Characteristics of the adsorption of immunoglobulin M onto Q Sepharose® Fast Flow ion-exchangers

Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow(based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate(RMM 950000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.This revised version was published online in October 2005 with corrections to the Cover Date.     

Confocal laser scanning microscopy as an analytical tool in chromatographic research

In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.     

Effects of ionic strength on lysozyme uptake rates in cation exchangers. I: Uptake in SP Sepharose FF

Fluorescence scanning confocal microscopy was used in parallel with batch uptake and breakthrough measurements of transport rates to study the effect of ionic strength on the uptake of lysozyme into SP Sepharose FF. In all cases the adsorption isotherms were near-rectangular. As described previously, the intraparticle profiles changed from slow-moving self-sharpening fronts at low salt concentration, to fast-moving diffuse profiles at high salt concentration, and batch uptake rates correspondingly increased with increasing salt concentration. Shrinking core and homogeneous diffusion frameworks were used successfully to obtain effective diffusivities for the low salt and high salt conditions, respectively. The prediction of column breakthrough was generally good using these frameworks, except for low-salt uptake results. In those cases, the compressibility of the stationary phase coupled with the shrinking core behavior appears to reduce the mass transfer rates at particle-particle contacts, leading to shallower breakthrough curves. In contrast, the fast uptake rates at high ionic strength appear to reduce the importance of mass transfer limitations at the particle contacts, but the confocal results do show a flow rate dependence on the uptake profiles, suggesting that external mass transfer becomes more limiting at high ionic strength. These results show that the complexity of behavior observable at the microscopic scale is directly manifested at the column scale and provides a phenomenological basis to interpret and predict column breakthrough. In addition, the results provide heuristics for the optimization of chromatographic conditions.     

Competitive adsorption of labeled and native protein in confocal laser scanning microscopy

Confocal laser scanning microscopy has been previously applied to the study of protein uptake in porous chromatography resins. This method requires labeling the protein with a fluorescent probe. The labeled protein is then diluted with a large quantity of native protein so that the fluorescence intensity is a linear function of the labeled protein concentration. Ideally, the attachment of a fluorescent probe should not affect the affinity of the protein for the stationary phase; however, recent experimental work has shown that this assumption is difficult to satisfy. In the present study, we present a mathematical model of protein diffusion and adsorption in a single adsorbent particle. The differences in adsorption behavior of labeled and native protein are accounted for by treating the system as a two-component system (labeled and native protein) described by the steric mass action isotherm (SMA). SMA parameters are regressed from experimental linear gradient elution data for lysozyme and lysozyme-dye conjugates (for the fluorescent dyes Cy3, Cy5, Bodipy FL, and Atto635). When the regressed parameters are employed in the model, an overshoot in the labeled lysozyme concentration is predicted for Cy5- and Bodipy-labeled lysozyme, but not for Atto635-labeled lysozyme. The model predictions agree qualitatively well with recent work showing the dependence of the concentration overshoot on the identity of the attached dye and provide further evidence that the overshoot is likely caused by the change of binding characteristics due to the fluorescent label.     

Computerized quantification of immunofluorescence-labeled axon terminals and analysis of co-localization of neurochemicals in axon terminals with a confocal scanning laser microscope

The confocal scanning laser microscope (CSLM) offers improved optical resolution and contrast, high photometric precision, and the ability to make optical sections. These benefits were explored for use in quantitative analysis of immunofluorescence-labeled axon terminals. Guidelines were obtained for adjustments of the CSLM parameters. In the present applications, bleaching of the fluorescence did not represent a serious obstacle to analysis with the CSLM. A method was developed to distinguish the background fluorescence from the specific fluorescence labeling. This procedure made way for the development of automated quantification of immunolabeled axon terminals. The automated procedures substantially reduced the man-hour expenditure for analysis and provided highly reproducible quantifications compared with manual methods. The increased resolution and contrast of the CSLM allowed measurements of the fluorescence signal strength of individual axon terminals. The CSLM also allowed detection of co-localized neurochemicals in axon terminals.     

