-domain antibodies from Camelidae yielded a completely pure and functional ice structuring protein from Lolium perenne, which modifies ice crystal growth and therefore has potential application in medicine, biotechnology, agriculture and (frozen) foods. The results display that highly pure proteins can be recovered from biological material in a single-step process (CitationVerheesen et al., 2003).

Conclusions and perspectives

In this review, purification and isolation of antibodies and antigens by IAC have been discussed. Because of the specific binding between antibodies and antigens or proteins, antigens or proteins A, G and L are used to separate various antibodies (MAbs or polyclonal antibodies) such as IgGs, IgM, IgE, IgA, IgY, and so on, as well as Fab fragments of antibodies and so on, while antibodies are commonly used in affinity chromatography as versatile and specific means for isolating target molecules from complex mixtures. However, purification of antigens with antibodies should choose low-affinity antibodies to avoid denaturation of most proteins. With respect to the cost and safety, prompted research activities have being focused on novel synthetic ligands (synthetic mimic ligands of proteins A and L) with improved properties such as lower cost, avoidance of the risk of contamination associated with natural ligands of human or animal origin to isolate antibodies (CitationD’Agostino et al., 2008). What’s more, affinity chromatography (one of the protein separation approaches) on immobilized antibodies with specific binding to proteins plays an important role in the proteomic (phosphoprotemics or glycoproteomics) and genomic field.

The specific binding between antigen and antibody is the principle for their isolation by IAC but is also the limitations for the high affinity, leading to inactivation of the protein product during elution from the immobilized antibody. Therefore, approaches of selection of low-affinity antibodies or using polyol-responsive MAbs have been investigated to solve this problem (CitationThompson & Burgess, 2001). It is anticipated that the improvements of IAC will have impact not only on large-scale production of antibodies but also on the generation of new affinity-based methods for the increasing number of proteins and antibody derivatives available by protein engineering and the proteomics revolution.

Acknowledgment

We thank Miss Trista Talbot (Department of Chemistry, Brandeis University) for language editing of the manuscript.

Declaration of interest

The authors report no declarations of interest.

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