Diagnostic Performance of Tuberculosis-Specific IgG Antibody Profiles in Patients with Presumptive Tuberculosis from Two Continents.

BACKGROUND: Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. METHODS: Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. RESULTS: Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. CONCLUSIONS: Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries.

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.

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 CCß 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.

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 CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß 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.

Rapid multiplexed immunoassay for simultaneous serodiagnosis of HIV-1 and coinfections.

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.

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.

A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion.

This paper describes a new bioassay surface chemistry that effectively inhibits non-specific biomolecular and cell binding interactions, while providing a capacity for specific immobilization of desired biomolecules. Poly(ethylene glycol) (PEG) as the primary component in nonfouling film chemistry is well-established, but the multicomponent formulation described here is unique in that it (1) is applied in a single, reproducible, solution-based coating step; (2) can be applied to diverse substrate materials without the use of special primers; and (3) is readily functionalized to provide specific attachment chemistries. Surface analysis data are presented, detailing surface roughness, polymer film thickness, and film chemistry. Protein non-specific binding assays demonstrate significant inhibition of serum, fibrinogen, and lysozyme adsorption to coated glass, indium tin oxide, and tissue culture polystyrene dishes. Inhibition of S. aureus and K. pneumoniae microbial adhesion in a microfluidic flow cell, and inhibition of fibroblast cell adhesion from serum-based cell culture is shown. Effective functionalization of the coating is demonstrated by directing fibroblast adhesion to polymer surfaces activated with an RGD peptide. Batch-to-batch reproducibility data are included. The in situ cross-linked PEG-based coating chemistry is unique in its formulation, and its surface properties are attractive for a broad range of in vitro bioassay applications.

High-sensitivity detection of DNA hybridization on microarrays using resonance light scattering.

The application of resonance light scattering (RLS) particles for high-sensitivity detection of DNA hybridization on cDNA microarrays is demonstrated. Arrays composed of approximately 2000 human genes ("targets") were hybridized with colabeled (Cy3 and biotin) human lung cDNA probes at concentrations ranging from 8.3 ng/microL to 16.7 pg/microL. After hybridization, the arrays were imaged using a fluorescence scanner. The arrays were then treated with 80-nm-diameter gold RLS Particles coated with anti-biotin antibodies and imaged in a white light, CCD-based imaging system. At low probe concentrations, significantly more genes were detected by RLS compared to labeling by Cy3. For example, for hybridizations with a probe concentration of 83.3 pg/microL, approximately 1150 positive genes were detected using RLS compared to approximately 110 positive genes detected with Cy3. In a differential gene expression experiment using human lung and leukemia RNA samples, similar differential expression profiles were obtained for labeling by RLS and fluorescence technologies. The use of RLS Particles is particularly attractive for detection and identification of low-abundance mRNAs and for those applications in which the amount of sample is limited.