Adsorption kinetics and equilibria of bovine serum albumin on rigid ion-exchange and hydrophobic interaction chromatography matrices in a stirred cell

Rigid adsorbents have advantages over soft gel media for downstream processing of proteins. The adsorption of bovine serum albumin (BSA) has been investigated on a rigid adsorbent based on a wide-pore, hydrophilically coated, silica-gel matrix. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction chromatography) and particle size have been studied on the physical properties of the adsorbent and on the adsorption equilibria and adsorption kinetics. The rates of adsorption of BSA have been measured in a stirred cell and are found to be satisfactorily described by a two-step theoretical model, in which the mass transfer involves a pore diffusion resistance and an extra-particle film resistance. On the anion exchanger, the effective pore diffusivity decreases substantially with increasing protein concentration, approximately halving as the initial concentration rises from 0.7 to 2g/l. In the hydrophobic interaction chromatography medium, the pore diffusivity is less sensitive to protein concentration and is also reduced by a factor of about 4 by aggregation of the protein. Effective pore diffusivities with the "wide-pore" silica adsorbents in anion-exchange form are 36-94 times lower than the diffusivity in free solution and are comparable with the lower of the wide range of values published for soft gels.     

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.     

Immunological Approach to the Detection of Taurine and Immunocytochemical Results

An immunological approach to the detection of taurine resulted in antibodies specific enough to be used for immunocytochemical studies. The experimental conditions were similar to those previously described for raising antibodies against some small-sized neurotransmitter molecules: antisera were obtained from rabbits immunized with taurine conjugated to carrier proteins via glutaraldehyde and purified by adsorption on the glutaraldehyde-treated protein carriers. Antibody affinity and specificity were determined in competition experiments between conjugated taurine and other conjugated amino acids or derivatives by enzyme-linked immunosorbent assay. The resulting cross-reactivity ratios, calculated at half-displacement, showed conjugated taurine to be the best recognized compound. Given the molecular structure of taurine and the method used to prepare the conjugate, it seemed necessary to perform an oxidation step. However, adsorption of antisera on reoxidized or nonreoxidized taurine conjugates suggested that reoxidation did not make a significant difference. Immunocytochemical application of the sera revealed populations of strongly immunopositive nerve cells in the cerebellum, striatum, and septum. The results confirmed that antitaurine antibodies can be used as specific tools for a better understanding of the role of taurine in the central nervous system.     

Direct observation of intraparticle equilibration and the rate-limiting step in adsorption of proteins in chromatographic adsorbents with confocal laser scanning microscopy

The adsorption of different proteins in a single biospecific and hydrophobic adsorbent particle for preparative protein chromatography has been observed directly by confocal laser scanning microscopy as a function of time at a constant bulk concentration c(b). The bulk concentration was in the non-linear part of the adsorption isotherm. At all times the concentration of free protein at the particle surface was almost equal to the bulk content indicating that external mass transfer resistance is not rate limiting for the adsorption under these conditions. Inside the particles a distinct maximum in adsorbed and free protein concentration that moved inside to a distance of approximately 0.2 R (R particle radius) from the particle surface, was observed. This is due to a decreasing solid-phase density and adsorptive capacity in the particle between 0.8 R and R indicating that the fraction of macropores (or void space) is larger in the outer than in the inner part of the adsorbent particles. By increasing the bulk concentration by a factor of 10 the equilibration time was reduced by about the same magnitude. This is in agreement with the concentration dependence of the effective pore diffusion coefficient D(p,eff)=D(p)/[epsilon(p)[1+nK/(K +c)(2)]] derived from the mass conservation relations describing the adsorption process. The time dependence protein adsorption up to approximately 90% of the equilibration value q* could be described by a bilinear free driving force model. The rapid equilibration in the outer part of the particle with a half-life time of approximately 100 s in the studied systems accounted for 0.3-0.4 q*. The slower equilibration with a up to ten times longer half-life time, was the adsorption in the inner part of the particle that outside 0.5 R accounts for 0.5-0.6 q*. These data were compared with literature data for batch adsorption of proteins in biospecific, hydrophobic and ion-exchange adsorbents. They could also be described by a bilinear free driving force model, with about the same quantitative results as obtained for similar conditions in the single particle experiments. The static adsorption parameters, maximum binding site concentration n, and dissociation constant for the protein binding to a binding site K, were determined from Scatchard plots. For the same protein-adsorbent system the plots changed from linear to non-linear with increasing n. This change occurred when the average distance between adjacent binding sites become of the same order of magnitude as the size of the binding site or adsorbed protein. This causes a shielding of free binding sites increasing with n and the concentration of adsorbed protein, yielding a concentration dependence in K. These results show that for a high throughput and rapid adsorption in preparative chromatography, the adsorption step should be carried out in the non-linear part of the adsorption isotherm with concentrations up to c(b) where q*/c(b)>/=10 to obtain high protein recoveries. To avoid tailing due to the flow of adsorbed proteins in the inner part of the particles further into the particles at the start of the desorption, and to speed up desorption rates, protein adsorption in the particle within 0.5 R from the particle center should be avoided. This requires the further development of suitable pellicular particles for preparative protein chromatography that meet this requirement.     

