Evaluation of an Electrochemiluminescent SARS-CoV-2 Antibody Assay.

BACKGROUND: Little is known about the performance of the Roche novel severe acute respiratory syndrome coronavirus 2 antibody (anti-SARS-CoV-2) assay. We provide an extensive evaluation of this fully automated assay on Cobas e801/e602 immunoassay analyzers. METHODS: We assessed the linearity, precision, and throughput of the Roche anti-SARS-CoV-2 assay. Sensitivity was calculated from 349 SARS-CoV-2 polymerase chain reaction (PCR) positive samples; specificity was determined from 715 coronavirus disease 2019 (COVID-19)-naive samples. We examined cross-reactivity against other antibody positive samples [syphilis, rheumatoid factor (RF), antinuclear antibody (ANA), double-stranded DNA (ds-DNA), influenza, dengue, hepatitis B (HBV), hepatitis C (HCV)] and the anti-SARS-CoV-2 kinetics. RESULTS: The assay cut-off index (COI) was linear up to 90.8. The interassay precision was 2.9% for a negative control (COI = 0.1) and 5.1% for a positive control (COI = 3.0). Assay time is 18 min and results are available 1 min later; throughput for 300 samples was 76 min. Only 1 case positive for HBsAg tested falsely positive; specificity was 99.9%. The assay has a sensitivity of 97.1% 14 days after PCR positivity (POS) and 100% at ≥21 days POS; 48.2% of cases had anti-SARS-CoV-2 within 6 days POS. In 11 patients in whom serum was available prior to a positive antibody signal (COI ≥1.0) the interval between the last negative and first positive COI (time to "seroconversion") on average is 3 days (range 1-6 days) and 4 more days (range 1-7) for the anti-SARS-CoV-2 to plateau. CONCLUSION: The Roche anti-SARS-CoV-2 assay shows excellent performance with minimal cross-reactivity from other viral and confounding antibodies. Antibody development and seroconversion appears quite early.

Impact of the Sofia® Influenza A+B FIA rapid diagnostic test in a pediatric emergency department.

OBJECTIVE: The objective of this study was to evaluate the impact of a rapid diagnostic test for influenza (the Sofia® Influenza A+B FIA rapid diagnostic test [RDT]) in a pediatric emergency department (PED). METHODS: A retrospective, observational, cross-sectional study was conducted in the PED of the Lille University Hospital between 2013 and 2015. All patients under 18 years of age for whom influenza RDT was administered were included. Clinical data, management, and related hospitalizations were compared between positive and negative RDT groups. The length of stay in the PED (main outcome) and the number of additional tests (biological and radiographic tests) between the two groups were compared. RESULTS: A total of 238 tests were reported: 119 positive, 110 negative, nine invalid. The mean length of stay in the PED was significantly lower in the positive RDT group: 4.0h vs. 7.4h (P<10-6). Patients with positive RDT had significantly fewer biological tests (20% vs. 56%; P<10-7) and radiographs (23% vs. 52%; P<10-5). The prevalence of hospitalizations in a short-stay unit was significantly lower in patients with positive RDT (0.8% vs. 9.1%; P=0.009). CONCLUSIONS: This study showed a significant medical impact of the use of Sofia® Influenza RDT A+B FIA in a PED regarding the length of stay and the number of additional explorations.

Performance of a time-resolved fluorescence immunoassay for measuring varicella-zoster virus immunoglobulin G levels in adults and comparison with commercial enzyme immunoassays and Merck glycoprotein enzyme immunoassay.

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR).

The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine. Synthetic peptides (residues 84-92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3(1-277)) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84-88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3(93-274). The assay is linear at 0-30 ng/ml, with an intra-assay CV of 10% (n=20), inter-assay CV of 15% (n=9) and a recovery of D2D3(84-274) added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3(84-274) was found in two groups of tumor patients versus healthy volunteers (p

Measurement of vasoactive intestinal peptide using a competitive fluorescent microsphere immunoassay or ELISA in human blood samples.

