Protein-binding Membrane Research Articles

Transmission of BSA during cross-flow microfiltration: influence of pH and salt concentration

In many downstream processes microfiltration (MF) is used as the first step to separate the micro-organisms from the product-containing broth. The transmission of proteins through these MF membranes is of great importance, especially when a protein is the desired product. Usually, the largest fraction of the proteins retained in the membrane during the separation of micro-organisms is retained in the filter cake of micro-organisms that is built up on the membrane surface. However, this is not always the case. In some cases the transmission of proteins can decrease below 100% even though there are no micro-organisms present in the broth. In the present study the transmission of BSA through two different MF membranes (a nylon membrane, Pall Filtron Ultipore, Nylon 66, 0.2 μm, and a membrane made of polyether sulphone, Pall Filtron, Omega, 0.16 μm) was investigated. The transmission of BSA was highest for the membrane that was said to be a low protein binding membrane (Omega) and it was affected by the pH in the solution. At pH 5, which is close to the IEP of the protein, the transmission was 100%, while at pH 3 and 7 the transmission was much lower (sometimes as low as 40%). However, when the ionic strength in the solution was increased the transmission of BSA through both MF membranes increased dramatically (at pH 3 and 7). This shows that the electrostatic interactions between the protein and the filter cake (made up of protein aggregates) and between the protein and the membrane affect the transmission. The increase in transmission when the protein is close to its IEP (at pH 5), is believed to be due to the lack of electrostatic repulsion between the proteins in the solution and filter cake of protein aggregates formed on the membrane. At high ionic strength the charged protein molecules are shielded from each other and from the membrane and the filter cake, by the ions in the solution. Due to this shielding effect the protein molecules act as if they were uncharged and the transmission of BSA is thus increased.

Two-dimensional electrophoresis replica blotting: a valuable technique for the immunological and biochemical characterization of single components of complex extracts.

The combination of the high resolution electrophoresis (2-DE) with subsequent transfer onto a protein-binding membrane (blotting), immunological detection, and/or N-terminal sequencing is a powerful tool to identify and characterize single components of complex protein mixtures. Direct comparison of protein staining, immunological detection, and biochemical characterization of single protein spots was achieved by the replica blotting technique. The proteins were transferred from one two-dimensional gel onto several blotting membranes one after another. A canon of methods has been employed to identify and characterize allergens from different allergen sources. We have studied single major allergens as well as related major allergens from different grass species ("allergen groups") using patients' sera and allergen-specific monoclonal antibodies. The biochemical structure of the allergenic components has been analyzed by N-terminal and internal protein sequencing, precise mass determinations by matrix-assisted laser desorption/ionization mass spectrometry and investigations on post-translational modifications such as glycosylation. Here, we give a general survey of methods, and we describe an array of techniques suitable for characterization and identification of components of complex extracts, even if there is little or no previous information available.

Solid-phase immunoassay detection of peptides from complex matrices without a separation

A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps. Both direct and indirect immunoassay methods are demonstrated; the indirect is more sensitive and features a 1 pmol detection limit of neat dynorphin A solutions. To further increase the immunoassay sensitivity, a novel capillary blotting system with hydrophilic membranes was designed where optimized sample volumes of 167 nL were deposited for each spot. The area-reduced blotting method shows a 1000-fold improved, 1.3 fmol spot(-1) detection limit of a dynorphin A diluted in a buffered solution of 150 mg L(-1) of casein. Low-flow push-pull perfusates with volumes of 1 microL sampled from the striatum of the rat were assayed for dynorphin A by the method of standard addition. The detection limit was estimated to be 1.9 fmol in the low-flow push-pull perfusates. These data demonstrate a solid-phase near infrared immunofluorescence strategy for the study of peptides directly blotted from chemically complex biological fluid matrices.