Evaluation of the immunocytochemical method for amino acids

Free amino acids can be coupled to proteins by glutaraldehyde. Rabbits immunised with a bovine serum albumin-glutaraldehyde-amino acid conjugate form antibodies that recognise similar conjugates with brain proteins in glutaraldehyde-fixed tissue. Antisera raised against conjugated GABA (gamma-aminobutyrate), glutamate, aspartate, taurine, glutamine, or glycine were tested against a variety of small molecular compounds that had been fixed by glutaraldehyde to brain protein and immobilised on cellulose ester filters for processing together with the brain sections. This system permitted closely similar conditions for testing and immunocytochemistry. After removing antibodies against the carrier used for immunisation and against cross reacting amino acid conjugates the antisera showed a high specificity. The specific nature of the antisera was corroborated by solid phase adsorption to the homologous antigens and by inhibition experiments with free amino acids and amino acid-glutaraldehyde fixation complexes. After transection of the striatonigral pathway the ipsilateral substantia nigra was almost depleted of GABA-like immunoreactivity; this observation lends additional support to the selectivity of the GABA antiserum. A semiquantitative relation was established between the concentration of amino acid before fixation in a model system and the subsequent intensity of immunostaining. Similar model experiments suggested that the conjugation of an amino acid to brain protein with glutaraldehyde, and the immunoreactivity of the conjugates, may be significantly inhibited in the presence of high concentrations of other amino compounds.     

In situ fabrication of a microfluidic device for immobilised metal affinity sensing

In this work a novel microfluidic device was constructed in situ containing the smallest microscopic co-polymeric immobilised metal affinity (IMA) adsorbent yet documented. This device has for the first time allowed the microlitre scale chromatographic assay of histidine-tagged proteins in a biological sample. To enable this approach, rather than using a high capacity commercial packed bed column which requires large sample volumes and would be susceptible to occlusion by cell debris, a microgram capacity co-polymeric chromatographic substrate suitable for analytical applications was fabricated within a microfluidic channel. This porous co-polymeric IMA micro-chromatographic element, only 27μl in volume, was assessed for the analytical capture of two different histidine-tagged recombinant fusion proteins. The micro-chromatographic adsorber was fabricated in situ by photo-polymerising an iminodiacetic acid (IDA) functionalised polymer matrix around a template of fused 100μm diameter NH(4)Cl particles entirely within the microfluidic channel and then etching away the salt with water to form a network of interconnected voids. The surface of the micro-chromatographic adsorber was chemically functionalised with a chelating agent and loaded with Cu(2+) ions. FTIR and NMR analysis verified the presence of the chelating agent on the adsorbent surface and its Cu(2+) ion binding capacity was determined to be 2.4μmol Cu(2+) (ml of adsorbent)(-1). Micro-scale equilibrium adsorption studies using the two different histidine-tagged proteins, LacI-His(6)-GFP and α-Synuclein-His(8)-YFP, were carried out and the protein binding capacity of the adsorbent was determined to be 0.370 and 0.802mg(g of adsorbent)(-1), respectively. The dynamic binding capacity was determined at four different flow rates and found to be comparable to the equilibrium binding capacity at low flow rates. The sensing platform was also used to adsorb LacI-His(6)-GFP protein from crude cell lysate. During adsorption, laser scanning confocal microscopy identified locations within the adsorbent where protein adsorption and desorption occurred. The findings indicate that minimal channelling, selective product capture and near quantitative elution of the captured (adsorbed) product could be achieved, supporting the application of this new device as a high-throughput process analytical tool (PAT) for the in-process monitoring of histidine-tagged proteins in manufacturing.     

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.     

Adsorption of rutin with a novel β-cyclodextrin polymer adsorbent: Thermodynamic and kinetic study

The adsorption properties toward rutin of a cyclodextrin polymer adsorbent CroCD-TuC 3 have been studied. The adsorption capacity is reduced as temperature and pH of solution rises, but increases with the increase of solvent polarity. Compared with Sephadex? G-15 dextran gel beads, CroCD-TuC 3 shows dramatically higher isosteric enthalpy due to a significant contribution of rutin/β-cyclodextrin inclusion complex formation in CroCD-TuC 3 skeleton. A highlight in our study is that the pore diffusion model has been employed to describe the mass transfer inside the adsorbent pores. It reveals that the diffusion inside the pores is the rate-restricting step in the whole adsorption process. The effective pore diffusivity of rutin in CroCD-TuC 3 calculated is much lower than the diffusivity in diluted solution. The pore diffusion model is an available tool to investigate the profile of mass transfer inside the pores, and provides an effective method to describe adsorption kinetics.     

