Evaluation of the time resolved fluorescence immunoassay (TRFIA) for the detection of varicella zoster virus (VZV) antibodies following vaccination of healthcare workers

Determination of varicella zoster virus (VZV) immunity in healthcare workers without a history of chickenpox is important for identifying those in need of vOka vaccination. Post immunisation, healthcare workers in the UK who work with high risk patients are tested for seroconversion. To assess the performance of the time-resolved fluorescence immunoassay (TRFIA) for the detection of antibody in vaccinated as well as unvaccinated individuals, a cut-off was first calculated. VZV-IgG specific avidity and titres six weeks after the first dose of vaccine were used to identify subjects with pre-existing immunity among a cohort of 110 healthcare workers. Those with high avidity (≥60%) were considered to have previous immunity to VZV and those with low or equivocal avidity (<60%) were considered naive. The former had antibody levels ≥400mIU/mL and latter had levels <400mIU/mL. Comparison of the baseline values of the naive and immune groups allowed the estimation of a TRFIA cut-off value of >130mIU/mL which best discriminated between the two groups and this was confirmed by ROC analysis. Using this value, the sensitivity and specificity of TRFIA cut-off were 90% (95% CI 79-96), and 78% (95% CI 61-90) respectively in this population. A subset of samples tested by the gold standard Fluorescence Antibody to Membrane Antigen (FAMA) test showed 84% (54/64) agreement with TRFIA.

Immunoassays, methods for carrying out immunoassays, immunoassay kits and method for manufacturing immunoassay kits

The invention relates to immunoassays, methods for carrying out immunoassays, immunoassay kits and methods for manufacturing immunoassay kits. In particular, the invention has relevance to capillary (especially microcapillary) immunoassay technology.

Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase

Background: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. Objectives: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. Methods: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. Results: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. Conclusions: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.

Tachykinins regulate the function of platelets

Evidence has been mounting for peripheral functions for tachykinins, a family of neuropeptides including substance P (SP), neurokinin A, and neurokinin B, which are recognized for their roles in the central and peripheral nervous system. The recent discovery of 4 new members of this family, the endokinins (EKA, B, C, and 13), which are distributed peripherally, adds support to the notion that tachykinins have physiologic/endocrine roles in the periphery. In the present study we report a fundamental new function for tachykinins in the regulation of platelet function. We show that SP stimulates platelet aggregation, and underlying this is the intracellular mobilization of calcium and degranulation. We demonstrate the presence of the tachykinin receptors NK1 and NK3 in platelets and present evidence for the involvement of NK1 in SP-mediated platelet aggregation. Platelets were found to contain SP-like immunoreactivity that is secreted upon activation implicating SP-like substances in the autocrine/paracrine regulation of these cells. Indeed, NK1-blocking antibodies inhibited aggregation in response to other agonists. Of particular note is the observation that EKA/B cross-react in the SP immunoassay and are also able to stimulate platelet activation. Together our data implicate tachykinins, specifically SP and EKA/B, in the regulation of platelet function. (C) 2004 by The American Society of Hematology.

Multimetallic ruthenium(II) complexes as electrochemiluminescent labels

Two homometallic complexes containing two and three ruthenium polypyridyl units linked by amino acid lysine (Lys) and the related dipeptide (LysLys) were synthesized and their electrochemical, spectroscopic, and electrochemiluminescence (ECL) properties were investigated. The electrochemical and photophysical data indicate that the two metal complexes largely retain the electronic properties of the reference compound for the separate ruthenium moieties in the two bridged complexes, [4-carboxypropyl-4'-methyl-2,2'-bipyridine]bis(2,2'-bipyridine)ruthenium(II) complex. The ECL studies, performed in aqueous media in the presence of tri-n-propylamine as co-reactant, show that the ECL intensity increases by 30% for the dinuclear and trinuclear complexes compared to the reference. Heterogeneous ECL immunoassay studies, performed on larger dendritic complexes containing up to eight ruthenium units, demonstrate that limitations due to the slow diffusion can easily be overcome by means of nanoparticle technology. In this case, the ECL signal is proportional to the number of ruthenium units. Multimetallic systems with several ruthenium centers may, however, undergo nonspecific bonding,to streptavidin-coated particles or to antibodies, thereby increasing the background ECL intensity and lowering the sensitivity of the immunoassay.

