Michael J. Lochhead

Rapid Multiplexed Immunoassay for Simultaneous Serodiagnosis of HIV-1 and Coinfections

ABSTRACT Diagnosis of opportunistic infections in HIV-infected individuals remains a major public health challenge, particularly in resource-limited settings. Here, we describe a rapid diagnostic system that delivers a panel of serologic immunoassay results using a single drop of blood, serum, or plasma. The system consists of disposable cartridges and a simple reader instrument, based on an innovative implementation of planar waveguide imaging technology. The cartridge incorporates a microarray of recombinant antigens and antibody controls in a fluidic channel, providing multiple parallel fluorescence immunoassay results for a single sample. This study demonstrates system performance by delivering antibody (Ab) reactivity results simultaneously for multiple antigens of HIV-1, Treponema pallidum (syphilis), and hepatitis C virus (HCV) in a collection of clinical serum, plasma, and whole-blood samples. By plotting antibody reactivity (fluorescence intensity) for known positive and negative samples, empirical reactivity cutoff values were defined. The HIV-1 assay shows 100% agreement with known seroreactivity for a collection of 82 HIV Ab-positive and 142 HIV Ab-negative samples, including multiple samples with HCV and syphilis coinfection. The treponema-specific syphilis assay correctly identifies 67 of 68 T. pallidum Ab-positive and 100 of 102 T. pallidum Ab-negative samples, and the HCV assay correctly identifies 59 of 60 HCV Ab-positive and 120 of 121 HCV Ab-negative samples. Multiplexed assay performance for whole-blood samples is also demonstrated. The ability to diagnose HIV and opportunistic infections simultaneously at the point of care should lead to more effective therapy decisions and improved linkage to care.

Detection of the cyanobacterial toxin, microcystin-LR, using a novel recombinant antibody-based optical-planar waveguide platform

Microcystins are a major group of cyanobacterial heptapeptide toxins found in freshwater and brackish environments. There is currently an urgent requirement for highly-sensitive, rapid and in-expensive detection methodologies for these toxins. A novel single chain fragment variable (scFv) fragment was generated and is the first known report of a recombinant anti-microcystin avian antibody. In a surface plasmon resonance-based immunoassay, the antibody fragment displayed cross-reactivity with seven microcystin congeners (microcystin-leucine-arginine (MC-LR) 100%, microcystin-tyrosine-arginine (MC-YR) 79.7%, microcystin-leucine-alanine (MC-LA) 74.8%, microcystin-leucine-phenylalanine (MC-LF) 67.5%, microcystin-leucine-tryptophan (MC-LW) 63.7%, microcystin-arginine-arginine (MC-RR) 60.1% and nodularin (Nod) 69.3%, % cross reactivity). Following directed molecular evolution of the parental clone the resultant affinity-enhanced antibody fragment was applied in an optimized fluorescence immunoassay on a planar waveguide detection system. This novel immuno-sensing format can detect free microcystin-LR with a functional limit of detection of 0.19 ng mL(-1)and a detection range of 0.21-5.9 ng mL(-1). The assay is highly reproducible (displaying percentage coefficients of variance below 8% for intra-day assays and below 11% for inter-day assays), utilizes an inexpensive cartridge system with low reagent volumes and can be completed in less than twenty minutes.

Current Microarray Surface Chemistries

In almost all microarray technologies that are currently used, some type of surface chemistry serves as the interface between immobilized biomolecules and the solid support. Factors such as probe loading, spot morphology, and signal-to-noise ratio are all intimately linked to surface chemistry. Surface chemistry also significantly impacts important performance parameters such as three-dimensional structure of the immobilized biomolecules and nonspecific assay backgrounds. Here, an overview of the major types of surface chemistries currently used in printed microarrays is provided, with an emphasis on standard glass slide formats. The first part of this chapter focuses on DNA array surface chemistries, including both commercially fabricated and custom-made arrays. The second part of the chapter focuses on emerging protein, peptide, and carbohydrate array techniques. The intent is to provide the molecular biology researcher and bio-analytical or diagnostic specialist with a guide to the surface chemistry state-of-the-art for established and emerging array technologies.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCβ were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCβ and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L.

Development of a planar waveguide microarray for the monitoring and early detection of five harmful algal toxins in water and cultures.