Peptide-and Biotin-Oligonucleotide-Pyrrole Conjugates for Electrochemical Addressing on Silicon Chip

Abstract

The syntheses of pyrrole-oligonucleotide-peptide conjugates and pyrrole-oligonucleotide-biotin conjugates were described. The oligonucleotide moiety acted as an ?active linker? which allowed the easy purification and quantitation of the conjugates and in turn controlled the grafting. The peptide conjugates were immobilised on silicon array and their immunoreactivity was tested using biotinylated antibodies and a phycoerythrin-streptavidin staining. The biotin conjugate provided a fluorescence scale.     

The influence of particle size distribution and operating conditions on the adsorption performance in fluidized beds

The influence of matrix properties and operating conditions on the performance in fluidized-bed adsorption has been studied using Streamline diethyl-aminoethyl (DEAE), an ion exchange matrix based on quartz-weighted agarose, and bovine serum albumin (BSA) as a model protein. Three different particle size fractions (120-160 mum, 120-300 mum, and 250-300 mum) were investigated. Dispersion in the liquid phase was reduced when particles with a wide size distribution were fluidized compared to narrow particle size distributions. When the mean particle diameter was reduced, the breakthrough capacities during frontal adsorption were enlarged due to a shorter diffusion path length within the matrix. At small particle diameters the effect of film mass transfer became more relevant to the adsorption performance in comparison to larger particles. Therefore matrices designed for fluidized-bed adsorption should have small particle diameter and increased mean particle density to ensure small diffusion path length in the particle and a high interstitial velocity to improve film mass transfer. Studies on the influence of sedimented matrix height on axial mixing showed an increased Bodenstein number with increasing bed length. Higher breakthrough capacities were also found for longer adsorbent beds due to reduced dispersion and improved fluid and particle side mass transfer. With increasing bed height the influence of flow rate on breakthrough capacity was reduced. For a settled bed height of 50 cm breakthrough capacities of 80% of the equilibrium capacity for flow rates varying from 3 to 9 cm/min could be achieved. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 54-64, 1997.     

Removal of herbicide paraquat from an aqueous solution by adsorption onto spent and treated diatomaceous earth

A spent diatomaceous earth from the beer brewery has been tentatively activated by sodium hydroxide at about 100 degrees C. The resulting product was used as a novel adsorbent for the adsorption of herbicide paraquat from an aqueous solution in a continuously stirred adsorber and batch flasks, respectively. The results showed that the adsorption process could be well described by the pseudo-second-order reaction model. From the view of the negatively charged surface of diatomaceous earth and cationic property of paraquat, the results were also reasonable to be explained by physical adsorption in the ion-exchange process under the effects of pH and temperature. Further, it was found that the Freundlich model appeared to fit the isotherm data better than the Langmuir model.     

The use of dextran as a blocking agent on nitrocellulose membrane in the analysis of sporozoite antigens of Plasmodium vivax

Dextran (molecular weight, 71,200) has been found to block the unbound sites of the nitrocellulose membrane, to which antigens have been electroblotted from acrylamide gel, for use in assaying monoclonal antibodies. The use of polysaccharide as a blocking agent allows the antigens on the nitrocellulose membrane to be digested with pronase and subsequently reacted with monoclonal antibodies. Sporozoite antigens of Plasmodium vivax, after being digested with pronase, completely lost their antigenicity to bind to the sporozoite-specific monoclonal antibodies, thus suggesting that they are proteins or protein conjugates in nature. The method described here for qualitative determination of protein antigens requires as little as 2000 sporozoites for each assay.     

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.     

Albumin recovery with centrifugal adsorption technology (CAT)

Centrifugal adsorption technology (CAT) is a new compact, countercurrent technology for efficient adsorption from large liquid streams by using adsorbent particles in the micrometer range. CAT seems particularly suited for the recovery of macromolecules at low concentrations, because the small particle dimensions lead to fast mass transfer rates. In this work, the potential of CAT for protein recovery is studied by model and experiment. A predictive model for the separation performance of CAT is presented, incorporating mass transfer resistance and axial dispersion transport in the liquid and the adsorbent phases. The model calculations were compared to experimental data for the adsorption of bovine serum albumin (BSA) on a standard commercial anion-exchange resin with particle diameter d(p) = 50 microm in a pilot-scale CAT apparatus. The model calculations accurately predicted the separation efficiency of CAT. The experimental set-up is shown to be mass transfer limited for the conducted experiments, which agrees with the model predictions. The model was also used to estimate the dimensions and performance of a CAT apparatus for the large-scale recovery of human serum albumin (HSA) from fermentation broth at the scale of 40 tons per year. The resulting equipment dimensions proved to be very small indeed, making CAT a potentially very attractive technology.     

Hydrophobic interaction adsorption of hen egg white proteins albumin, conalbumin, and lysozyme

Hydrophobic adsorption equilibrium data of the hen egg white proteins albumin, conalbumin, and lysozyme were obtained in batch systems, at 25 degrees C, using the Streamline Phenyl resin as adsorbent. The influence of three types of salt, NaCl, Na(2)SO(4), or (NH(4))(2)SO(4), and their concentration on the equilibrium data were evaluated. The salt Na(2)SO(4) showed the higher interaction with the studied proteins, thus favoring the adsorption of proteins by the adsorbent, even though each type of salt interacted in a distinct manner with each protein. The isotherm models of Langmuir, Langmuir exponential, and Chen and Sun were well fitted to the equilibrium data, with no significant difference being observed at the 5% level of significance. The mass transfer model applied simulated correctly adsorption kinetics of the proteins under the studied conditions.     

Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.     

Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials

A range of studies were carried out to investigate the underlying reason for differences in dynamic binding capacities observed with various antibodies and Fc-fusion proteins during Protein A chromatography. Dynamic binding capacities were determined for these biomolecules using different protein A stationary phase materials. SEC was carried out to determine the relative sizes of the antibodies and fusion proteins. Pore diffusivities and static binding capacities were also determined on these Protein A resin materials. Trends in the dynamic binding capacities for these molecules did not correlate with differences in pore diffusion coefficients as might be expected for a mass transfer limited system. Instead, dynamic binding capacities were seen to follow the same trends as the static binding capacities and the apparent size of the molecules. Differences in static binding capacities were attributed to be due to differences in steric factor between the molecules. Solution binding stoichiometry studies were employed to estimate intra-Protein A steric effects while binding to the various domains within a Protein A ligand. In addition, steric hindrance was also found to exist between adjacent immobilized Protein A ligands on the chromatographic surface. The combination of intra and inter Protein A steric hindrances can explain differences in binding capacities observed between various antibody and Fc fusion proteins. The effect of Protein A ligand density on these supports was also examined and the results indicate that increasing Protein A ligand density leads to a situation of diminishing returns for binding capacity due to increased steric hindrance on the resin surface. The results presented in this paper show that steric hindrances can dominate over mass transfer effects in causing capacity variation between different molecules on the same stationary phase. This can lead to the development of more cost-efficient chromatographic stationary phases as well as provide information during the selection of Protein A media for preparative purification of monoclonal antibodies and Fc fusion proteins.     

Development of a one-step immunochromatographic strip test for the detection of sennosides A and B

An immunochromatographic strip test was developed to detect sennoside A (1) and sennoside B (2) using anti-1 and anti-2 monoclonal antibodies. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of colloidal gold particles coated with the respective sennoside antibodies. The capture reagents were 1- and 2-human serum albumin (HSA) conjugates immobilised on a nitrocellulose membrane on the test strip. The sample containing 1 and 2, together with detector reagent, passed over the zone where the capture reagents had been immobilised. The analytes in the sample competed for binding to the limited amount of antibodies in the detector reagent with the immobilised 1- and 2-HSA conjugates on the membrane and hence positive samples showed no colour in the capture spot zone. Detection limits for the strip test were 125 ng/mL for both sennosides. The assay system is useful as a rapid and simple screening method for the detection of 1 and 2 in plants, drugs and body fluids.     

Visualizing two-component protein diffusion in porous adsorbents by confocal scanning laser microscopy.

The use of confocal scanning laser microscopy (CSLM) has recently been described for the visualization of intraparticle protein profiles during single-protein finite bath uptake experiments. By coupling of fluorescent molecules to proteins the penetration of porous media by labeled macromolecules could be detected by scanning single adsorbent particles for fluorescence emission after laser excitation. Thus the internal protein distribution profile, which is a central element in modeling of protein transport in porous adsorbents, became experimentally accessible. Results from the simultaneous visualization of two proteins by this technology are shown here. The use of two different fluorescent dyes for protein labeling and two independent detectors in the CSLM allowed for the first time ever the direct observation of a two-component diffusion process within a porous stationary phase. The finite bath uptake of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) to two different ion exchange adsorbents (SP Sepharose Fast Flow and Source 30S) and to an affinity adsorbent (Protein A Sepharose) was measured using Cy5 and Oregon Green as labels. Single adsorbent particles were scanned for intensity distribution of fluorescence emission from the two fluorophors. The intraparticle profiles obtained from the confocal images were translated into a relative protein concentration thus allowing the calculation of protein uptake kinetics from direct measurement in the stationary phase. The confocal technique may prove to be a very powerful means of data generation for modeling of multi-component mass transfer phenomena in protein adsorption.     

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.