Evaluation of a novel agarose-based synthetic ligand adsorbent for the recovery of antibodies from ovine serum

This paper evaluates a prototype agarose-based affinity adsorbent utilizing a bound synthetic ligand designed to replace Protein A as an IgG-affinity capture resin and compares its purification characteristics with four commercially available matrices for the recovery of polyclonal antibodies from crude hyperimmune ovine serum. The novel adsorbent was found to show the highest dynamic capacity (29.2 mg/mL) of all matrices under evaluation--30% higher than the other commercial adsorbents evaluated. When using a post-load caprylic acid wash, IgG yields of over 85% and purities of over 90% were achieved consistently over multiple loading cycles. To evaluate bead diffusion, inverted confocal microscopy was used to visualise fluorescent antibody binding on to individual adsorbent beads in real time. The results indicate that the binding characteristics of the prototype adsorbent are similar to those obtained with Protein G Sepharose. This study indicates that the high-capacity prototype matrix is a feasible and potentially cost-effective alternative for the direct capture of antibodies from crude ovine serum and may therefore also be applicable to the purification of other complex industrial feedstocks such as transgenic milk or monoclonal antibodies expressed using recombinant technologies.     

Bioseparations with permeable particles

Permeable large-pore particles have many applications, in particular in perfusion chromatography for bioseparations. The objective of this paper is to elucidate the mass transport mechanisms in two commercial adsorbents-POROS Q/M and Q Hyper D- and to answer the question if intraparticle convection is present as a mass transfer mechanism. The paper contains three sections. In the first part, mass transfer inside porous particles is discussed. The mass transfer mechanism which allows improved performance of perfusion chromatography is intraparticle convection. The combined effect of intraparticle convection and diffusion is an “augmented” effective diffusivity. This is the key concept to explain the peak sharpening and modified Van Deemter plots found with large-pore particles. In the second part, column efficiencies in terms of HETP as a function of bed superficial velocity are experimentally measured for a non-retained protein (bovine serum albumine, BSA) in two adsorbents: POROS Q/M (PerSeptive Biosystems) and Q Hyper D (BioSepra). In the third section breakthrough curves for both materials are measured for a test protein (BSA) from which useful capacities and productivities as a function of flow-rate are calculated. Experimental results indicate that intraparticle convection plays indeed an important role in both adsorbents.     

Factor analysis of confocal image sequences of human papillomavirus DNA revealed with fast red in cervical tissue sections stained with TOTO-iodide

OBJECTIVE: To visualize and localize specific DNA sequences by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus (HPV) DNA was identified in cervical tissue sections with biotinylated DNA probes recognizing the whole genome of HPV DNA types 18 and 16, and DNA-DNA hybrids were revealed by streptavidin-alkaline phosphatase and Fast Red (FR). Cell nuclei were counterstained with TOTO-iodide. Image sequences were obtained using successive dynamic or spectral sequences of images on different optical slices from CLSM. The location of fluorescent signals inside tissue preparations was determined by FAMIS and/or selection of filters at emission. Image sequences were summarized into a reduced number of images, called "factor images," and curves, called "factors." Factors estimate spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between FR and nucleus staining in HPV DNA hybridization signals by taking into account differences in their spectral patterns and improved visualization by taking into account differences in their focus (depth emission profiles). CONCLUSION: FAMIS, together with CLSM, made possible the detection and characterization of HPV DNA sequences in cells of cervical tissue sections.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

Tubular GFP uptake pattern in the rat and frog kidneys

The renal tubular uptake of green fluorescent protein (GFP) after its bolus intravenous injection was studied in both frogs and rats. GFP fluorescence in the proximal tubule (PT) was revealed by fluorescent and confocal microscopy. Granular GFP fluorescence was observed nearby in the apical membrane of PT cells featuring distribution over the cytoplasm. GFP was internalized into endosomes and lysosomes as determined by immunocytochemistry in frogs. The tubular uptake and accumulation of GFP were dose- and time-dependent in both rats and frogs. Intralymphatic sac injection of arginine vasotocin (AVT) decreased the uptake of GFP in hydrated frogs. A high negative correlation between the AVT dose and the uptake of GFP was revealed. The effect of AVT was inhibited by a V(1)-receptor antagonist. A noted decrease in the average number of fluorescent PT profiles per kidney section and their irregular distribution after AVT injections suggest that not all of the glomeruli or preglomerular vessels are equally responsive to AVT. GFP may serve as a good marker for tubular uptake and intracellular traffic in the amphibian kidney for use in in vivo studies.     

