Analytical Performance of a Multiplexed Microarray Assay for Rapid Identification and Quantification of a Multivalent mRNA Vaccine.

mRNA vaccines were highly effective in response to the COVID-19 pandemic, making them an attractive platform to address cancers and other infectious diseases. Many new mRNA vaccines in development are multivalent, which represents a difficulty for the standard assays commonly used to characterize the critical quality attributes of monovalent formulations. Here, we present a multiplexed analytical tool with nucleic acid microarray technology using the VaxArray platform that measures the identity and quantity of mono- and multivalent mixtures of naked mRNA and mRNA encapsulated in lipid nanoparticle formulations in under 2 h without any additional preparation steps, such as extraction or RT-PCR.

30-Minute Highly Multiplexed VaxArray Immunoassay for Pneumococcal Vaccine Antigen Characterization.

Pneumonia accounts for over 20% of deaths worldwide in children aged 1 to 5 years, disproportionately affecting lower- and middle-income countries. Effective, highly multivalent pneumococcal vaccines are available to decrease disease burden, with numerous new vaccines currently under development to serve a variety of worldwide markets. However, pneumococcal conjugate vaccines are among the hardest biologics to manufacture and characterize due to their complexity and heterogeneity.

Rapid Identity and Quantity CQA Test for Multivalent mRNA Drug Product Formulations.

The COVID-19 pandemic highlighted mRNA as a promising platform for vaccines and therapeutics. Many of the analytical tools used to characterize the critical quality attributes of mRNA are inherently singleplex and are not necessarily optimal from a labor and cost perspective. Here, we demonstrate the feasibility of a multiplexed platform (VaxArray) for efficient identity verification and concentration determination for both monovalent and multivalent mRNA formulations.

VaxArray immunoassay for the multiplexed quantification of poliovirus D-antigen.

Next generation poliovirus vaccines are critical to reaching global poliovirus eradication goals. Recent efforts have focused on creating inactivated vaccines using attenuated Sabin strains that maintain patient safety benefits and immunogenicity of conventional inactivated vaccines while increasing manufacturing safety and lowering production costs, and on developing novel oral vaccines using modified Sabin strains that provide critical mucosal immunity but are further attenuated to minimize risk of reversion to neurovirulence. In addition, there is a push to improve the analytical tools for poliovirus vaccine characterization.

Multiplexed VaxArray immunoassay for rapid antigen quantification in measles and rubella vaccine manufacturing.

Measles-containing vaccines (MCV), specifically vaccines against measles and rubella (MR), are extremely effective and critical for the eradication of measles and rubella diseases. In developed countries, vaccination rates are high and vaccines are readily available, but continued high prevalence of both diseases in developing countries and surges in measles deaths in recent years have highlighted the need to expand vaccination efforts. To meet demand for additional vaccines at a globally affordable price, it is highly desirable to streamline vaccine production thereby reducing cost and speeding up time to delivery.

Evaluation of a multiplexed coronavirus antigen array for detection of SARS-CoV-2 specific IgG in COVID-19 convalescent plasma.

Mitigation of the COVID-19 pandemic requires an understanding of the antibody response to SARS-CoV-2. However, throughout the development of SARS-CoV-2 IgG antibody assays during the past year, cross-reactivity to other coronaviruses remained a question. To address these issues, we evaluated IgG in COVID-19 convalescent plasma samples for reactivity against three SARS-CoV-2 antigens including full-length spike, receptor binding domain, and the proximal extracellular fusion domain, and spike antigens from other coronaviruses (SARS-CoV, MERS-CoV, hCoV-HKU1, hCoV-OC43, hCoV-229E and hCoV-NL63) using the VaxArray Coronavirus SeroAssay which is a multiplexed antigen assay developed by InDevR Inc.

Multiplexed, microscale, microarray-based serological assay for antibodies against all human-relevant coronaviruses.