Endothelin-1 directly modulates its own secretion: studies utilising the cell immunoblot technique

Endothelin-1 is an important factor in vasoregulation and circulating levels of the peptide are increased in a number of cardiovascular disorders. However, control of endothelin-1 secretion is only sketchily understood. The possibility that endothelin-1 influences its own release was investigated. A cell immunoblot method, which can detect local secretion of peptide from individual human vascular endothelial cells, was employed. Cells were dispersed onto a protein-binding membrane. Endothelin-1 in cells or secreted and adhering to the protein-binding membrane outside the cells was detected using immunohistochemical techniques. The numbers of cells that contained endothelin-1 and secreted endothelin-1 were counted after the cells had been incubated in control conditions, or with added endothelin-1, angiotensin-II, or endothelin receptor antagonists, bosentan and BQ788. Endothelin-1 and angiotensin-II increased the numbers of cells that secreted endothelin-1. On the other hand, bosentan and BQ788 caused a reduction in the numbers of endothelin-1-secreting cells. These results indicate that human endothelial cells contain a pathway by which endothelin-1 induces its own release. The receptor antagonists, bosentan and BQ788, inhibited basal secretion of endothelin-1.

Allergen detection from 11 fungal species before and after germination

Background: Allergens dispersed by airborne fungal spores play an important but poorly understood role in the underlying cause and exacerbation of asthma. Previous studies suggest that spores of Alternaria and Aspergillus release greater quantities of allergen after germination than before germination. It is unknown whether this is true of other allergenic fungi. Objective: Our purpose was to investigate the release of allergen from a range of individual fungal spores before and after germination. Methods: Allergen expression from spores of Alternaria alternata , Cladosporium herbarum , Aspergillus fumigatus , Botrytis cinerea , Epicoccum nigrum , Exserohilum rostratum , Penicillium chrysogenum , Stemphylium botryosum, Curvularia lunata, Trichoderma viride , and Bipolaris spicifera was examined by halogen immunoassays through the use of pooled serum IgE from patients allergic to fungus. Spores were deposited onto protein-binding membranes direct from culture. To germinate spores, samples were incubated in high humidity at room temperature for 48 hours. Ungerminated and germinated samples were then laminated with an adhesive film and immunostained by the halogen assay. The samples were examined by light microscopy, and positive counts (haloed particles) were expressed as percentages of total spores. Results: For 9 of 11 species, between 5.7% and 92% of spores released allergen before germination. Spores of Penicillium and Trichoderma did not release detectable allergen. After germination, all spores that germinated had allergen elution from their hyphae. Eight of 11 species showed a significant increase (P < .05) in the percentage of spores eluting detectable allergen. Localization of allergen along the hyphae varied with species, such that some eluted allergen mainly from hyphal tips and septal junctions whereas others eluted allergen along the entire length. Conclusions: Increased elution of allergen after germination might be a common feature of many species of allergenic fungi. Although allergens from both spores and hyphae were recognized by human IgE, the extent to which human exposure occurs to allergens eluted from inhaled spores or from hyphae that germinate after deposition in the respiratory tract remains to be explored. The patterns of allergen expression might affect the clinical response to such exposure. (J Allergy Clin Immunol 2003;111:285-9.)

Incorporation of Protein-Binding Kinetics and Carrier-Mediated Membrane Transport into a Model of Chemical Kinetics in the Isolated Perfused Rat Liver

Nonlinear processes present difficult problems in extrapolating toxicological kinetics from one dose or species to another. A model that includes the details of these processes will aid in identifying those conditions under which a linear extrapolation is not valid. Previously, a kinetic model of the perfused rat liver system was presented that included the effects of equilibrium protein binding, diffusion-based and saturable membrane transport, metabolism, and biliary excretion. This model has been extended here to account for two complex processes. First, the kinetics of chemical association and dissociation from binding sites in various compartments are explicitly included to allow for the possibility that the binding of the compound is not in equilibrium. Second, mediated transport via a simple four-state carrier in the membrane has been included at both the sinusoidal and biliary membranes of the liver cell. This enables inclusion of carrier-specific transport processes (such as selective transport against a concentration gradient) in modeling the kinetics in perfused rat liver experiments. Simulations demonstrating the effects of each of these processes on observable state variables have been conducted. Physiological conditions that elicit nonlinear behavior have been identified.

