Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography

We explored the possibility to apply single-domain antibodies from Camelidae for immunoaffinity purification of the ice structuring protein (ISP) from Lolium perenne, which modifies ice crystal growth and therefore has potential application in medicine, biotechnology, agriculture and (frozen) foods. Using phage display together with an appropriate selection method, a group of candidate fragments was isolated from a llama-derived immune library. Affinity chromatography using a purposely selected antibody coupled to a matrix yielded a completely pure and functional ISP. Due to the extreme refolding capabilities and physical stability of single-domain antibodies, the affinity matrix could be regenerated more than 2000 times without loss of capacity, while the fragment's monomeric nature permitted an efficient elution of antigen. The results of this study show that highly pure proteins can be recovered from biological material in a single-step process.     

Affinity chromatography system for parallel purification of recombinant protein samples

In protein engineering, the tasks of generating and testing a large number of variants of a molecule and of optimizing expression conditions for one distinct molecule create the need for purification methods that can handle a large number of samples simultaneously. We describe the development and some application results of a simple affinity chromatography system that can be used for the parallel purification of 24 protein samples, yielding sufficient quantities for biochemical and functional analysis. Advantages of this system over existing systems are as follows. Compared with commercially available complete chromatography systems, the costs of this system are minimal. In comparison with vacuum systems with various outlets, and with batch purification systems where centrifugation is necessary, this system allows gentler processing of the samples. This could be important for proteins that are easily damaged.     

Rapid purification of phospholamban by monoclonal antibody immunoaffinity chromatography

Monoclonal antibodies have been raised against canine phospholamban purified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Four of twenty-four antibodies were purified to close to homogeneity from mouse ascites. All four antibodies could react with isolated bovine cardiac sarcoplasmic reticulum (SR) to result in the stimulation of ATP-dependent Ca2+ pump activity and blocking of phospholamban phosphorylation by cAMP-dependent protein kinase. Relative efficiencies of antibodies in Ca2+ pump stimulation and on phospholamban phosphorylation were not correlated. An immunoabsorbent prepared by conjugating antibody Al to Affi-Gel 10 was used for the purification of phospholamban. Isolated bovine cardiac SR was solubilized in a buffer containing deoxycholate and the soluble fraction was applied to the immunoaffinity column. After washing the column with a series of detergent-containing buffer solutions, the column-bound protein which contained essentially pure phospholamban was eluted by a buffer containing 2.8 M MgCl2. The phospholamban recovery from the immunoaffinity column was close to 100%; the overall yield of purification from SR vesicles was about 70%. SDS-PAGE analysis showed that purified phospholamban consisted of a 25 and 5 kilodalton (kDa) protein species. Upon brief boiling (20 s) of the sample in SDS-PAGE sample buffer, five molecular species ranging from 5 to 25 kDa could be detected by immunotransblotting following SDS-PAGE. This observation supports the notion that phospholamban is composed of five 5-kDa polypeptides. The pure phospholamban could be phosphorylated maximally by cAMP-dependent protein kinase to 1-1.5 mol phosphate/mol phospholamban (25,000 g). This stoichiometry of phosphorylation could be increased to about 5 upon addition of the immunoaffinity column flow through fraction.     

A highly efficient preparative-scale generic immunoaffinity chromatography method for the purification of antibodies to hydrophobic haptens: purification procedure and monitoring tests

A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50-150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS-PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.     

Single framework recombinant antibody fragments designed for protein chip applications

High-throughput proteomics, based on the microarray platform, requires stable, highly functional components that will yield a highly sensitive read-out of low abundance proteins. Although antibodies are the best characterized binding molecules for this purpose, only a fraction of them appear to behave satisfactorily in the chip format. Therefore, high demands need to be placed on their molecular design. In the present study, we have focused on recombinant antibody design based on a single framework for protein chip applications, aiming at defining crucial molecular probe parameters. Our results show that engineered human recombinant scFv antibody fragments that displayed appropriate biophysical properties (molecular [functional] stability in particular) can be generated, making them prime candidates for high-density antibody arrays. In fact, a superior framework that displays both multifaceted adsorption properties and very high functional stability over several months on chips (stored in a dried-out state) was identified. Taken together, designed scFv fragments based on a single molecular scaffold, readily accessible in large phage display libraries, can undoubtedly meet the requirements of probe content in antibody microarrays, particularly for global proteome analysis.     