A novel multiplex microarray has been developed for the detection of five groups of harmful algal and cyanobacterial toxins found in marine, brackish, and freshwater environments including domoic acid (DA), okadaic acid (OA, and analogues), saxitoxin (STX, and analogues), cylindrospermopsin (CYN) and microcystins (MC, and analogues). The sensitivity and specificity were determined and feasibility to be used as a screening tool investigated. Results for algal/cyanobacterial cultures (n = 12) and seawater samples (n = 33) were compared to conventional analytical methods, such as high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Detection limits for the 15 min assay were 0.37, 0.44, 0.05, 0.08, and 0.40 ng/mL for DA, OA, STX, CYN, and MC, respectively. The correlation of data obtained from the microarray compared to conventional analysis for the 12 cultures was r(2) = 0.83. Analysis of seawater samples showed that 82, 82, 70, 82, and 12% of samples were positive (>IC20) compared to 67, 55, 36, 0, and 0% for DA, OA, STX, CYN, and MC, respectively, for conventional analytical methods. The discrepancies in results can be attributed to the enhanced sensitivity and cross-reactivity profiles of the antibodies in the MBio microarray. The feasibility of the microarray as a rapid, easy to use, and highly sensitive screening tool has been illustrated for the five-plex detection of biotoxins. The research demonstrates an early warning screening assay to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying harmful toxins in water samples.

Performance evaluation of the MBio Diagnostics point-of-care CD4 counter.

The measurement of the absolute CD4 T-cell count is critical in the initial evaluation and staging of HIV-infected persons, yet access to this technology remains limited in many low resource settings where disease burden is highest. Here we evaluate the performance of a prototype point-of-care device (POC) to quantify CD4 T cells from MBio Diagnostics, Inc. Whole blood samples, both venous and capillary (finger stick), were collected from known HIV-infected participants at the University of California, San Diego Antiviral Research Center, and tested using the MBio system and conventional flow cytometry. A total of 94 venipuncture and 52 capillary samples were processed and statistical analyses included comparison to flow cytometry results. For the venipuncture samples, Bland-Altman analysis resulted in a mean bias of -10 cells/μL (-23 to +3 cells/μL, 95% CI), and limits of agreement (LOA) of -132 and +112 cells/μL. For the capillary samples, Bland-Altman resulted in a mean bias of -4 cells/μL (-31 to +23 cells/μL, 95% CL), and LOA of -195 and +186 cells/μL. For the San Diego study cohort, the prototype MBio system showed negligible quantitative bias relative to flow cytometry. Higher variability was observed in the capillary samples relative to venipuncture, but system precision for both capillary and venipuncture samples was good. There was also close agreement between results from the same participant when tested with two different systems, different operators and different locations. This preliminary evaluation suggests that the MBio CD4 device holds promise as a POC system for quantitation of CD4 T cells in limited-resource settings.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10 min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78 ng mL(-1) and the CCβ to be 1 ng mL(-1). Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R(2)=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1 ng mL(-1), and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1 ng mL(-1). This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1 μg L(-1). This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Use of biosensors for the detection of marine toxins.

Increasing occurrences of harmful algal blooms (HABs) in the ocean are a major concern for countries around the globe, and with strong links between HABs and climate change and eutrophication, the occurrences are only set to increase. Of particular concern with regard to HABs is the presence of toxin-producing algae. Six major marine biotoxin groups are associated with HABs. Ingestion of such toxins via contaminated shellfish, fish, or other potential vectors, can lead to intoxication syndromes with moderate to severe symptoms, including death in extreme cases. There are also major economic implications associated with the diverse effects of marine biotoxins and HABs. Thus, effective monitoring programmes are required to manage and mitigate their detrimental global effect. However, currently legislated detection methods are labour-intensive, expensive and relatively slow. The growing field of biosensor diagnostic devices is an exciting area that has the potential to produce robust, easy-to-use, cost-effective, rapid and accurate detection methods for marine biotoxins and HABs. This review discusses recently developed biosensor assays that target marine biotoxins and their microbial producers, both in harvested fish/shellfish samples and in the open ocean. The effective deployment of such biosensor platforms could address the pressing need for improved monitoring of HABs and marine biotoxins, and could help to reduce their global economic impact.

Low-cost fluorescence microscopy for point-of-care cell imaging

Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

Low-cost, multiplexed biosensor for disease diagnosis

Cost-effective disease diagnosis in resource-limited settings remains a critical global health challenge. Qualitative rapid tests based on lateral flow technology provide valuable screening information, but require relatively expensive confirmatory tests and generally lack quantitation. We report on a fluorescence technology that combines low cost instrumented readout with passive pumping in a disposable cartridge. The detection system utilizes a novel waveguide illumination approach in conjunction with commercial CMOS imagers. Total instrument cost in production are projected to be around