Effects of endothelin-1 on release of adrenomedullin and C-type natriuretic peptide from individual human vascular endothelial cells.

Regulation of cardiovascular system activity involves complex interactions amongst numerous factors. Three of these vasoactive factors are adrenomedullin, C-type natriuretic peptide (CNP) and endothelin-1 (ET-1), each of which is claimed to have important local effects. To investigate paracrine/autocrine regulation of the secretion of these peptides we used a cell immunoblot method. We postulated that basal release of adrenomedullin and CNP by endothelial cells is modulated by ET-1. Dispersed human aortic endothelial cells were attached to a protein binding membrane and incubated for 1 or 4 h with control medium or with ET-1, endothelin receptor antagonists or antibody to ET-1, and then submitted to immunohistochemical staining. Peptides (adrenomedullin, CNP and ET-1) within individual cells were stained, as was peptide secreted and adjacent to the cell. It was demonstrated that adrenomedullin, CNP and ET-1 can be contained within the same cell. In addition, we observed that individual endothelial cells can secrete all three peptides. The endothelin ET-A/ET-B receptor antagonist, bosentan, the ET-B receptor antagonist, BQ-788, and anti-ET-1 serum decreased the percentage of endothelial cells that secreted adrenomedullin and CNP relative to control. Conversely, the addition of ET-1 induced an increase in the number of endothelial cells that secreted adrenomedullin and CNP. These results provide strong evidence that endogenous ET-1, from human vascular endothelial cells, acts in a paracrine/autocrine manner to modulate the basal release of adrenomedullin and CNP. Our observations of this modulation suggest that vascular endothelial cells of humans constitute an important component of a self-responsive vasoregulatory system.

The influence of different growth media and different isolates of Stachybotrys chartarum on monoclonal antibody (Mab) reactivity

potent when few spores are detected in the air. The purpose of this study was to investigate a wide range of genera and examine the differences in allergen released from spores preand post-germination. Allergen expression from spores of Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, Botrytis cinerea, Epicoccum nigrum, Exserohilum rostratum, Penicillium chrysogenum, Stemphylium botryosum and Bipolaris spicifera, were detected using immnnoassay techniques (HALOgen assayTM), by use of serum IgE from fungal allergic subjects. Spores were deposited onto protein binding membranes direct from culture. To germinate spores, samples were incubated in high humidity at room temperature for 48 hours. Ungerminated and germinated samples were then laminated with an adhesive film and immunostained by the Halogen assay. The samples were examined by light microscopy and positive counts (haloed particles) were expressed as percentages of total spores. The percentage of ungerminated spores releasing allergen was variable across the genera, with Alternaria alternata (67.7_+2.3), Epicoccum nigrum (92-+0.6), Stemphylium botryosum (87.3-+2.4) and Cladosporium herbarum (40.8-+7.4) recording the highest percentages. The remaining species eluted allergen from fewer spores (< 15%) with Penicillium chrysogenum (0-+0) recording the least. Across all genera, all spores that germinated exhibited allergen elution along their hyphae. Post-germination, six of the nine genera showed a significant increase in the percent of spores eluting allergen (p<0.0001). Localization of allergen along the hyphae appeared to vary with the genera, such that Exserohilum rostratum and Stemphylium botryosum eluted allergen from hyphal tips and septal junctions, whereas Alternaria alternata, Aspergillus fumigatus, Penicillium chrysogenum and Epicoccum nigrum eluted allergen along the entire length of their hyphae. This study further supports the hypothesis that greater amounts of allergen may be released upon germination of fungal spores. This is the first study to identify the nature of allergen release from spores and hyphae of these genera. Further studies are required to identify if this phenomenon occurs in the respiratory tract, potentially leading to allergic reactions.

