Preparative-scale fractionation by isoelectric trapping under nondenaturing conditions: separation of egg white protein isoforms on a modified Gradiflow unit.

pH-biased isoelectric trapping was used to separate proteins from egg white at the preparative level (80 mg), into discrete protein fractions based on isoelectric point. The problems of isoelectric precipitation that are common for the separation of complex protein mixtures under isoelectric conditions were mitigated by using single-component isoelectric buffers within the sample separation compartments. This combined with the mild process conditions of the Gradiflow unit that was modified for binary isoelectric trapping separations, ensured that biological activity was maintained.

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology.

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies.

The purification of IgY from chicken egg yolk by preparative electrophoresis.

Chicken IgY has been purified from egg yolk by preparative electrophoresis on the Gradiflow, a system which has been employed for the purification of a wide range of proteins with high recovery and biological activity. Protein purification on the Gradiflow utilises electrophoresis with selected combinations of porous membranes and buffers. The purification of IgY was achieved by initial PEG lipid precipitation, then a single step Gradiflow run by a strategy based on the relatively high pI range of IgY compared to other egg yolk proteins.

Preparation of monoclonal antibodies using the electrophoresis separation instrument, Gradiflow.

Gradiflow, a preparative electrophoresis separation device, was utilized to develop and test generic protocols for the preparation of monoclonal antibodies (MAbs) from tissue culture supernatant and ascites fluid. The charge based protocol separated the high pI antibodies from the lower isoelectric points (pI) contaminants by either moving the antibody (ascites fluid) or contaminants (tissue culture supernatant) through a polyacrylamide separation membrane. A total of 60 separations were performed with tissue culture supernatant, and a further 30 separations with ascites fluid.

Design of a new, twelve-channel electrophoretic apparatus based on the Gradiflow technology.

The Gradiflow technology, originally designed to carry out binary, size-based and charge sign-based electrophoretic protein separations, has been extended to simultaneously obtain multiple protein fractions from a single electrophoretic separation. The separation unit of the new apparatus houses the anode and cathode compartments and up to twelve shallow separation compartments through which the background electrolyte solution that contains the separated protein fractions is recirculated. The separation compartments are formed from grids as thin as 1.

Depletion of the highly abundant protein albumin from human plasma using the Gradiflow.

Analysis of complex protein samples by two-dimensional electrophoresis (2-DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions.

Preparative-scale isoelectric trapping separations using a modified Gradiflow unit.

The Gradiflow BF200 preparative electrophoretic unit (Gradipore), which has been developed for size-based and charge-sign-based protein separations and in which the hydraulic flow path of the recirculating sample stream in the separation cartridge is orthogonal to the electric field, has been modified to carry out binary protein separations using the principles of isoelectric trapping. The disposable separation cartridge contained three isoelectric membranes which, along with the cartridge holder, formed the anode and cathode compartments and the anodic and cathodic separation compartments. The utility of the modified instrument was demonstrated by effecting a binary separation of chicken egg white across an isoelectric point 5.

Gradiflow as a prefractionation tool for two-dimensional electrophoresis.

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions.

Purification of the basic protein avidin using Gradiflow technology.

The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, from chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.

Preparative electrophoresis: a general method for the purification of polyclonal antibodies.

Antibodies were purified from normal rabbit, sheep, goat, rat, human and bovine serum using preparative electrophoresis on a Gradiflow in a single-step process using an asymmetrical cartridge with three different pore size polyacrylamide membranes. Recoveries in each case were over 80% and were higher than those obtained using affinity chromatography on protein A, protein G or protein L. Degree of purity was at least comparable with these methods.

Electrophoretic transfer of proteins across polyacrylamide membranes.

The electrophoretic transfer of purified proteins has been examined in a Gradiflow "Babyflow BF100" unit. A number of factors affect protein separation within this preparative electrophoresis system. We established that the rate of protein transfer was proportional to the applied voltage.

Detecting APC-resistant factor V: a functional method without plasma dilution.

A method for detecting activated protein C (APC)-resistant factor V, especially factor V Leiden, is described, which uses reagents containing two unfractionated snake venoms. The procedure can be used for testing plasma samples from patients receiving oral anticoagulant therapy, heparin therapy and patients with lupus anticoagulant, and does not require the use of factor-V-deficient plasma. The sample plasma is first incubated with dilute venom from Agkistrodon contortrix contortrix (Southern Copperhead) which activates the endogenous protein C and then a dilute Russell's viper venom time test is performed.

Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis.

Four monoclonal antibodies (including Ig subclasses, G1, G2a, and G2b) were purified from murine ascitic fluid by a preparative electrophoresis system using a charge- and size-based strategy. Most of the smaller contaminating proteins were removed at pH 8.3 when the ascitic fluid was passed through a cartridge containing a separating membrane with a pore size of M(r) 100,000.

Rapid detection of D-dimer using a fiber optic biosensor.

We describe a rapid and sensitive method for detection and quantification of D-dimer and other crosslinked fibrin degradation products (XL-FDPs), which are present in elevated concentrations in patients with sepsis and thrombotic disorders. The method utilizes a sandwich fluoroimmunoassay immobilized in the sensing region of an evanescent wave biosensor. Physiological concentrations of D-dimer and high molecular weight XL-FDP could be determined in buffer and plasma samples on calibrated fibers in 11 min.

The importance of antibody specificity in measuring cross-linked fibrin degradation products by ELISA.

We and others have previously shown that plasma concentrations of XL-FDPs are accurately characterized with an enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody DD-3B6/22, which is specific for D-dimer, and a pan-specific tag antibody (DD-4D2/182) in patients with thrombotic disorders. However, in patients treated with fibrinolytic agents, increases in non-cross-linked fibrin(ogen) degradation products are detected by the pan-specific tag antibody due to formation of complexes between non-cross-linked derivatives and XL-FDPs. Assays based on the use of fibrin degradation product-specific tag antibodies appear to be more specific, but whether they would be clinically more informative is unclear.

Application of a bedside whole blood D-dimer assay in the diagnosis of deep vein thrombosis.

Cross-linked fibrin degradation products have been used to detect venous thrombosis. While the sensitivity of plasma D-dimer measured by ELISA in the diagnosis of deep vein thrombosis (DVT) is high, the utility of ELISA methods is limited in a clinical setting. This study analysed the diagnostic value of a rapid D-dimer assay performed on whole blood samples (SimpliRed D-dimer) compared with latex and ELISA in 86 patients suspected of having DVT.

A rapid test for endotoxin in whole blood.

A rapid whole blood agglutination test has been developed for the detection of endotoxin. The test reagent consists of polymyxin B (PmB) conjugated to the Fab fragment of the anti-glycophorin antibody 1C3/86. After addition of reagent to whole blood, red cell agglutination occurs within two minutes in samples from endotoxaemic patients or with the addition of either whole Gram negative bacteria, supernatants from Gram negative bacterial cultures or purified endotoxin.

The detection of D-dimer in plasma by enzyme immunoassay: improved discrimination is obtained with a more specific signal antibody.

Most commercial D-dimer enzyme immunoassays employ two antibodies, a fibrin specific capture monoclonal and a less specific antibody cross-reacting with epitopes present on fibrinogen. Two different ELISA systems were compared to test whether this cross-reaction can lead to overestimation of the true levels of cross-linked fibrin derivatives. Both assays used the same D-dimer specific antibody for capture, DD-3B6/22, but utilized signalling antibodies which differed in fibrinogen reactivity.

Technetium-99 m labelling of DD-3B6/22 antifibrin monoclonal antibody fragment Fab' for thrombus imaging.

The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99 m (99mTc) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature of intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo.

Automated determination of cross-linked fibrin derivatives in plasma.

Automated assays for the measurement of cross-linked fibrin derivatives in plasma (XL-FbDP) have been developed utilizing latex beads coated with anti-D dimer monoclonal antibody (DD-3B6/22) for both the Cobas Fara Chemistry Centrifugal and the Cobas Mira analysers (Roche, Basle, Switzerland). The analysers were programmed to mix plasma and latex reagent simultaneously and analyse absorbance changes over a 10-15 min period. Results were interpolated by the analyser from a standard curve derived from a polymer of D-dimer.

Evaluation of thrombus detection in a rabbit model using a technetium-99m-labelled anti-fibrin monoclonal antibody.

Technetium-99 m (99mTc)-labelled conjugates of an anti-fibrin monoclonal antibody, DD-3B6/22, have been assessed for their detection of vascular thrombi in a rabbit model. DD-3B6/22 binds to a D-dimer epitope present on cross-linked fibrin but absent from the fibrin monomer or fibrinogen. Injection of a 99mTc-labelled Fab' fragment of DD-3B6/22 allowed delineation of model thrombi as early as 30 min postinjection (p.

Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.

A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on agglutination of autologous red blood cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596).