Free PAPP-A: a Novel Marker in Acute Coronary Syndrome Patients

In recent years, one important objective of cardiovascular research has been to find new markers that would improve the risk stratification and diagnosis of patients presenting with symptoms of acute coronary syndrome (ACS). Pregnancy-associated plasma protein A (PAPP-A) is a large metalloproteinase involved in insulin-like growth factor signalling. It is expressed in various tissues and seems to be involved in many physiological and pathological processes, such as folliculogenesis, bone formation, wound healing, pregnancy and atherosclerosis. The aim of this thesis was to investigate PAPP-A in ACS patients. Circulating concentrations of PAPP-A had been previously shown to be elevated in ACS. In this study it was revealed that the form of PAPP-A causing this elevation was the free noncomplexed PAPP-A. Thus, the form of PAPP-A in the circulation of ACS patients differed from the complexed PAPP-A form abundantly present in the circulation during pregnancy. A point-of-care method based on time-resolved immunofluorometric assays was developed, which enabled the rapid detection of free PAPP-A. The method was found to perform well with serum and heparin plasma samples as well as with heparinized whole blood samples. With this method the concentrations of free PAPP-A in healthy individuals were shown to be negligible. When the clinical performance of the method was evaluated with serum samples from ACS patients, it was shown that the free PAPP-A concentration in the admission sample was an independent predictor of myocardial infarction and death. Moreover, as a prognostic marker, free PAPP-A was revealed to be superior to total PAPPA, i.e. the combination of free and complexed PAPP-A, which has been measured by the other groups in this field. As heparin products are widely used as medication in ACS patients, the effect of heparin products on free PAPP-A molecule and circulating concentrations were also investigated in this study. It was shown that intravenous administration of low molecular weight or unfractionated heparin elicits a rapid release of free PAPP-A into the circulation in haemodialysis patients and patients undergoing angiography. Moreover, the interaction between PAPP-A and heparin was confirmed in gel filtration studies. Importantly, the patients included in the clinical evaluation of the free PAPP-A detection method developed had not received any heparin product medication before the admission sample and thus the results were not affected by the heparin effect. In conclusion, free PAPP-A was identified as a novel marker associated with ACS. The point-of-care methods developed enable rapid detection of this molecule which predicts adverse outcome when measured in the admission sample of ACS patients. However, the effect revealed of heparin products on circulating PAPP-A concentrations should be acknowledged when further studies are conducted related to free or total PAPP-A in ACS.Siirretty Doriast

Novel sensitive cardiac troponin I immunoassay free from troponin I-specific autoantibody interference

BACKGROUND\nCardiac troponins (cTnI and cTnT) are the recommended biomarkers of myocardial infarction. As cTn-specific autoantibodies (cTnAAb) can interfere with the cTn detection by state-of-the-art cTnI assays, our objective was to develop a sensitive cTnI immunoassay free from this analytical interference.\nMETHODS\nThe assay used antibody-coated spots containing three capture Mabs/Fabs directed against the N-terminus, midfragment and C-terminus of cTnI and a europium chelate-labeled tracer Mab against the C-terminus. Following a 3-h sample incubation and washing, cTnI was quantified by time-resolved fluorometry.\nRESULTS\nThe limit of detection (LoD) was 2.9 ng/L and the assay was linear up to 50,000 ng/L. The total precision of 10% CV was not reached, but 20% CV was reached at 10 ng/L. Mean cTnI (10-50,000 ng/L) recoveries were 100% and 119% in three cTnAAb-positive and two cTnAAb-negative individuals, respectively, verifying the interference resistance of the antibody design used. On average, Architect hs-cTnI assay gave seven-fold higher cTnI concentrations than the new assay but the correlation between the assays was good (r=0.958). Of apparently healthy individuals (n=159), 18% had measurable cTnI values (&gt;LoD) and 10% were cTnAAb-positive. The proportion of measurable cTnI values, however, was significantly higher in cTnAAb-positive individuals (13/16, median cTnI 8.5 ng/L) than in cTnAAb-negative individuals (15/143, median cTnI</p

Sensitive and quantitative detection of Cardiac Troponin I with upconverting nanoparticle lateral flow test with minimized interference

Measurement of cardiac troponin I (cTnI) should be feasible for point-of-care testing (POCT) to diagnose acute myocardial infarction (AMI). Lateral flow immunoassays (LFIAs) have been long implemented in POCT and clinical settings. However, sensitivity, matrix effect and quantitation in lateral flow immunoassays (LFIAs) have been major limiting factors. The performance of LFIAs can be improved with upconverting nanoparticle (UCNP) reporters. Here we report a new methodological approach to quantify cTnI using UCNP-LFIA technology with minimized plasma interference. The performance of the developed UCNP-LFIA was evaluated using clinical plasma samples (n = 262). The developed UCNP-LFIA was compared to two reference assays, the Siemens Advia Centaur assay and an in-house well-based cTnI assay. By introducing an anti-IgM scrub line and dried EDTA in the LFIA strip, the detection of cTnI in plasma samples was fully recovered. The UCNP-LFIA was able to quantify cTnI concentrations in patient samples within the range of 30–10,000 ng/L. The LoB and LoD of the UCNP-LFIA were 8.4 ng/L and 30 ng/L. The method comparisons showed good correlation (Spearman’s correlation 0.956 and 0.949, p </p

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumorassociated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145-and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa-cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs

Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum

Background Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. Aim Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. Methods and results The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3-doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. Conclusions These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.</p

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2–10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs

Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Abstract Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9–9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays

Integrated acoustic sample preparation for rapid sepsis diagnostics

We present a novel, integrated microfluidic system based on acoustophoretic cell-handling that detects bacteria in whole blood in less than 2 hours, substantially faster than current standard methods for detection of sepsis-causing bacteria. Bacteria are acoustically separated from 1 ml blood, enriched by acoustic trapping and finally detected using a dry-reagent PCR-chip

Sensitive and quantitative detection of cardiac troponin I with upconverting nanoparticle lateral flow test with minimized interference

Abstract Measurement of cardiac troponin I (cTnI) should be feasible for point-of-care testing (POCT) to diagnose acute myocardial infarction (AMI). Lateral flow immunoassays (LFIAs) have been long implemented in POCT and clinical settings. However, sensitivity, matrix effect and quantitation in lateral flow immunoassays (LFIAs) have been major limiting factors. The performance of LFIAs can be improved with upconverting nanoparticle (UCNP) reporters. Here we report a new methodological approach to quantify cTnI using UCNP-LFIA technology with minimized plasma interference. The performance of the developed UCNP-LFIA was evaluated using clinical plasma samples (n = 262). The developed UCNP-LFIA was compared to two reference assays, the Siemens Advia Centaur assay and an in-house well-based cTnI assay. By introducing an anti-IgM scrub line and dried EDTA in the LFIA strip, the detection of cTnI in plasma samples was fully recovered. The UCNP-LFIA was able to quantify cTnI concentrations in patient samples within the range of 30–10,000 ng/L. The LoB and LoD of the UCNP-LFIA were 8.4 ng/L and 30 ng/L. The method comparisons showed good correlation (Spearman’s correlation 0.956 and 0.949, p &lt; 0.0001). The developed UCNP-LFIA had LoD suitable for ruling in AMI in patients with elevated cTnI levels and was able to quantify cTnI concentrations in patient samples. The technology has potential to provide simple and rapid assay for POCT in ED setting