An Enzyme Electrode for Extended Linearity Citrate Measurements Based on Modified Polymeric Membranes

Pyruvate oxidase (POD), oxaloacetate decarboxylase (AOCD), and citrate lyase (CL) were coimmobilized (or immobilized separately) in a series of polymeric membranes for the construction of amperometric bisensors of pyruvic, oxaloacetic, or citric acid in concentrated samples. For oxaloacetic acid, POD, and OACD were coimmobilized on dialysis membranes and were used in a multimembrane configuration with an inner spin-coated cellulose acetate (CA) membrane modified with isopropyl myristate (IPM) and an outer diffusion restricted membrane of cellulose acetate modified with a cationic surfactant. These membranes were also tested in a POD laminate containing the cofactors FAD and TPP for monitoring the response stability with duration of exposure to an external electrolyte and found to be effective in reagentless mode, with good pyruvate response stability over 3 h without compromise of signal size. Coimmobilization of POD/OACD and CL on different types of high protein binding membrane such as mixed cellulose ester (HA) was investigated. The relative optimum concentrations of several activators (divalent cations) and cofactors such as FAD and thiamine pyrophosphate (TPP) were investigated with the probe assembled as a pyruvate biosensor. An extended linearity up to 100 mM citric acid was achieved.

Efficient purification of DNA fragments using a protein binding membrane.

A novel and efficient method has been developed for isolation of correctly digested DNA fragments without the use of classic size-dependent electrophoretic separation methods. To achieve this, DNA fragments are end-labelled by haptens. After specific endonuclease digestion of the hapten-labelled DNA, the DNA is incubated with a protein that specifically binds to the hapten. The incubation mixture is then passed through a cartridge containing a protein-binding membrane that does not bind DNA. Undigested and partly digested DNA are retained on the membrane, while correctly digested DNA is selectively recovered for use in further downstream applications.

The nasal air sampler: a device for sampling inhaled aeroallergens

The object is to design, develop, and test a personal aerosol sampling device consisting of impaction samplers worn just inside the nostrils, driven by the wearer's respiration. The device provides a novel and unique measure of individual exposure to aeroallergens. It was conceived as an integral part of an allergen diagnostic system, in which collected aerosols are immunostained with monoclonal antibodies or the patient's IgE and associated particles positively identified using techniques of image analysis. Each sampler comprises a slot impactor with a detachable impaction plate covered with either a specially developed medical adhesive or a protein-binding membrane. Sampler performance has been validated by rig tests of aerodynamic resistance and collection efficiency of different sized particles at various flow rates. There have also been field trials with human subjects which show that the sampler can be comfortably worn for periods of up to 4 hours. This is sufficient to gather a representative sample of inhaled allergens in most environments. The sampler collects an increasing proportion of particles in the inhalable range at and above 5 microm. This includes most bioaerosols of interest to allergists. Sampler prototypes have been built by CNC mill and stereolithography. Batches of samplers have been molded in biocompatible materials for field and clinical trials. The device successfully collects aeroallergens from a patient's own respiration. While developed specifically as a vehicle for the allergen diagnostic system, it can be adapted for studies of other aspects of air quality or for prophylactic use.

A new method for simultaneous immunodetection and morphologic identification of individual sources of pollen allergens

Background: Exposure to outdoor allergens has commonly been estimated by collecting airborne particles with a Hirst-type spore trap and then using morphologic criteria to identify the intact pollen grains and fungal spores that are recognized as allergen sources. Several antibody-based blotting or fixation methods have also been developed that enable the counting of amorphous airborne particles carrying allergen, but none of these methods allow the ready association of the released allergen with the morphologically identifiable particle of origin. A method has been developed that uses pressure-sensitive adhesive tape to sample the airborne particles and then allows the immunoidentification of the specific particles that are the allergen sources. Objective: Our purpose was to visualize and immunostain the particles carrying pollen allergen that are collected with a volumetric spore trap. Methods: A Burkard sampler was used to collect airborne particles onto pressure-sensitive adhesive tapes. The particles were permanently fixed between the tape and a protein-binding membrane when the tape was laminated with the membrane. Allergens that elute from the particles onto the membrane were detected with a range of antibodies. Both the particle and associated immunostained allergen were viewed through the transparent tape for final microscopic identification. Results: Polyclonal and monoclonal antibodies and IgE from allergic patients stained allergens in the periphery of particles collected on the tapes. Individual pollen grains and paucimicronic particles were seen with halos of immunostained allergen surrounding them. When IgE was used, the density of immunostaining in the halo surrounding Lolium perenne pollen grains was found to be proportional to the level of Lolium -specific IgE. The method is highly sensitive and can be used to detect different airborne particles that carry allergen. Both the particle and the immunostaining can be subjected to a range of simple measurement techniques. Conclusion: Individual particles carrying allergens and antigens were visualized. These particles included intact pollen grains, paucimicronic particles, and fungal spores. (J Allergy Clin Immunol 2000;105:725-31.)