Grafting of protein L-binding activity onto recombinant antibody fragments

Recombinant antibody fragments consisting of variable domains can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free form or complexed with their antigen. Protein L (PpL) is a cell wall protein isolated from Peptostreptococcus magnus, which has been reported to interact with the V-KAPPA chain of some, but not all, antibodies. Here we grafted the V-KAPPA framework region 1 (FR1) sequence of a high-affinity PpL-binding antibody onto single-chain antibody fragments (scFvs), which have no reactivity with PpL. This substitution made it possible to purify and detect scFvs using PpL conjugates. It did not hinder scFv folding and expression in recombinant bacteria, and it did not interfere with their antigen-binding function. We also identified residue 12 as being potentially able to alter PpL binding. This study, therefore, suggests a way of engineering a PpL-binding site on any scFv without interfering with its function. This could provide a universally applicable method both for the rapid purification of functional recombinant antibody fragments and for their detection even when complexed with their antigen without requiring fusion to an epitope Flag.     

Protein A chromatography for antibody purification

Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.     

Double chip protein arrays using recombinant single-chain Fv antibody fragments

Protein arrays permit the parallel analysis of many different markers in a small sample volume. However, the problem of cross-reactivity limits the degree of multiplexing in parallel sandwich immunoassays (using monoclonal antibodies (mAbs)), meaning antibodies must be prescreened in order to reduce false positives. In contrast, we use a second chip surface for the local application of detection antibodies, thereby efficiently eliminating antibody cross-reactions. Here, we illustrate the potential advantages of using single-chain Fv fragments rather than mAbs as capture and detection molecules with this double chip technology.     

Site-specific antibodies to human erythropoietin: immunoaffinity purification of urinary and recombinant hormone

The 19-amino acid domain Ala111----Pro129 of human erythropoietin was identified as an accessible surface antigen based on the binding of radio-iodinated and of unmodified hormone to antibodies prepared against a synthetic peptide of homologous sequence. The specificity and affinity of this binding was sufficient to provide for the use of anti-peptide antibodies in the preparation of an immunosorbent for the purification of urinary, and of recombinant human erythropoietin. Immobilization of anti-peptide antibodies using agarose activated either with CNBr or with N-hydroxysuccinimido groups largely inactivated binding sites for erythropoietin. In contrast, antibodies crosslinked to N-acetyl-DL-homocysteine agarose through the hetero-bifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate retained their antigen-binding capacity virtually completely and provided a superior immunosorbent for hormone. Urinary erythropoietin with a specific bioactivity of 100 U/A280 was prepared initially by chromatography on phenyl-Sepharose. Subsequent immunoaffinity chromatography resulted in a 350-fold purification with 46.2% recovery yielding erythropoietin with a specific bioactivity of 35,200 U/A280 (44,300 U/mg). Radioiodination of this purified protein and subsequent SDS-polyacrylamide gel electrophoresis indicated that this preparation contained a single major component (Mr 30,000) which co-migrated in gels with unmodified biologically active hormone. Recombinant erythropoietin, which was prepared by the cloning of the human erythropoietin gene and its expression in COS cells using the SV40-derived vector pSV2, was purified by the same scheme. Chromatography on phenyl-Sepharose of medium derived from transfected cells (400 U/ml, 170 U/A280) provided for a 3.6-fold purification of recombinant hormone with an apparent recovery of 122%. This erythropoietin bound to the anti-peptide antibody gel and was purified to a specific bioactivity of 10,370 U/A280 with 55% recovery. The procedure described here for attaching antibodies to a solid support maximizes their antigen-binding capacity and is generally applicable. The development of an anti-peptide immunosorbant for human erythropoietin provides a valuable means for isolating hormone for use in studies of its receptor and its presently unresolved mechanism of action.     

Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli]

We have fused the variable domains of a mouse antibody to the C-terminal end of the maltose-binding protein (malE), at the genetic level. The hybrid proteins were expressed in E. coli under control of the malEp promoter, and exported to the periplasm, at low temperature. They were purified by affinity chromatography on cross-linked amylose. When the two variable domains were fused together through a peptide link, the hybrid displayed similar affinity and specificity to the antigen as the native antibody.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.     

