Evaluation of anti-activated protein C antibody development in patients with severe sepsis from four clinical studies with drotrecogin alpha (activated).

BACKGROUND: Drotrecogin alpha (activated) (DAA) is a recombinant human activated protein C (APC), which is an antithrombotic protein. OBJECTIVES: To evaluate the development of anti-APC antibodies in severe sepsis patients in DAA clinical studies. PATIENTS AND METHODS: Serum and plasma samples were collected in placebo-controlled studies (PROWESS, ADDRESS) and studies where all patients were DAA-treated (ENHANCE, XPRESS). An enzyme-linked immunosorbent assay detecting anti-APC IgA/IgG/IgM antibodies was used. IgG isolated from plasma of positive samples was tested for neutralizing activity against DAA-induced prolongation of activated partial thromboplastin time. RESULTS: The proportions of patients with negative baseline but positive postbaseline anti-APC antibodies were 1.5% (27/1855) and 1.6% (24/1493) in the DAA and placebo cohorts, respectively (P = 0.72 for the difference). Of the 27 DAA and 24 placebo patients with positive anti-APC antibodies, all but one (DAA) were alive at day 28, and all but seven (four DAA and three placebo) were alive at hospital discharge, including eight (five DAA and three placebo) patients who tested positive for anti-APC neutralizing antibodies. Two of the 51 patients who tested positive for the development of anti-APC antibodies experienced a thrombotic event (one DAA, one placebo). In ADDRESS, no anti-APC antibody was detected in the six DAA-treated patients who had received a previous course of DAA therapy. CONCLUSIONS: The proportion of patients with anti-APC antibodies was low and was similar between DAA-treated and placebo-treated patients. No relationship between anti-APC antibody development and adverse reactions was observed. There was no evidence that the anti-APC antibodies detected represented a specific immune response to DAA therapy.

The influence of matrix type, diurnal rhythm and sample collection and processing on the measurement of plasma beta-amyloid isoforms using the INNO-BIA plasma Abeta forms multiplex assay.

OBJECTIVE: The aim of the study was to determine the extent to which plasma matrix types, diurnal rhythm and sample collection and processing procedures contribute to overall variability of measurements with the INNO-BIA plasma Abeta forms assay. METHODS: Plasma samples from healthy volunteers were collected at BARC-CRI. Analyte concentrations from various plasma matrix types (EDTA, heparin, fluoride) were compared to serum after collection of blood in commercial plastic and glass tubes. Sample processing variables including time and temperature before and after centrifugation, centrifugal force and plasma dilution factor were also investigated. Diurnal variability in plasma Abeta isoforms was determined in 29 healthy volunteers by analysis of EDTA plasma specimens serially collected over 24 hours and stored frozen following oral administration of a placebo treatment. All plasma samples from a given individual and experiment were analyzed in a single analytical run. RESULTS: Highest Abeta levels were obtained using EDTA-plasma samples (in contrast to serum, heparin, citrate, or fluoride). Addition of aprotinin to EDTA plasma had no effect on Abeta peptide recovery. The elapsed time and temperature exposure, before and after sample processing affects the recovery of Abeta isoforms. Analyte recovery was not significantly affected by the presence of platelets in plasma samples. At the subject level, analysis of serially collected EDTA plasma specimens from healthy volunteers revealed no evidence of diurnal variation in any of the Abeta isoforms investigated and results from samples collected on a monthly basis showed only very limited intra-individual variation. CONCLUSIONS: Optimal recovery of Abeta peptides was obtained from blood drawn into EDTA tubes and processed within 4 h. Plasma that was refrigerated after separation and analysed within 4 h gave comparable results to samples immediately processed and frozen at -70 degrees C.

Structural analysis of bovine somatotropin using monoclonal antibodies and the conformation-sensitive immunoassay.

Bovine somatotropin was studied with respect to thermal stability, quantitative thermal denaturation kinetics, and refolding potential following thermal denaturation using a panel of 6 monoclonal antibodies and the Conformation-Sensitive Immunoassay (CSI). The antibody panel consisted of 4 conformation-dependent and 2 sequence-specific antibodies. Each of the antibodies revealed unique thermal stability profiles for their respective epitopes suggesting that they each recognize different antigenic determinants. Comparing the thermal stability profiles generated with these antibodies allowed the stability of bovine somatotropin to be "dissected" based on individual structural features. The degree to which bovine somatotropin is stabilized by disulfide bonds was examined using CSI-based quantitative thermal denaturation kinetics profiles generated under reducing and nonreducing conditions. All of the conformational epitopes unfolded faster under reducing conditions indicating that the two disulfide bonds within the somatotropin molecule impart some degree of global stabilization. The ability of bovine somatotropin to refold after reducing or nonreducing thermal denaturation was also examined using the antibody panel and the CSI. The results show that, although significant refolding was evident for some epitopes, bovine somatotropin cannot refold to the native state following thermal denaturation under either reducing or nonreducing conditions.

Comparative polyclonal antithymocyte globulin and antilymphocyte/antilymphoblast globulin anti-CD antigen analysis by flow cytometry.

Polyclonal antithymocyte globulin (ATG)/antilymphocyte and antilymphoblast globulins (ALG) antibodies have been used successfully in transplantation, aplastic anemia and graft-versus-host disease. Flow cytometry has been used to analyze peripheral blood lymphocyte populations in transplant patients receiving polyclonal ATG/ALG preparations for immunosuppression. Recent reports have indicated clinical dose adjustment based on levels of patient's cells expressing various CD antigens. In vitro analysis of individual polyclonal ATG/ALG CD antigen specificity could identify appropriate antigens for clinical monitoring as well as provide useful in vitro activity data. Therefore, a flow cytometry based assay to characterize and compare activities to specific CD antigens found on the surface of peripheral blood lymphocytes has been developed. Activities found in four lots each of horse ATG (ATGAM, Upjohn), rabbit and horse ATG (thymoglobulin and lymphoglobulin, Merieux), horse ALG (Minnesota), and rabbit ATG (Fresenius) have been compared for CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD18, CD28, CD44, CD45, and TCR-alpha/beta antigens. Quantitation is achieved by measuring the concentration of ATG/ALG required to give 50% inhibition of antigen specific fluorescent-labeled monoclonal antibody relative to buffer controls. The three horse products tested have similar activity to most antigens tested. However, Fresenius rabbit ATG has the lowest activity for almost all antigens tested whereas the Merieux rabbit ATG has activities closer to the horse products. This technique allows for rapid in vitro comparison of reactivities to individual lymphocyte antigens as well as in vitro analysis of consistency.

The conformation-sensitive immunoassay: a membrane based ELISA system for identifying antibodies sensitive to alterations of protein conformation.

A Conformation-Sensitive Immunoassay (CSI) has been developed for identification of antibodies that are sensitive to alterations in protein conformation. The method involves covalently coupling proteins to an activated hydrophilic membrane support. The membrane bound proteins are then treated under conditions known to alter protein conformation, immobilized in a non-native state via additional covalent interactions with the support, and subsequently probed using conventional ELISA techniques. This method has been validated using several well characterized conformation-sensitive antibodies to horse cytochrome c and sperm whale myoglobin. A panel of monoclonal antibodies directed against bovine somatotropin (bSt) has been partially characterized using this validated CSI procedure. Each of these antibodies to bSt has been shown to detect conformational alterations of bSt structure. Data are also presented that demonstrates that the accuracy of CSI analysis is superior to that of Western blotting for characterizing conformation-sensitive antibodies.