New immunodiagnostic system

We have developed a new immunodiagnostic system whichmeasures personal allergen exposure and which can beused to identify allergens.Personal exposure is directly sampled using inertialimpaction filters which fit just inside the nose andcollect particles (mainly >5 µm) inhaled duringnormal respiration. These samplers provide both anindex of personal exposure as well as being aninexpensive and portable sampling system.The particles are captured on adhesive tapes which arethen laminated with a protein-binding membrane. Theallergens eluting from the particles are bound by the membrane in theperiphery of each particle. The systemthen uses either allergen-specific monoclonalantibodies or the subject's IgE as primary probes toimmuno-label the `halo' of allergen around individualallergen-containing particles. Such an assay is verysensitive and can detect a single particle carryingallergens. In addition, the system providesinformation on the size, shape and allergen content ofthe particles. Because the particles carryingallergens can be seen, high resolution video images ofpollen grains and fungal spores can be subjected to atraditional morphological study or a range of featureextraction routines. This information can be comparedto a database of some known allergenic pollen grainsand fungal spores which we have also assembled tofacilitate their identification. When using monoclonal antibodies as the probe, thesystem determines the amount of allergen the subjectis exposed to and the characteristics of the particles(size, shape, etc). When using the subject's IgE as theprobe, the system allows visualisation of the allergensources that an individual is allergic to. The systemmay have clinical applications in quantifying personalexposure as well as identifying allergens anddetermining exposure to unsuspected allergens.

GnRH and oxytocin have nonidentical effects on the cellular LH response by gonadotrophs at pro-oestrus.

For full fertility in the female ovulation is necessary, which is dependent on the production of a surge of LH. An understanding of the processes which result in the high levels of LH requires delineation of the activities of individual component cells. In this study the responses of gonadotrophs to two signalling hypothalamic peptides, GnRH and oxytocin, were investigated. A cell immunoblot method was used to identify and distinguish between cells which secrete LH and those which contain LH but do not secrete the glycohormone. Rats were killed on the morning of pro-oestrus, the pituitary collected and the cells dispersed onto a protein-binding membrane for study. Cells were then incubated with GnRH and oxytocin, after which the membranes including the attached cells were stained by immunocytochemistry for LH. GnRH increased the total number of immunopositive cells which were present in a concentration-dependent manner. The most prominent change after 2 h incubation was in the number of secreting cells, whereas after 4 h there was also a marked increase in numbers of nonsecreting cells. Oxytocin also increased the total number of immunopositive cells in a concentration-responsive manner, however the profile of action of oxytocin was different from that observed for GnRH. Oxytocin had a relatively greater effect on numbers of immunopositive nonsecreting cells. Thus, the results reveal the potential for gonadotrophs to be flexibly and appropriately modulated by selected hypothalamic peptides. When cells were preincubated with oxytocin prior to GnRH there was not an additive increase in the numbers of immunopositive cells, suggesting that the two agonists act, in a nonidentical manner, on similar cells. The increase in the total number of immunopositive cells implies that there was a production of LH or post-translational processing, induced by exposure to GnRH or oxytocin. The results confirmed the heterogeneity of gonadotrophs and the existence of functionally distinguishable subpopulations, and revealed a difference between the effects of GnRH and oxytocin on expression and secretion of LH.

Detection of inhaled Der p 1.

