Evaluation of an IgM/IgG sensitive enzyme immunoassay and the utility of index values for the screening of syphilis infection in a high-risk population.

BACKGROUND: Increasing interest in the use of enzyme immunoassays (EIA) for syphilis screening has generated a considerable need for data on the performance of such tests. METHODS: We compared the performance of 1 EIA, the TREP-SURE EIA to that of the Venereal Disease Research Laboratory (VDRL) and Treponema pallidum particle agglutination assay (TPPA) in the detection of infection with Treponema pallidum. In total, 674 specimens were tested by VDRL and EIA (356 VDRL-nonreactive and 318 VDRL-reactive). All specimens that were found to be reactive by either the VDRL or EIA were subsequently analyzed by TPPA. RESULTS: We found that the TREP-SURE EIA was marginally less sensitive than the VDRL test for screening, but was significantly more specific. All EIA-TPPA discordant specimens were analyzed by multiple tests, including Immunoglobulin M- and G-specific Western blots and an IgM-specific EIA. Signal-to-cutoff ratios (index values) generated by the TREP-SURE EIA were also investigated. It was found that these values may be instructive regarding the interpretation of test results, as they were found to correlate strongly with the probability of positivity on a TPPA assay. Specimens that reacted positively on the EIA with very high index values were found overwhelmingly to be reactive by TPPA, perhaps obviating the need for the testing of most EIA positive specimens with a secondary treponemal test. CONCLUSIONS: An IgM/IgG sensitive EIA would be an effective alternative to VDRL for syphilis screening. Using the EIA index values may provide additional, helpful information to the diagnostic process.

Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis.

INTRODUCTION: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays. METHODS: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support. RESULTS: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10µL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less. DISCUSSION: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.

Highly stable resistive switching on monocrystalline ZnO.

We report on the achievement of planar memristive devices on monocrystalline ZnO substrates using Ti/Al and Pt/Au contacts with dimensions of 100 x 100 microm(2) and spacings of approximately 60 microm. Effects of both thermal and electro-forming processes on the switching characteristics are investigated. It is observed that the thermally formed devices exhibit an extremely large R(OFF)/R(ON) value of approximately 20 000. The electrically formed devices, on the other hand, demonstrate an exceptional switching stability, with R(OFF)/R(ON) variations of < 2% for durations of over 10(5) s and more than 1800 switching cycles. The dependence of the switching characteristics on the formation processes, as well as the metal electrodes, could be explained by an oxygen vacancy formation/annihilation and migration model.

Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates.

The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.

Interactions of human P-glycoprotein with simvastatin, simvastatin acid, and atorvastatin.

PURPOSE: In this study, P-glycoprotein (P-gp) mediated efflux of simvastatin (SV), simvastatin acid (SVA), and atorvastatin (AVA) and inhibition of P-gp by SV, SVA, and AVA were evaluated to assess the role of P-gp in drug interactions. METHODS: P-gp mediated efflux of SV, SVA, and AVA was determined by directional transport across monolayers of LLC-PK1 cells and LLC-PK1 cells transfected with human MDR1. Inhibition of P-gp was evaluated by studying the vinblastine efflux in Caco-2 cells and in P-gp overexpressing KBV1 cells at concentrations of SV, SVA, and AVA up to 50 microM. RESULTS: Directional transport studies showed insignificant P-gp mediated efflux of SV, and moderate P-gp transport [2.4-3.8 and 3.0-6.4 higher Basolateral (B) to Apical (A) than A to B transport] for SVA and AVA, respectively. Inhibition studies did not show the same trend as the transport studies with SV and AVA inhibiting P-gp (IC50 -25-50 microM) but SVA not showing any inhibition of P-gp. CONCLUSIONS: The moderate level of P-gp mediated transport and low affinity of SV, SVA, and AVA for P-gp inhibition compared to systemic drug levels suggest that drug interactions due to competition for P-gp transport is unlikely to be a significant factor in adverse drug interactions. Moreover, the inconsistencies between P-gp inhibition studies and P-gp transport of SV, SVA, and AVA indicate that the inhibition studies are not a valid means to identify statins as Pgp substrates.