A competitive inhibition ELISA as a probe for studying the refolding of human serum albumin.

The refolding of iodoacetic acid-blocked human serum albumin (HSA) was studied using a modified competitive inhibition ELISA. A maximum of 89% native activity was detected 24 hours after initiating refolding using an albumin concentration of 600 micrograms/mL. The presence of both monomer and polymer HSA was studied using native polyacrylamide gel electrophoresis of thiol-blocked HSA samples. Monomer HSA was not detected until 2.5 hours after initiating refolding. Fractionated polymer and monomer HSA from a sample trapped at 72 hours after initiating refolding was determined to have 40% and 87% native activity respectively. Both polymer and monomer HSA fractions contribute to the overall immunological activity detected by the ELISA, at various times. The ELISA assay was able to detect the changing HSA conformation associated with refolding of totally reduced HSA.

Extraction of circulating gastrointestinal cancer antigen using solid-phase immunoadsorption system of monoclonal antibody-coupled membrane.

An immunoadsorption system of monoclonal antibody immobilized on a polyolefin alloy fiber is described for extraction of serum gastrointestinal cancer antigen (GICA). Continuous circulation or single passage of plasma from gastrointestinal cancer patients through this antibody-fiber matrix resulted in 90% depletion of circulating GICA in 2 h using 0.6 mg immobilized antibody, and 90% depletion in 5 min using 8 mg antibody. Continual circulation resulted in total GICA removal in both cases. Desorption of antibody or of antibody-containing complexes was minimal. This methodology provides a selective and convenient means of removing any targeted substance by monoclonal antibody from the serum, and thus overcomes many of the shortcomings associated with conventional plasmapheresis.

Membrane plasma exchange: principles and application techniques.

Membrane plasmapheresis was introduced in 1978 as a new method for performing therapeutic plasma exchange. Its principal advantages over traditional techniques include speed, ease of performance, and ready adaptability to clinical centers already performing routine extracorporeal therapy. The appearance of a membrane plasmapheresis circuit (vascular access, anticoagulation, connectology) is similar to that of hemodialysis and especially hemofiltration; the operating protocols (treatment time, filtration rates, pressures, pharmacokinetics) are quite different. Particular attention must be paid to avoiding operating conditions that lead to hemolysis. In clinical use membrane plasma separation is as effective as centrifugal plasma exchange in removing plasma proteins. The sieving coefficients for proteins with a molecular weight (MW) ranging from 67,000 (albumin) to 2,400,000 (beta-lipoprotein) daltons are unity. An exchange of one patient plasma volume has been shown to cause a 55% reduction of the serum levels of intravascular proteins. There are no significant differences between membrane and centrifugal plasmapheresis in substitution fluid requirements (human albumin or fresh frozen plasma), indications for treatment and complications. The next major advance in plasmapheresis technology will almost certainly be development of a "closed loop" circuit in which filtered plasma is treated to remove the offending moiety and returned to the patient. This would eliminate both the cost and the possible side effects of replacement fluid. Membrane-based systems are already available for removing cryoglobulins or proteins with MW of at least 900,000 daltons.

Mass transfer in membrane plasma exchange.

In vitro and in vivo sieving coefficients (SC) have been determined for a spectrum of proteins ranging in molecular weight from 66,500 daltons (albumin) to 2.4 million (beta-lipoprotein) daltons for three commercially available membrane plasma separation devices: the Plasmaflo 0.1, Plasmaflo 02, and Plasmaflux. A model relating serum level of a protein to pretherapy level, plasma volume, plasma filtration rate, membrane SC, and duration of treatment has been used to investigate the influence of SC on exchange efficiency. Comparison of predicted and clinically obtained reductions in serum solute levels demonstrated the validity of the model. The results of the analysis suggest that all three plasma separators are capable of delivering equally acceptable therapy. The model further demonstrates the decreasing effectiveness, and increased cost in terms of replacement fluid per unit of solute removed, with prolonged treatment times.

Sorbent membrane dialysis in uremia.

The inclusion of activated charcoal within hemodialysis membranes offers potentially improved plasma clearance of creatinine and middle molecules. However, the carbon becomes saturated with continued use and beyond 1 h removal of solutes is by dialysis alone. Two independently conducted crossover studies, to assess the efficacy of sorbent membrane dialysis (SMD) in the treatment of uremia, found predialysis urea levels increased by approximately 15%, creatinine by 10-15%, and inorganic phosphate levels by 10-18% on SMD compared to conventional hemodialysis. One study also observed "middle molecule' (peak "b') levels elevated. No differences were observable in the clinical status of patients. The results suggest that the charcoal content of the SMD device is too small to effect any advantages over conventional dialysis.