Measurement of personal exposure to Der p 1 aeroallergen has previously been limited by the low quantity of material collected by sampling systems and the assay sensitivity. This has meant that exposure could only be detected if long sampling periods were used or reservoir dust was artificially disturbed. We have developed a sampling method to sample true personal exposure and combined it with a novel method which is sensitive enough to measure allergen exposure over shorter time frames. To describe normal domestic exposure to dust mite allergen during a range of activities in houses in Sydney, Australia. Inhaled particles containing mite allergen Der p 1 were collected using a nasal air sampler which impacts particles (> approximately 5 microm) onto a protein-binding membrane coated with a thin, porous, adhesive film. The allergen is bound to the membrane in the immediate vicinity of the particle and detected by immunostaining with monoclonal antibodies specific for Der p 1. In addition, samples were collected using a standard Institute of Occupational Medicine (IOM) personal air sampler and the amount of eluted Der p 1 was assayed by ELISA. The median number (range) of inhaled particles containing Der p 1 collected in each 10-min sampling period was: dust raising 5 (2-10); lying in bed, 0 (0-2); sitting on the bed, 1 (0-2); walking around the bedroom, 0 (0-2). This represented 0-5.1% of all particles captured. The Der p 1 concentration of floor and bed dust was 19.4 and 55.1 microg/g, respectively. The standard IOM personal sampler and ELISA were unable to detect Der p 1 for any of the activities performed. We were able to count individual allergen-carrying particles inhaled over short time periods, during different domestic exposure situations. This will offer new insight into several aspects of personal allergen exposure.

A Serum-Free System for Primary Cultures of Human Pituitary Adenomas.

We report the successful use of a serum-free culture system for primary cultures of human pituitary adenomas. The system utilizes histiotypic suspension culture with low protein-binding membrane inserts that enable cells to retain their three-dimensional tissue configuration, closely mimicking the growth pattern in vivo. A serum-free defined medium was developed with CMRL-1969 (Connaught, Willowdale, Ontario, Canada) supplemented with 0.375% albumin bovine Fraction V, 5 ug/mL insulin, 5 ug/mL transferrin, 5 ng/mL sodium selenite, 30 ug/mL putrescine, 6.85 x 10(11)M hydrocortisone, and 3.7 x 10'(11)Mtri-iodothyronine (T(3)). We analyzed eight surgically resected human pituitary adenomas. Basal pituitary hormone secretion measured by radioimmunoassay of pituitary hormones was compared with hormone hypersecretion in vivo and with control cells of the same tumors cultured in CMRL-1969 with 10% fetal calf serum. The light microscopic, immunocytochemical and ultrastructural morphology of cells cultured in this serum-free histiotypic system was compared with cells cultured in serum-supplemented media and with cells cultured on collagen-coated plastic; all cultured cells were compared with the morphology of surgically resected tissues of the same specimens. Basal pituitary hormone secretion during 24-hour incubations correlated with the clinical patterns of hormone excess; the data were similar in serum-enriched and serum-free cultures, however, hormone secretion decreased less rapidly in the serum-free cultures. Cells maintained in the histiotypic culture system closely resembled the corresponding surgically resected tumor using the morphologic parameters and were better preserved than those plated in collagen-coated plastic wells. This comparative study indicates that this serum-free histiotypic culture system provides an ideal method of examining pituitary adenomas in vitro without altering the profile of hormone secretion and cell morphology documented in vivo. This system can be used to examine the production and effects of a wide range of hormones and growth factors that have been implicated as causative agents in pituitary tumorigenesis.