Human recombinant laminin-binding protein: isolation, purification, and crystallization

The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.     

Expression and immunoaffinity purification of recombinant dengue virus 2 NS1 protein as a cleavable SUMOstar fusion

Dengue virus (DENV) encoded nonstructural one (NS1) is a 352 amino acid protein that exists in multiple oligomeric states and is conserved within the flavivirus family. Although NS1 has been heavily researched for its diagnostic utility, there is a gap in the understanding of its role in a range of viral processes, including replication and development of clinical pathologies such as vascular leakage. Many of these functions involve unknown interactions with viral and host proteins. This study describes the generation of a mouse monoclonal antibody (mAb 56.2) that reacts with NS1 from DENV1 and 2, and the expression of recombinant SUMOstar-tagged DENV2 NS1 (DENV2 S^∗-NS1) in baculovirus. This is the first time dengue NS1 has been produced as a SUMOstar fusion with the S^∗-tag increasing protein solubility and secretion compared with a non-S^∗-tagged NS1 construct. The protein was readily purified using a mAb 56.2 immunoaffinity column and untagged NS1 was obtained by treatment with tobacco etch virus protease to remove the S^∗-tag. Size exclusion chromatography and glycosylation assays showed that both secreted S^∗-NS1, and cleaved NS1, are hexameric and glycosylated, and will be useful tools in elucidating dengue NS1 protein interactions and functions.     

Latex-based thin-layer immunoaffinity chromatography for quantitation of protein analytes.

A rapid immunochromatographic method for qualitative and quantitative analysis of protein antigens is described. The method is based on the "sandwich" assay format using monoclonal antibodies (Mabs) of two distinct specificities. Mabs of one specificity are covalently immobilized to a defined detection zone on a porous membrane while Mabs of the other specificity are covalently coupled to blue latex particles which serve as a label. The sample is mixed with the Mab-coated particles and allowed to react. The mixture is then passed along a porous membrane by capillary action past the Mabs in the detection zone, which will bind the particles which have antigen bound to their surface, giving a blue color within this detection zone with an intensity logarithmetrically proportional to the antigen concentration in the sample. Analysis is complete in less than 10 min, requires a minimum amount of sample (4 microliters), and has a detection limit below the nanomolar range for the antigen we studied, human chorionic gonadotropin.     

Application of recombinant antibody fragments for troponin I measurements

The cardiac isoform of troponin I is a reliable biomarker of damaged cardiomyocytes that accompanies such severe cardiovascular diseases as myocardial infarction. Monoclonal antibody 19C7 recognizes troponin I in the blood-stream with high affinity and specificity. Recombinant antibodies can be used to improve detection systems based on monoclonal antibodies produced with hybridoma technology. In the present study, we compare the properties of monoclonal anti-body 19C7 and its recombinant fragments. It is shown that the recombinant antibody fragments demonstrate similar affinity values as monoclonal antibodies and can be applied for troponin I detection.     

Affinity chromatography as a tool for antibody purification

The global antibody market has grown exponentially due to increasing applications in research, diagnostics and therapy. Antibodies are present in complex matrices (e.g. serum, milk, egg yolk, fermentation broth or plant-derived extracts). This has led to the need for development of novel platforms for purification of large quantities of antibody with defined clinical and performance requirements. However, the choice of method is strictly limited by the manufacturing cost and the quality of the end product required. Affinity chromatography is one of the most extensively used methods for antibody purification, due to its high selectivity and rapidity. Its effectiveness is largely based on the binding characteristics of the required antibody and the ligand used for antibody capture. The approaches used for antibody purification are critically examined with the aim of providing the reader with the principles and practical insights required to understand the intricacies of the procedures. Affinity support matrices and ligands for affinity chromatography are discussed, including their relevant underlying principles of use, their potential value and their performance in purifying different types of antibodies, along with a list of commercially available alternatives. Furthermore, the principal factors influencing purification procedures at various stages are highlighted. Practical considerations for development and/or optimizations of efficient antibody-purification protocols are suggested.     

A calcium-dependent antibody for identification and purification of recombinant proteins

We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in a single chromatographic step. The marker peptide could subsequently be removed by proteolysis with the enzyme enterokinase.     

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.