Analysis of aluminium content and iron homeostasis in nipple aspirate fluids from healthy women and breast cancer-affected patients

Aluminium is not a physiological component of the breast but has been measured recently in human breast tissues and breast cyst fluids at levels above those found in blood serum or milk. Since the presence of aluminium can lead to iron dyshomeostasis, levels of aluminium and iron-binding proteins (ferritin, transferrin) were measured in nipple aspirate fluid (NAF), a fluid present in the breast duct tree and mirroring the breast microenvironment. NAFs were collected noninvasively from healthy women (NoCancer; n = 16) and breast cancer-affected women (Cancer; n = 19), and compared with levels in serum (n = 15) and milk (n = 45) from healthy subjects. The mean level of aluminium, measured by ICP-mass spectrometry, was significantly higher in Cancer NAF (268.4 ± 28.1 μg l−1; n = 19) than in NoCancer NAF (131.3 ± 9.6 μg l−1; n = 16; P < 0.0001). The mean level of ferritin, measured through immunoassay, was also found to be higher in Cancer NAF (280.0 ± 32.3 μg l−1) than in NoCancer NAF (55.5 ± 7.2 μg l−1), and furthermore, a positive correlation was found between levels of aluminium and ferritin in the Cancer NAF (correlation coefficient R = 0.94, P < 0.001). These results may suggest a role for raised levels of aluminium and modulation of proteins that regulate iron homeostasis as biomarkers for identification of women at higher risk of developing breast cancer. The reasons for the high levels of aluminium in NAF remain unknown but possibilities include either exposure to aluminium-based antiperspirant salts in the adjacent underarm area and/or preferential accumulation of aluminium by breast tissues.

A simple device for multiplex ELISA made from melt-extruded plastic microcapillary film

We present a simple device for multiplex quantitative enzyme-linked immunosorbant assays (ELISA) made from a novel melt-extruded microcapillary film (MCF) containing a parallel array of 200µm capillaries along its length. To make ELISA devices different protein antigens or antibodies were immobilised inside individual microcapillaries within long reels of MCF extruded from fluorinated ethylene propylene (FEP). Short pieces of coated film were cut and interfaced with a pipette, allowing sequential uptake of samples and detection solutions into all capillaries from a reagent well. As well as being simple to produce, these FEP MCF devices have excellent light transmittance allowing direct optical interrogation of the capillaries for simple signal quantification. Proof of concept experiments demonstrate both quantitative and multiplex assays in FEP MCF devices using a standard direct ELISA procedure and read using a flatbed scanner. This new multiplex immunoassay platform should find applications ranging from lab detection to point-of-care and field diagnostics.

Circulating insulin-like factor 3 (INSL3) in healthy and infertile women

STUDY QUESTION: How does insulin-like factor 3 (INSL3) concentration in blood vary across the menstrual cycle in women? SUMMARY ANSWER: INSL3 is secreted by the theca interna cells of growing antral follicles and is phasic in its expression. WHAT IS KNOWN ALREADY: The relaxin-like hormone INSL3 is known to be expressed in follicles of several mammal species, and was recently shown in cows to be specifically secreted into the bloodstream by growing antral follicles, corresponding to follicular waves. In males INSL3 is known to be acutely independent of the hormones of the hypothalamic-pituitary-gonadal axis, suggesting that in women INSL3 might be a novel biomarker for antral follicle recruitment and development. STUDY DESIGN, SIZE, DURATION: Two cohorts of women were studied. First, 18 healthy women of reproductive age were followed longitudinally for one and a half cycles, with blood sampling and hormone measurement every 2-3 days. A second cohort comprised a cross-sectional study of 909 women attending an infertility clinic, with a single blood sample taken at entry, together with other clinical and hormonal parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples from both retrospective cohorts were analyzed for INSL3 using a highly sensitive time-resolved fluorescent immunoassay, and data were analyzed in comparison with other clinical and hormonal parameters. MAIN RESULT AND THE ROLE OF CHANCE: For young healthy women of reproductive age, we showed a phasic expression of INSL3 corresponding to antral follicle growth in both the follicular and luteal phases of the cycle, which was significantly (P < 0.05) elevated compared with that during menses. For women attending an infertility clinic, those with diagnosed polycystic ovarian syndrome indicated significantly (P < 0.0005) greater circulating INSL3 levels and those with low ovarian reserve showed significantly (P < 0.002) decreased INSL3 values. LIMITATIONS, REASONS FOR CAUTION: These were retrospective studies and the results were obtained from natural cycles only, with their inherent variability. WIDER IMPLICATIONS OF THE FINDINGS: We show for the first time that INSL3 in women does vary across the menstrual cycle, and appears to reflect the number of growing antral follicles recruited within both follicular and luteal phases. STUDY FUNDING/COMPETING INTEREST(S): The present retrospective study was largely supported by departmental funds. There were no competing interests.

