Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of varicella-zoster virus immunoglobulin G antibodies in fingerstick blood.

Varicella zoster virus (VZV) causes childhood chickenpox, becomes latent in sensory ganglia and reactivates years later to cause shingles (Zoster) and postherpetic neuralgia in the elderly and immunosuppressed individuals. Serologic IgG tests can be used to determine if a person has antibodies to VZV from past varicella infection or had received varicella or zoster (shingles) vaccination. Commercial enzyme-linked immunosorbent assays (ELISAs) are currently used for the detection of VZV IgG antibodies in patient serum samples. However, ELISA tests require collection and processing of blood samples in a CLIA laboratory to separate serum or plasma for further testing. In this paper, we describe the development and testing of an antibody based Lateral Flow Immunochromatographic assay (LFA) device for the detection of VZV IgG in fingerstick whole blood. Analytical and clinical analyses were performed to compare the performance characteristics of the Viro VZV IgG LFA (VZV LFA) and the Diamedix VZV IgG ELISA. Analytical studies demonstrated the higher sensitivity of the VZV LFA compared to the ELISA by testing dilutions of the WHO VZV IgG serum International Standard. Clinical performance characteristics of the VZV LFA fingerstick whole blood assay were assessed at three point of care (POC) facilities by untrained users testing samples from 300 prospectively enrolled study subjects. VZV LFA results were compared with results obtained by testing serum samples obtained from the same study participants by the Diamedix VZV IgG ELISA. Two specimens with invalid results by the LFA assay were not included in the LFA performance calculations and nine equivocal ELISA results were included as positive for IgG results. The results from all three POC clinical sites demonstrated the higher sensitivity/positive percent agreement (PPA) (99.26%, 95% CI: 97.34-99.80) of the VZV LFA compared to the Diamedix VZV IgG ELISA (94.08%, 95% CI: 90.72-96.27). The specificity/negative percent agreement (NPA) of the VZV LFA compared to the ELISA test was calculated initially to be 39.29% (95% CI: 23.57-57.59) with 19 discordant test results out of 298 test results between the two assays (17 LFA positive/ELISA negative and two LFA negative/ELISA positive). The PPA and true NPA of the VZV LFA were determined by testing all 298 samples, including the discordant (19) and all concordant negative and positive (279) study subject serum samples, before and after blocking VZV gE antibody sites in the samples by spiking with VZV LFA gE capture antigen. The NPA improved to 100% (95% CI: 74.12-100) after the procedure when compared to the ELISA test results. The comparator ELISA PPA based on the spiking/blocking study remained as 94.08%, (95% CI: 90.72-96.27), comparable to test results from untreated samples. The VZV LFA has been demonstrated to be simple and sufficiently robust for use in CLIA-waived POC facilities by untrained healthcare professionals and to detect VZV IgG in 20 min from fingerstick whole blood. The VZV LFA therefore provides a fast, reliable, and highly sensitive method of determining prior VZV viral infection or varicella and zoster vaccination status.

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings.

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid-protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (K D ) and kinetics (kon and koff ). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached.

Evaporative edge lithography of a liposomal drug microarray for cell migration assays.

Lipid multilayer microarrays are a promising approach to miniaturize laboratory procedures by taking advantage of the microscopic compartmentalization capabilities of lipids. Here, we demonstrate a new method to pattern lipid multilayers on surfaces based on solvent evaporation along the edge where a stencil contacts a surface called evaporative edge lithography (EEL). As an example of an application of this process, we use EEL to make microarrays suitable for a cell-based migration assay. Currently existing cell migration assays require a separate compartment for each drug which is dissolved at a single concentration in solution. An advantage of the lipid multilayer microarray assay is that multiple compounds can be tested on the same surface. We demonstrate this by testing the effect of two different lipophilic drugs, Taxol and Brefeldin A, on collective cell migration into an unpopulated area. This particular assay should be scalable to test of 2000 different lipophilic compounds or dosages on a standard microtiter plate area, or if adapted for individual cell migration, it would allow for high-throughput screening of more than 50,000 compounds per plate.

Lipid multilayer microarrays for in vitro liposomal drug delivery and screening.

Screening for effects of small molecules on cells grown in culture is a well-established method for drug discovery and testing, and faster throughput at lower cost is needed. Small-molecule arrays and microfluidics are promising approaches. Here we introduce a simple method of surface-mediated delivery of drugs to cells from a microarray of phospholipid multilayers (layers thicker than a bilayer) encapsulating small molecules. The multilayer patterns are of sub-cellular dimensions and controllable thickness and were formed by dip-pen nanolithography. The patterns successfully delivered a rhodamine-tagged lipid and drugs only to the cells directly over them, indicating successful encapsulation and no cross-contamination to cells grown next to the patterns. We also demonstrated multilayer thickness-dependant uptake of the lipids from spots with sub-cellular lateral dimensions, and therefore the possibility of delivering different dosages from different areas of the array. The efficacies of two drugs were assayed on the same surface, and we were able to deliver dosages comparable to those of solution based delivery (up to the equivalent of 30 µg/mL). We expect our method to be a promising first step toward producing a single high-throughput liposome-based screening microarray plate that can be used in the same way as a standard well plate.

Stability of varicella-zoster virus open reading frame 63.

The stability of varicella-zoster virus (VZV) open reading frame (ORF) 63 was analyzed by sequential passage of a virus strain in cell culture. VZV was propagated in culture for 1,206 passages. ORF63 from six passages (18, 220, 516, 730, 1060, and 1,206) was selected and sequenced. Among the six passages, only passage 1,206 showed point mutations at three locations: 551, 618 and 661. In addition, western blot analysis with anti-ORF63 monoclonal antibodies showed no discernable difference in the size of the ORF63 gene product from passage 18 and that from passage 1,206. These results indicate the stability of VZV ORF63 gene in culture over 1,206 passages.