Alterations in Erythrocyte Rheology in Patients with Severe Peripheral Vascular Disease: 1. Cell Volume Dependence of Erythrocyte Rigidity

The erythrocyte rigidity of patients suffering from severe peripheral vascular disease (PVD) was measured by a filtration method using 3 microns pore size filters. Filtration pressures for both PVD patients and normal subjects showed a cell volume dependency, and patient filtration pressures were normalized to cell volume to evaluate intrinsic, ie, nonvolume dependent, abnormalities in erythrocyte deformability. A significant (p less than 0.001) increase in cell rigidity was found in 44 of 54 PVD patients in comparison with volume-matched normal controls. No significant difference was found between patient mean corpusculer hemoglobin (MCH) and normal MCH at any given mean corpuscular volume (MCV), indicating that observed increases in erythrocyte rigidity are not attributable to changes in patient MCH. Therefore, the mechanism of increase in erythrocyte rigidity for PVD patients still needs further investigation into such parameters as levels of adenosine triphosphate,2,3-DPG, and membrane fluidity (calcium- and/or protein-binding membrane, cholesterol and phospholipid content of membrane, etc), as well as other aspects of erythrocyte physiology.

Identification of the Species of Origin of Residual Blood on Lithic Material

The examination of stone-tool edges for blood-antigen residue is a relatively new technique in the archaeological analysis of lithic material. To date, a number of different methods have been employed to determine the species of origin of residual blood, such as Ouchterlony double-diffusion (Ouchterlony 1968) and radioimmunoassays (RIAs) (Lowenstein 1985, 1986). These techniques have been of limited use due to problems of sensitivity, cost, and applicability to archaeological field conditions. Enzyme immunoassays (EIAs) utilizing a nitrocellulose protein-binding membrane have shown that it is possible to retain both specificity and sensitivity in a technique that can endure the rigors of archaeological field work and the vicissitudes of differential preservation (Tersak and Hyland 1988). However, due to cross-reactivity, species identification of blood residue using immunological techniques can be problematic when it is necessary to distinguish individual species within a single genus or family. Recent work has shown that careful refinement of antisera can eliminate cross-reactivity, thereby increasing specificity (Berkeley Antibody Company, personal communication 1987). This refinement is accomplished by absorbing any antibodies from the antisera that may cross-react with antigenic sites held in common by closely related species. The ramifications of the successful implementation of this technique are discussed in terms of Paleoindian artifact function as well as paleoeconomic, paleoenvironmental, and paleodietary reconstruction using a case study from the Shoop site in central Pennsylvania.

Changes in cell phenotype during regeneration of junctional epithelium of human gingiva in vitro

The relationship between cell attachment and the phenotype of the attached oral epithelial cells was studied by comparing junctional epithelium (JE) with a culture model for JE in which epithelial cells form an equivalent organization of tissues. Gingival explants were cultured on either a high or a low protein-binding membrane. The cut edge of epithelium and connective tissue was placed on the membrane; epithelial cells migrated to form a sheet of tissue between the explant and the membrane substratum. Cells which grew in contact with the high protein-binding membrane attached to the substratum and assumed a cuboidal shape. With time in culture these cells showed a decrease in reactivity with antibodies to psi-3 antigen (an antigen associated with epithelial migration) and an increase in reactivity with antibodies to cytokeratin 19 (a marker for JE). Cells grown on the low protein-binding membrane did not exhibit changes in shape or antigens. Because similar features were found in the JE in vivo, it was concluded that the junctional cells which are directly attached to the tooth (DAT cells) have a nonmigratory phenotype that develops as a response to the tooth surface. Because the cells are in contact with a metabolically inert material the changes appear to be largely self-induced. The culture method allows studies on putative inducer molecules and on mechanisms which may control the phenotype of epithelial cells at the dentogingival interface.

Effects of chemical modifications of membranes on transmembrane potential

The role of membrane surface substances on the generation of transmembrane potential was studied. Several functional groups such as amino, epoxy, and carboxyl groups were covalently introduced to a bromoacetyl cellulose membrane. These functional groups caused a marked change in the surface potential of the membrane. The transmembrane potential shift caused by the chemical modification was attributed to the charge of the functional groups. Several proteins were covalently immobilized to the modified membrane. The modification process was followed through the transmembrane potential. The transmembrane potential of the protein-binding membranes showed that lysozyme and egg albumin at the membrane surface produced a positive and a negative charge, respectively. It was concluded that attachment of protein to the surface of the membrane affects a change in the charge density of the membrane surface with a resulting change in transmembrane potential.