A Lab-in-a-briefcase for rapid prostate specific antigen (PSA) screening from whole blood

We present a new concept for rapid and fully portable Prostate Specific Antigen (PSA) measurement, termed “Lab-in-a-Briefcase”, which integrates an affordable microfluidic ELISA platform utilising a melt-extruded fluoropolymer Micro Capillary Film (MCF) containing 10 bore, 200 μm internal diameter capillaries, a disposable multi-syringe aspirator (MSA) plus a sample tray pre-loaded with all required immunoassay reagents, and a portable film scanner for colorimetric signal digital quantitation. Each MSA can perform 10 replicate microfluidic immunoassays on 8 samples, allowing 80measurements to be made in less than 15 minutes based on semi-automated operation and norequirement of additional fluid handling equipment. An assay was optimised for measurement of a clinically relevant range of PSA from 0.9 to 60.0 ng/ml in 15 minutes with CVs in the order of 5% based on intra-assay variability when read using a consumer flatbed film scanner. The PSA assay performance in the MSA remained robust in the presence of undiluted or 1:2 diluted human serum or whole blood, and the matrix effect could simply be overcome by extending sample incubation times. The PSA "Lab-in-a-briefcase" is particularly suited to a low-resource health setting where diagnostic labs and automated immunoassay systems are not accessible, by allowing PSA measurement outside the laboratory using affordable equipment.

Portable smartphone quantitation of prostate specific antigen (PSA) in a fluoropolymer microfluidic device

We present a new, power-free and flexible detection system named MCFphone for portable colorimetric and fluorescence quantitative sandwich immunoassay detection of prostate specific antigen (PSA). The MCFphone is composed by a smartphone integrated with a magnifying lens, a simple light source and a miniaturised immunoassay platform, the Microcapillary Film (MCF). The excellent transparency and flat geometry of fluoropolymer MCF allowed quantitation of PSA in the range 0.9 to 60 ng/ml with < 7 % precision in 13 minutes using enzymatic amplification and a chromogenic substrate. The lower limit of detection was further improved from 0.4 to 0.08 ng/ml in whole blood samples with the use of a fluorescence substrate. The MCFphone has shown capable of performing rapid (13 to 22 minutes total assay time) colorimetric quantitative and highly sensitive fluorescence tests with good %Recovery, which represents a major step in the integration of a new generation of inexpensive and portable microfluidic devices with commercial immunoassay reagents and off-the-shelf smartphone technology.

Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves.

Multiplexed femtomolar quantitation of human cytokines in a fluoropolymer microcapillary film

Sensitive quantitation of multiple cytokines can provide important diagnostic information during infection, inflammation and immunopathology. In this study sensitive immunoassay detection of human cytokines IL-1β, IL-6, IL-12p70 and TNFα is shown for singleplex and multiplex formats using a novel miniaturized ELISA platform. The platform uses a disposable plastic multi-syringe aspirator (MSA) integrating 8 disposable fluoropolymer microfluidic test strips, each containing an array of ten 200 mean i.d. microcapillaries coated with a set of monoclonal antibodies. Each MSA device thus performs 10 tests on 8 samples, delivering 80 measurements. Unprecedented levels of sensitivity were obtained with the novel fluoropolymer microfluidic material and simple colorimetric detection in a flatbed scanner. The limit of detection for singleplex detection ranged from 2.0 to 15.0 pg/ml, i.e. 35 and 713 femtomolar for singleplex cytokine detection, and the intra- and inter-assay coefficient of variation (CV) remained within 10%. In addition, a triplex immunoassay was developed for measuring IL-1β, IL-12p70 and TNFα simultaneously from a given sample in the pg/ml range. These assays permit high sensitivity measurement with rapid <15 min assay or detection from undiluted blood serum. The portability, speed and low-cost of this system are highly suited to point-of-care testing and field diagnostics applications.