High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth

Three-dimensional images can be assembled by piling up consecutive confocal fluorescent images obtained by confocal scanning laser microscopy. The present work was based on three-dimensional (50-microns-deep) images at high (x, y) resolution obtained with an MRC-500 after en bloc staining of thick slices of rat liver by chromomycin A3 for nuclear DNA. The results of studies on bleaching, fluorescence excitation and emission intensities at various depths of histologic preparations are described. These effects could be evaluated separately by acquiring piled-up ("brick-stepping") and non-piled-up ("side-stepping") (x, y) images at consecutive depths and also (x, z) images. Empirical equations allowed the fitting of experimental plots of bleaching versus time, at different laser intensities and at different depths, and of fluorescence emission intensity versus depth. The main conclusions were that under our experimental conditions: (1) there was no attenuation by depth of the fluorochrome penetration, (2) there was no attenuation of the exciting beam intensity up to at least 50 microns deep, (3) there was an attenuation of the fluorescence emission intensity by depth, (4) bleaching happened equally on all planes above and below any confocal plane being studied, and (5) the fluorescence bleaching half-life was independent of depth. A mathematical correction scheme designed to compensate for bleaching and for attenuation of fluorescence emission in depth is presented. This correction is required for obtaining three-dimensional images of better quality, for optimal three-dimensional image segmentation and for any quantitative analysis based upon voxel-discretized emission intensities (gray levels)--e.g., estimating, by confocal image cytometry, textural chromatin parameters and nuclear DNA amounts.     

Dual fluorescence confocal imaging of the accessibility and binding of F(ab′)2 to an EBA resin with various immobilised antigen densities

Dual fluorescence confocal laser scanning microscopy has been used to visualise the binding of a fluorescently labelled polyclonal ovine anti-fluorescein F(ab′)₂ antibody to immobilised fluorescein. The fluorescent ligand was immobilised on a Streamline quartz base agarose matrix; a resin used industrially for expanded bed chromatography, using two different fluorescein initial concentrations in order to obtain two batches of immunogen-affinity adsorbent with different immobilised ligand densities. The fluorescein specific F(ab′)₂ were purified from anti-fluorescein serum pepsin digest by adsorption on immobilised antigen chromatographic resin, followed by conjugation to the fluorescent probe Alexa Fluor 660. The dual fluorescence signals from the immobilised antigen and the immuno-specific F(ab′)₂ were used to map the progressive depth of the bound F(ab′)₂ layer within individual adsorbent beads. In addition, the labelled anti-fluorescein F(ab′)₂ was diluted to identical antigen binding activity concentrations in crude serum digest and in blank buffer and the resulting fluorescence intensity profiles were comparatively assessed for any detectable differences in binding patterns that might be caused by processing the more complex mixture of crude serum digests. It was observed that the relative immobilised ligand utilisation was higher when using the immuno-adsorbent with lower immobilised antigen density. Furthermore, the progression of the adsorbed F(ab′)₂ front inside the immuno-adsorbent beads displayed closer agreement with the postulates of the shrinking core mechanism (SCM) when the immuno-adsorbent with lower immobilised antigen was used. The confocal images did not reveal any differences between the depth of the adsorption fronts of crude serum digest and pre-purified F(ab′)₂ samples.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed by five leucine-rich repeats, a proline-rich region, a single transmembrane region and an intracellular kinase domain. The AtSERK1 cDNA was fused to two different variants of green fluorescent protein (GFP), a yellow-emitting GFP (YFP) and a cyan-emitting GFP (CFP), and transiently expressed in both plant protoplasts and insect cells. Using confocal laser scanning microscopy it was determined that the AtSERK1-YFP fusion protein is targeted to plasma membranes in both plant and animal cells. The extracellular leucine-rich repeats, and in particular the N-linked oligosaccharides that are present on them appear to be essential for correct localization of the AtSERK1-YFP protein. The potential for dimerization of the AtSERK1 protein was investigated by measuring the YFP/CFP fluorescence emission ratio using fluorescence spectral imaging microscopy. This ratio will increase due to fluorescence resonance energy transfer if the AtSERK1-CFP and AtSERK1-YFP fusion proteins interact. In 15 % of the cells the YFP/CFP emission ratio for plasma membrane localized AtSERK1 proteins was enhanced. Yeast-protein interaction experiments confirmed the possibility for AtSERK1 homodimerization. Elimination of the extracellular leucine zipper domain reduced the YFP/CFP emission ratio to control levels indicating that without the leucine zipper domain AtSERK1 is monomeric.     

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein–protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).     

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.     

Effective Purification of Bioproducts by Fast Flow Affinity Chromatography

The performance of affinity adsorbents, such as HiPAC, Toyopearl, and Sepharose 4B coupled with protein A and anti-BSA, was compared. The HiPAC column, which is packed with rigid porous silica particles of 30 μm diameter, showed the highest value of D_pore/r² because of the small particle diameter and rigidity of the adsorbent particles. Further, it was operable at higher flow rates than the others. Thus, experimental data and calculation on the purification process show that fast and efficient purification is possible by use of the HiPAC column.     

Biotinylation and characterization of Cryptococcus neoformans cell surface proteins

AIMS: To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation. METHODS AND RESULTS: Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls. CONCLUSIONS: A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.