Rapid, sensitive, and precise multiplexed assays for serological analysis during candidate COVID-19 vaccine development would streamline clinical trials. The VaxArray Coronavirus (CoV) SeroAssay quantifies IgG antibody binding to 9 pandemic, potentially pandemic, and endemic human CoV spike antigens in 2 h with automated results analysis. IgG antibodies in serum bind to the CoV spike protein capture antigens printed in a microarray format and are labeled with a fluorescent anti-species IgG secondary label.

Clinical validation of the FluChip-8G Influenza A+B Assay for influenza type and subtype identification.

Background: The FluChip-8G Influenza A+B Assay is a multiplexed influenza RT-PCR and microarray-based assay with same day turnaround time, developed to subtype seasonal A viruses (H1N1pdm2009 and H3N2), distinguish B viruses as Yamagata or Victoria lineage, and is the only FDA cleared assay capable of positive identification of a wide variety of A subtypes as "non-seasonal" A viruses from human nasal specimens.

Objective: To evaluate clinical performance of the FluChip-8G Influenza A+B Assay for detection of seasonal influenza viruses in nasal and nasopharyngeal swab specimens, and to evaluate performance for detection of non-seasonal influenza viruses using contrived samples.

Study Design: For seasonal viruses, a multisite study of the FluChip-8G Influenza A+B Assay using prospectively and retrospectively collected nasal and nasopharyngeal swabs was performed using the FDA-cleared CDC Human Flu Dx Panel as the comparator assay.

Analytical evaluation of the microarray-based FluChip-8G Influenza A+B Assay.

Background: Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8G Influenza A+B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time.

A neuraminidase potency assay for quantitative assessment of neuraminidase in influenza vaccines.

Neuraminidase (NA) immunity leads to decreased viral shedding and reduced severity of influenza disease; however, NA content in influenza vaccines is currently not regulated, resulting in inconsistent quality and quantity of NA that can vary from manufacturer to manufacturer, from year to year, and from lot to lot. To address this problem, we have developed an assay for NA quantification that could be used by the industry to move toward developing influenza vaccines that induce a predictable immune response to NA. The VaxArray Influenza Seasonal NA Potency Assay (VXI-sNA) is a multiplexed sandwich immunoassay that relies on six subtype-specific monoclonal antibodies printed in microarray format and a suite of fluor-conjugated "label" antibodies.

VaxArray potency assay for rapid assessment of "pandemic" influenza vaccines.

The VaxArray Influenza Pandemic HA (VXI-pHA) potency assay is a multiplexed sandwich immunoassay that consists of nine broadly reactive yet subtype-specific monoclonal capture antibodies printed in microarray format and a suite of fluor-labeled secondary antibodies that were selected to probe conserved HA epitopes. VXI-pHA was designed to optimize the probability that the ready-to-use assay would work for the most concerning, emergent influenza A strains, eliminating the need for the time-consuming process of reference reagents production. The performance of this new potency test was evaluated using a panel of 48 potentially pandemic strains of influenza viruses and vaccines spanning 16 years of antigenic drift, including the most recent pre-pandemic vaccine being developed against the "5 wave" A/H7N9 virus.

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines.

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice.

VaxArray assessment of influenza split vaccine potency and stability.

Vaccine manufacturers require more rapid and accurate tools to characterize the potency and stability of their products. Currently, the gold standard for influenza vaccine potency is the single radial immunodiffusion (SRD) assay, which has inherent disadvantages. The primary objective of this study was to investigate the ability of the VaxArray Influenza (VXI) seasonal hemagglutinin (sHA) potency assay to accurately quantify potency and stability in finished vaccines as well as to quantify hemagglutinin protein (HA) within crude in-process samples.

Titer on chip: new analytical tool for influenza vaccine potency determination.

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process.

ampliPHOX colorimetric detection on a DNA microarray for influenza.

DNA microarrays have emerged as a powerful tool for pathogen detection. For instance, many examples of the ability to type and subtype influenza virus have been demonstrated. The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic.