The concentration of Vasoactive Intestinal Peptide (VIP) as measured by recycling immunoaffinity chromatography (RIC) has been reported to be elevated in the blood of patients with autism as compared with normal subjects. In this study, we have developed a "Competitive Fluorescent Microsphere Immunoassay" (cFMI) in which VIP competes with biotinylated VIP in binding to polyclonal antibodies on microspheres. The results were obtained using the Luminex100 system. We measured VIP in serum, plasma, and material eluted from dried blood spots on filter paper with both the cFMI and an ELISA procedure. We found that a purification procedure was necessary for obtaining useful results from plasma and serum, however, a preincubation step was required with the blood eluates. This newly developed cFMI was more sensitive (2.5 vs. 20.0 pg/ml), and more reproducible than the ELISA. To get accurate measurements of VIP in eluted material high sensitivity is especially important. Thus, the cFMI using the Luminex system has definite advantages over a conventional ELISA including the possibility that samples can be assayed at higher dilutions. We have determined that the VIP concentrations of serum, plasma, and dried blood spot eluate specimens as measured with the cFMI assay system were similar to those measured with ELISA. Thus, the new cFMI using Luminex system may be useful for detection of VIP in human blood samples.

A sensitive time-resolved fluorescent immunoassay for metallothionein protein.

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure MT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of anti-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.

Highly sensitive determination of plasma cytokines by time-resolved fluoroimmunoassay; effect of bicycle exercise on plasma level of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma).

A highly sensitive time-resolved fluoroimmunoassay of human plasma cytokines is described. The cytokines such as interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) are known to be acute inflammatory cytokines and it has been reported that these cytokines are secreted into blood by physical exercise. In this study, a sandwich-type immunoassay of cytokines was established using a europium chelate BHHCT-Eu3+ as a powerful labeling material. The minimum detection limits of cytokines, i.e. IL-1 alpha, TNF alpha, and interferon gamma (IFN gamma) were about 1/10 smaller than those of enzyme-linked immunosorbent assay currently used. By this immunoassay we investigated cytokine increase/decrease in plasma which was thought to derive from the myocytes damaged by bicycle exercise. Healthy young men performed two kinds of bicycle ergometer exercises, under conditions of an incremental and a constant loading. Blood samples were taken before, during, and after exercises, and the concentration levels of plasma IL-1 alpha, TNF alpha, and IFN gamma were determined. In the case of incremental exercise, IL-1 alpha increased significantly at the first stage but decreased to the basal level from the second stage, in spite of heavier exercise. In the case of 30 min constant exercise, the level of plasma IFN gamma increased in recovery period, 2 h after the light-exercise. TNF alpha level was significantly higher in a heavy-exercise. The concentration of IL-1 alpha peaked at the early stage of the incremental exercise; this fact has not been reported in previous studies. This cytokine is unique in showing a sudden increase during the early stage, while others increase after the exercise. Our highly sensitive assay made it possible to detect a slight change in plasma cytokines.

Continuous-flow fluoro-immunosensor for paclitaxel measurement.

A fluorescence-based continuous-flow immunosensor for sensitive, precise, accurate and fast determination of paclitaxel was developed. The sensor utilizes anti-paclitaxel antibody immobilized through its Fc region and crosslinked by dimethylpimelimidate to protein A attached covalently onto the silanized inner walls of a glass capillary column followed by saturation of the paclitaxel-binding sites with rhodamine-labeled paclitaxel. The assay is based on the displacement and detection downstream of the rhodamine-labeled paclitaxel, by a flow-through spectrofluorometer, as a result of the competition with paclitaxel introduced as a pulse into the stream of carrier buffer flowing through the system. The peak height of the fluorescence intensity profile of the displaced rhodamine-labeled paclitaxel was directly proportional to the concentration of paclitaxel applied and was a function of the carrier buffer flow rate. The sensitivity of the immunosensor response ranged from 0.31 relative fluorescence units (RFU)/ng/ml at a flow rate 0.1 ml/min to 0.52 RFU/ng/ml at 1 ml/min, while the lower detection limit ranged from 1 ng/ml at 0.1 ml/min to 4 ng/ml at 1 ml/min. The immunosensor response was very reproducible (RSD=4.8%; n=10) and linear up to 100 ng/ml. The assay time ranged from 2 min at 1 ml/min to 8 min at 0.1 ml/min. A technique developed to resaturate the antigen binding sites of the immobilized antibody with rhodamine-labeled paclitaxel was successful in regenerating the capillary column without affecting its performance, thus enhancing the economic viability of the immunosensor. The immunosensor was successfully applied for the determination of paclitaxel in human plasma.

Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence.

Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by beta-diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-specific binding of the streptavidin-coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to the conventional europium-labelled streptavidin tracer in the same assay format. Additionally, the streptavidin-coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin-biotin complex and a high number of binding sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated nanoparticles reacting with the surface-captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto- and histochemistry, multianalyte DNA-chip assays and single-particle assays.

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay.

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection and quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate. The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this time be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay.