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Biomedical subjects

D S Pepper

Publications and source records attributed to D S Pepper.

At least 19 recordsLinked to original sources
PubMed1998

Virus removal from bioproducts using ultrafiltration membranes modified with latex particle pretreatment.

Ultrafiltration is an attractive process for virus removal from bioproducts owing to its high throughput as well as the fact that the operation is carried out under ambient conditions (damage to proteins is highly limited). The principal concern regarding the adoption of conventional ultrafiltration membranes for virus removal is the possibility of the virus passing through abnormally large pores or surface imperfections on the membrane surface. The chief principle behind the present work is to pretreat the membrane by blocking the abnormally large pores using latex particles. Experimental work was conducted to validate this pretreatment using the bacteriophage phi x 174 as a model virus. The results attained were highly encouraging. Different sizes of latex particles were tested by treating a 100 KD molecular weight cut-off membrane, and the transmission of phage (suspended in buffer) through this membrane assessed. In the absence of any particle pretreatment, a virus clearance of 4.78 log reduction value was observed for this membrane. The transmission of phage through the membrane could be reduced by an order of magnitude using 0.11 micron latex particles, or two orders of magnitude using a combination of 0.11 and 0.50 micron particles. The application of latex particles did not hinder the transport of protein through the 100 KD membrane. Protein sieving coefficients obtained using this membrane were 91%, 16% and 2%, for lysozyme, HSA and IgG, respectively.

Bacteriophage phi X 174↗
PubMed1995

No detectable alterations in immunogenicity following terminal severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX (HP9) concentrates.

We used monoclonal antibody ELISAs, antigen molecular size distribution, competition ELISA and neonatal mouse immune tolerance methods to detect potential neoantigen formation and increased immunogenicity following severe dry-heat treatment of high-purity factor VIII (Liberate) and factor IX concentrates. To provide positive controls, concentrates were heated in solution (70 degrees C for 2 h) to produce denaturation on purpose. The competition ELISA applied to factor IX proved particularly useful for quantifying differences between the positive control and the dry-heated/unheated concentrates. None of the test systems employed by us indicated any detectable neoantigen formation or any alteration in immunogenicity following terminal severe dry-heat treatment of the high-purity concentrates, and this finding is supported by clinical experience so far.

Antigens↗
PubMed1994

Some alternative coupling chemistries for affinity chromatography.

Three different coupling chemistries that have been tried and tested for use in affinity chromatography are described. These methods are particularly recommended for use by workers who do not have access to, or do not wish to use, complex organic chemical synthetic procedures. They have been demonstrated repeatedly to be reliable, efficient, low cost, and easily scaleable up or down in size. The periodate oxidation method works best with Sephacryl type gels and uses only low toxicity reagents and couples well to proteins with both high efficiency and high capacity. The vinyl sulfone method is more reactive and couples both carbohydrates and proteins. The bis-epoxide method, although less reactive, can be used under more extreme conditions of pH to couple otherwise unreactive molecules, such as synthetic polymers, drugs, and so forth.

Acrylic Resins↗
PubMed1993

Interaction of immobilised unfractionated and LMW heparins with proteins in whole human plasma.

The profile of proteins bound to immobilised heparins in hirudin-anticoagulated human plasma was analysed. In molar terms, antithrombin III was the most abundant protein bound to therapeutic doses of unfractionated heparin (M(r) = 12,000), whereas heparin cofactor II constituted < 1% of the protein bound. Histidine-rich glycoprotein was the only plasma protein likely to influence anticoagulant activity by direct competition with antithrombin III, though significant quantities of complement Factor H, fibrinogen, fibronectin, vitronectin and apolipoprotein B were also detected. Only traces of von Willebrand factor, complement factor I, inter-alpha-trypsin inhibitor, alpha 2-macroglobulin, serum amyloid P and transferrin were identified, and neither thrombospondin nor platelet factor 4 were measurable. Binding of both antithrombin III and histidine-rich glycoprotein varied with the ratio of heparin to plasma. Clexane (M(r) = 4,500) also bound antithrombin III, but both histidine-rich glycoprotein and vitronectin were quantitatively significant neutralising proteins. Neutralising proteins dominated the binding profile for Oligo H (M(r) = 2,200).

Blood Proteins↗
PubMed1992

Human vascular endothelial cells catabolise exogenous glycosaminoglycans by a novel route.

Heparin and other anticoagulant glycosaminoglycans were radiolabelled with 125I and their catabolism by human vascular endothelial cells in culture was studied. Heparin, heparan sulphate and pentosan polysulphate were associated with the cellular fraction and incorporated into the subendothelial matrix, but dermatan sulphate was not found in either fraction. High molecular weight, fully desulphated carbohydrate chains were major catabolic products of all those glycosaminoglycans which were taken up by the cells. Pentosan polysulphate was not degraded further, but the catabolism of heparan sulphate, and to a lesser extent that of heparin, also yielded small oligosaccharides. Thus the first step in catabolism of exogenous glycosaminoglycans by human vascular endothelial cells appears to be complete desulphation, which destroys their biological activity, followed by depolymerisation of the carbohydrate chain. This alternative to the sequential action of lysosomal exoenzymes is dependent upon binding to the cell; thus dermatan sulphate, which is not associated with the cellular fraction, is not catabolised.

Binding Sites↗
PubMed1992

Direct dye binding--a quantitative assay for solid-phase immobilized protein.

A direct dye-binding procedure was established for the quantification of protein after its immobilization on a solid phase, using IgG and BSA as model proteins. The assay, which in the range 0-5 mg protein/ml gel correlates well with indirect protein determination by A280 as well as determination of protein hydrolyzed from the gel, is based on a modified Bradford dye-binding assay. As the protein coupled to the gel binds the dye, a decrease in A465 of the supernatant is measured. Three solid supports commonly used for protein immobilization (Sepharose, Sephadex, Sephacryl) were found to be compatible with the dye-binding assay while nonspecific dye binding was found to HEMA gels. Protein was coupled to Sephacryl S-1000 using three different activation methods (aldehyde, hydrazine, and adipic acid dihydrazide). Artifactual dye-binding was not observed using any of the three different "linkers." The assay is easily carried out and represents a useful tool, e.g., when optimizing procedures for protein immobilization.

Coloring Agents↗
PubMed1992

Inhibition of thrombin generation by heparin and low molecular weight (LMW) heparins in the absence and presence of platelet factor 4 (PF4).

The ability of several low molecular weight (LMW) heparins and unfractionated heparin (UFH) to inhibit thrombin generation, and their anti-Xa and anti-IIa activities, were measured in the absence and presence of platelet factor 4 (PF4). The LMW heparins studied were 2-5 times less potent, on a weight basis, than UFH as inhibitors of thrombin generation in platelet-poor plasma; the inhibition of thrombin generation by LMW heparins correlated better with their anti-IIa activity (r = 0.98) than with their anti-Xa activity (r = 0.69). At low concentrations of PF4, the activity of LMW heparins in the thrombin generation test was neutralized less than that of UFH, but at higher PF4 concentrations all their activities could be neutralized except in anti-Xa assays. These observations support the hypothesis that anti-IIa activity is important for inhibition of thrombin generation by LMW heparins in vitro. However, when all the anti-IIa activity of LMW heparins was neutralized by PF4, considerable inhibitory activity remained in thrombin generation and anti-Xa assays, indicating that a portion of the anti-Xa activity of LMW heparins also contributes towards inhibition of thrombin generation.

Factor Xa Inhibitors↗
PubMed1992

Development, optimization and use of an enzyme linked immunosorbent assay (ELISA) to measure factor VIII antigen utilizing monoclonal antibodies.

An enzyme linked immunosorbent assay (ELISA) has been developed to measure VIII:Ag in plasma and concentrates. The assay utilizes two commercially available monoclonal antibodies to VIII:Ag and provides an alternative to the established immunoradiometric assay (IRMA). It has the advantage of not requiring the use of radioactive material and human antibodies. The assay sensitivity is 0.006 u/ml and the interassay coefficient of variation is 6.3%. Forty-eight samples with VIII:Ag levels ranging from 0.006 to 1.5 u/ml were assayed by both ELISA and IRMA. The coefficient of correlation between the two assays was 0.89. In addition to measuring human VIII:Ag, it is also possible to detect antigen in several animal plasma and sera.

Antibodies, Monoclonal↗
PubMed1991

Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers.

A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

Buffers↗
PubMed1991

Hypotensive effects of stroma-free haemoglobin solutions attributable to adenine nucleotides.

Aqueous solutions of stroma-free human haemoglobin are being evaluated as potential oxygen-carrying resuscitation fluids. There are indications, however, that such solutions may produce toxic side-effects in vivo. Stroma-free haemoglobin solution produced a 50% fall in mean arterial pressure when infused into a small animal model despite containing very low levels of non-haem protein and phospholipid contaminants. This effect was not produced by haemoglobin solutions after extensive dialysis. Red cell-derived adenine nucleotides were found to be present in concentrations high enough to cause such a response (80-85 micrograms/ml). We have developed a chromatographic assay capable of predicting hypotension in our animal model and consider that the complete absence of adenine nucleotides must be confirmed in all studies concerning the possible toxic side-effects of stroma-free haemoglobin solutions.

Adenine Nucleotides↗
PubMed1990

High molecular weight aggregate content of heated and unheated factor VIII products determined by fast-protein liquid chromatography.

The molecular-weight distribution of proteins in factor VIII concentrates has been analysed by fast-protein liquid chromatography. The proportion of high-molecular-weight (HMW) aggregates in one product increased on freeze-drying and heating, with fibrinogen and fibronectin being the main protein components of the HMW peak. In all other concentrates, the HMW peak was less than or equal to 5% of the total protein content and there were no differences in HMW content according to purity or method of viral inactivation.

Chromatography, Liquid↗
PubMed1990

Immunomodulatory properties of platelet factor 4: prevention of concanavalin A suppressor-induction in vitro and augmentation of an antigen-specific delayed-type hypersensitivity response in vivo.

The immunomodulatory properties of platelet factor 4 (PF4) have been examined in vitro and in vivo. This agent prevented the induction of concanavalin A (Con A)-induced suppressor cells in vitro in a dose-dependent manner but it did not affect the function of established Con A suppressor cells. This effect was not due to an enhanced production of interleukin-2 (IL-2) by lymphocytes exposed to PF4. The delayed-type hypersensitivity (DTH) reaction to sheep erythrocytes (SRBC) was used as a model for the generation of antigen-specific suppression in vivo. PF4 enhanced the magnitude of the swelling following SRBC challenge 10 days after sensitization by the i.p. route or following sensitization by both the s.c. and i.p. routes. These studies show that PF4 has immunomodulatory activities in well-defined models of cell-mediated immunity and suggest that this agent has a potential use in the dissection of events in antigen-specific suppression.

Animals↗
PubMed1989

The absorption, clearance and metabolic fate of dermatan sulphate administered to man--studies using a radioiodinated derivative.

An iodinated derivative of dermatan sulphate was administered by the intravenous, subcutaneous and oral routes to healthy human volunteers in conjunction with unlabelled dermatan sulphate. Following intravenous injection clearance of radiolabel and concentration as measured by competitive binding assay were highly correlated and displayed complex kinetics which were not dose-dependent. Intact 125I-dermatan sulphate was absorbed following both subcutaneous and oral administration, though there appeared to be selective uptake by the gut of a subfraction comprising the smaller or less sulphated molecules. The intact material was subsequently excreted unchanged in the urine. Degradation products of dermatan sulphate were not detected by either gel filtration or affinity chromatography on Polybrene-Sepharose at any time in either plasma or urine, indicating that administered dermatan sulphate is not catabolised by man.

Absorption↗
PubMed1989

Quantitation of expression of idiotopes recognized by a panel of monoclonal antibodies in normal serum immunoglobulin: use of a novel 4-stage ELISA assay.

This paper describes the application of a sensitive ELISA assay detecting as little as 10 ng/ml of a specific idiotope. Using this assay we were able to divide monoclonal anti-idiotypic antibodies generated using a single IgG1 lambda paraprotein as the immunogen into three distinct groups. Seven antibodies detected determinants which were expressed only by the immunogen. The remaining seven antibodies all reacted with normal serum immunoglobulin. Four antibodies reacted with "public" idiotopes which were strongly expressed by serum immunoglobulin, and three antibodies reacted with "restricted public" idiotopes which were only weakly expressed by serum immunoglobulin. This last group may be of significant interest as agents for the immunotherapy of B-cell malignancies. The conservation of expression of these idiotopes in many individual sera suggests that they have a physiological role in immune regulation and would therefore be excellent targets for immunotherapy of B-cell tumours while their quantitatively low expression by circulating immunoglobulin is unlikely to interfere with tumour cell specific binding in vivo.

Animals↗
PubMed1987

Immunoaffinity purification of factor VIII complex.

Murine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII:C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R. cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII:C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibronectin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII:C and R. cof. in a single vial whilst using only one specific monoclonal antibody.

Antibodies, Monoclonal↗