Evaluation of the Virus Counter® for rapid baculovirus quantitation.

The utility of a new instrument for rapid virus quantitation, the Virus Counter, was evaluated in a blind study conducted at three sites. This instrument is a substantially improved version of the original academic research instrument described previously by Stoffel and Rowlen (2005a). The addition of hydrodynamic focusing, a self-contained fluidics system and customized software for system control and data analysis has resulted in a commercially viable and available design.

MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1.

Background: The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in <8 hours for under US$10.

Controlling the optical properties of plasmonic disordered nanohole silver films.

Disordered nanohole arrays were formed in silver films by colloidal lithography techniques and characterized for their surface-plasmon activity. Careful control of the reagent concentration, deposition solution ionic strength, and assembly time allowed generation of a wide variety of nanohole densities. The fractional coverage of the nanospheres across the surface was varied from 0.

Molecular detection of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel.

Molecular orientation of a model liquid crystal alignment layer.

An analytical methodology, involving the use of a combination of second harmonic generation (SHG) and linear dichroism, was utilized to probe the molecular orientation and angular distribution of a model liquid crystal (LC) alignment layer. In order to determine which film structure would be best suited for use as an alignment layer, the azo dye o-methyl red (MR) was covalently bound to a glass substrate using both monofunctional and trifunctional silane chemistry. The influence of solvent on the orientation and angular distribution of both thin films was also investigated.

Detection of adamantane-resistant influenza on a microarray.

Background: Influenza A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza therapeutics, thereby complicating treatment decisions.

Objective: To design a cost-effective low-density microarray for use in detection of influenza resistance to the adamantanes.

Study Design: We have taken advantage of functional genomics and microarray technology to design a DNA microarray that can detect the two most common mutations in the M2 protein associated with adamantane resistance, V27A and S31N.

Using polymeric materials to generate an amplified response to molecular recognition events.

Clinical and field-portable diagnostic devices require the detection of atto- to zeptomoles of biological molecules rapidly, easily and at low cost, with stringent requirements in terms of robustness and reliability. Though a number of creative approaches to this difficult problem have been reported, numerous unmet needs remain in the marketplace, particularly in resource-poor settings. Using rational materials design, we investigated harnessing the amplification inherent in a radical chain polymerization reaction to detect molecular recognition.

Evaluation of MChip with historic subtype H1N1 influenza A viruses, including the 1918 "Spanish Flu" strain.

The robustness of a recently developed diagnostic microarray for influenza, the MChip, was evaluated with 16 historic subtype H1N1 influenza A viruses (A/H1N1), including A/Brevig Mission/1/1918. The matrix gene segments from all 16 viruses were successfully detected on the array. An artificial neural network trained with temporally related A/H1N1 viruses identified A/Brevig Mission/1/1918 as influenza virus A/H1N1 with 94% probability.

Vapor deposition method for sensitivity studies on engineered surface-enhanced Raman scattering-active substrates.

The design and optimization of a vapor-phase analyte deposition method for limit of detection (LOD) studies on engineered surface-enhanced Raman scattering (SERS)-active substrates is presented. The vapor deposition method was designed to overcome current challenges in quantitative analysis of lithographically produced SERS substrates that are relatively small (hundreds of square micrometers). A custom-built flow cell was used to deposit benzenethiol from the vapor phase onto SERS-active Ag thin films, as the control substrates, and nanoaperture arrays that were generated by electron-beam lithography.

Diagnostic microarray for influenza B viruses.

The importance of global influenza surveillance using simple and rapid diagnostics has been frequently highlighted. For influenza type B, the need exists for discrimination between the two currently circulating major lineages, represented by virus strains B/Victoria/2/87 and B/Yamagata/16/88, as only one of these lineages is represented in seasonal influenza vaccines. Here, the development and characterization of a low-density DNA microarray (designated BChip) designed to detect and identify the two influenza B lineages is presented.