Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms.

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin-/PCR+, GDH+/toxin-/PCR-, and GDH-/toxin- samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.

Ultrasensitive quantification of cardiac troponin I by a Single Molecule Counting method: analytical validation and biological features.

AIM: To evaluate analytical and biological characteristics of the Singulex Clarity® cTnI assay, based upon Single Molecule Counting technology. METHODS: Assay's analytical sensitivity, precision, linearity, hook effect, cross-reactivity or interference by endogenous and exogenous substances, stability, 99th reference percentile [p99th] in EDTA plasma were evaluated in single or multi-site studies. RESULTS: Detection limit was 0.12 ng/L. Sensitivity was 0.14 ng/L at 20% CV (functional sensitivity) and 0.53 ng/L at 10% CV. Imprecision was 3.16%-10.0% in a multi-lot, single-site study, and 5.5%-12.0% in a single-lot, multi-site study; assay was linear from 0.08 to 25,000 ng/L. No hook effect was observed; any cross-reactivity/interference exceeded the 10%. Healthy subjects were recruited using clinical history, normal NT-proBNP and eGFR (n = 560) or plasma creatinine (n = 535) as inclusion criteria. cTnI was detectable in 96.8% of healthy subjects. The p99th were 8.01 (eGFR used) and 8.15 ng/L (plasma creatinine); both were measured with ≤5.7% CV. Median cTnI were significantly higher in older and male than in young and female subjects. CONCLUSIONS: The Singulex Clarity cTnI assay show analytical features and % detection in healthy subjects that improve the corresponding values of most of the existing high-sensitivity cTnI assays.

Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology.

Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

Immunomagnetic quantitative immuno-PCR for detection of less than one HIV-1 virion.

Methods that allow the accurate and reliable detection of ultra-low molecular levels of proteins using techniques such as quantitative immuno-PCR (qIPCR) have demonstrated numerous technical difficulties. Protein detection methods lose specificity when the protein target is immersed within a matrix of thousands of molecules having wide ranges of concentrations. In addition, sensitivities are limited because of high background signals. To validate the performance of an immunomagnetic bead qIPCR method designed to remove the 'matrix' effect for HIV-1 p24 antigen detection, regression analyses were performed using samples from patients infected with HIV-1 diluted to approximately 100-1000, 10-100, 1-10, and 0.1-1.0 HIV-1 p24 Ag molecules/reaction. The number of HIV-1 p24 Ag molecules was derived from quantified HIV-1 RNA determinations. The modified immunomagnetic qIPCR bead assay demonstrated a limit of quantification of 10-100 HIV-1 p24 molecules per reaction, with an average correlation coefficient of 0.948+/-0.028 over a 4-log dynamic range. This method detects less than one HIV-1 virion (a limit of detection unreported previously for HIV-1), and thus, has the potential to identify HIV-1 infection and monitor the dynamics of the disease course earlier than nucleic acid methods. The immunomagnetic qIPCR bead assay is a simple and inexpensive method for ultra-low protein detection of infectious agents, toxins, and cancer markers at a level unrecognized previously using any enzymatic or molecular method.

Immuno-polymerase chain reaction as a unique molecular tool for detection of infectious agents.

Theoretically, the immuno-polymerase chain reaction (IPCR) method is the most sensitive technique for the detection of proteins and gains its uniqueness through the exponential amplification of a signal-generating nucleic acid intermediate attached to a protein target. This method is similar to PCR for the detection of nucleic acid targets, and has now been shown to offer the ability to detect infectious agents where nucleic acids are not present. Although the technical development of IPCR has taken a torturous path down a winding avenue of encouraging advances, the method remains rarely utilized by the scientific community and completely unused as a clinical diagnostic test approved by a national accrediting agency. Although the use of real-time instrumentation has enhanced the performance of IPCR to higher levels of statistical accuracy and reproducibility, as compared with the conventional method, its application remains limited by the high standards required for clinical diagnoses of infectious diseases. This review summarizes experimental data published to date describing the utilization of the IPCR method as it relates to the detection and diagnosis of human infectious disease, and examines the progressive development of this method, as well as the factors impeding its universal application as a clinical diagnostic tool. With further standardization and validation, the IPCR method has the potential to become the most analytically sensitive method available for the detection of target proteins of infectious diseases.

SOS-red fluorescent protein (RFP) bioassay system for monitoring of antigenotoxic activity in plant extracts.

An optical antigenotoxicity assay using genetically engineered red fluorescent bacteria is presented. Exposure of Escherichia coli RS4U to genotoxicants [mitomycin C (MMC), nalidixic acid (NA) and hydrogen peroxide (HP)] resulted in phenotypic red fluorescence proportional to the concentration of the inducer. Except for tannic acid (TA), co-treatment of the genotoxicant-activated bacteria with ascorbic acid (AA) and aqueous plant extracts (Mangifera indica, Psidium guajava and Syzygium cumini) afforded protection against all three genotoxicants. TA was effective in suppressing the genotoxic effect of MMC and HP. The antigenotoxic effect is seen as inhibition of the genotoxicant-triggered red fluorescence. The IC50 of the plant extracts and AA varied with the genotoxicant used. Rec assay verified the antigenotoxic activity of the plant extracts. Folin-Ciocalteu test, FeCl3 test and DPPH assay confirmed the presence of polyphenolic compounds and hydrolyzable tannins in the plant extracts and the antioxidant capacity of the plant samples.

Fatty acid sensor for low-cost lifetime-assisted ratiometric sensing using a fluorescent fatty acid binding protein.

Elevated free fatty acid (FA) levels lead to insulin resistance, hypertension, and microangiopathy, all of which are associated with type 2 diabetes. On the other hand, deficiencies of FA are indicative of certain neurodegenerative diseases, including autism. Thus, free FA levels are a diagnostic indicator for a variety of disorders. Here we describe the use of a commercially available FA binding protein labeled with acrylodan (ADIFAB), which we modified with a ruthenium metal-ligand complex with the intention of creating a low-cost FA sensor. The dual-labeled FA binding protein was used in lifetime-assisted ratiometric sensing (LARS) of oleic acid. For both steady-state and time-resolved luminescence decay experiments, the protein is responsive to oleic acid in the range of 0.02-4.7 microM. The emission at 432 nm, which is associated with the acrylodan occupying the FA binding site, decreases in intensity and red shifts to 505 nm on the addition of oleic acid. The intensities of the 505-nm peak due to the acrylodan displaced from the binding site by FA and of the 610-nm emission peak of ruthenium remained nearly unchanged. Fitting of the fluorescence decay data using the method of least squares revealed three emitting components with lifetimes of approximately 0.60, 4.00, and 370 ns. Fractional intensities of the emitting species indicate that changes in modulation between 2 and 10 MHz on binding of the protein with oleic acid are due mainly to the 4.00-ns component. The 0.60- and 370-ns components are assigned to acrylodan (505 nm) and ruthenium, respectively. Note that because ruthenium has a lifetime that is two orders of magnitude longer than that of acrylodan, the FA measurements were carried out at excitation frequencies lower than what can be done with acrylodan alone. Thus, low-cost instrumentation can be designed for a practical FA sensor without sacrificing the quality of measurements.