Analytical Performance Of Assays Research Articles

Design and Analytical Evaluation of a Rapid Plasma Screening Assay for Circulating Human Papillomavirus DNA via Thermostable Enzyme Chemistries.

Infection with oncogenic strains of human papillomavirus (HPV), such as HPV-16 and HPV-18, can lead to malignant progression and tumorigenesis. As an adjunct to traditional invasive tissue sampling methods, the use of modern thermostable enzyme chemistries can aid in the development of innovative assay workflows to extract and detect circulating HPV DNA (cHPV-DNA) in liquid biopsies. In this work, we first successfully generated a model system to replicate fragmented cHPV-DNA in human plasma. Using this model system, we designed a novel thermostable enzyme chemistry-based cHPV-DNA assay for rapid clinical HPV screening and robustly evaluated its analytical assay performance. Our findings demonstrated that the use of thermostable enzymes provided faster cHPV-DNA extraction and amplification, leading to an overall three-fold improvement in overall assay time as compared to the current standard assay workflow and achieving clinically relevant levels of analytical specificity, sensitivity, and precision for accurate cHPV-DNA detection with excellent 100% sensitivity and specificity in contrived human plasma specimens. In summary, we have devised a rapid laboratory workflow to facilitate the emerging use of liquid biopsies for minimally invasive, rapid, and scalable HPV DNA testing. With facile assay modifications, our thermostable enzyme-based cHPV-DNA assay can be utilized for the detection of other clinically high-risk HPV genotypes.

Development and analytical performance testing of a novel sTREM2 ELISA method to support drug development

AbstractBackgroundThe triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor of microglia cells in the central nerve system and is associated with neuroinflammation. sTREM2 can be used as pharmacodynamic biomarker for inflammation or microglial activation in therapeutic development programs. There is a need for well characterized assays that are suitable for measurement of neuroinflammatory biomarkers in pre‐clinical and clinical drug development. This abstract describes the characterization of a new robust sTREM2 prototype immunoassay based on monoclonal recombinant rabbit antibodies(mAbs).MethodA colorimetric sandwich ELISA was developed using capture mAb B02 (code ADx601) and detector mAb C10 (code ADx602) reactive towards the sTREM2 extracellular ectodomain (aa 19‐172). The analytical assay performance will be reported: the repeatability and reproducibility was determined using a neat CSF based QC panel (n=3; 6 test runs; duplicate testing). Accuracy was assessed by testing parallelism and calibrator point back calculated concentrations. Passing Bablok regression analysis was used to demonstrate consistency of sample testing results (43 samples; duplicate testing; 3 test runs). Finally clinical routine CSF samples were analysed (19 patients diagnosed with AD and 20 controls based upon biomarker profile).ResultA prototype sTREM2 ELISA assay was used to generate the assay characterization data: The %CV for repeatability respectively reproducibility were <12.2% and 15.4%. Accuracy: a slope value of 0.992 was obtained after Deming regression between the theoretical and experimentally obtained levels of 8 samples diluted over the measuring range (25‐3500 pg/mL) while the accuracy of the calibrator points was between 0 and 12.6% difference in back calculated concentration over 6 runs. Testing of the lot consistency (Figure 1) resulted in a slope and intercept value of respectively 1.07 (95%CI: 0.90‐1.17) and ‐80.9 (95%CI: ‐294.70‐154.30).Testing of clinical samples showed an increased sTREM2 concentration in AD versus controls (p<0.01).ConclusionA novel CSF prototype ELISA for sTREM2 was developed and characterized including lot consistency, precision, accuracy and others. The generated data indicate a robust sTREM2 immunoassay for use in drug development programs. The next step is the transfer to a manufacturing environment to make the assay available as a commercial test kit (RUO).

Analytical Sigma metrics: A review of Six Sigma implementation tools for medical laboratories.

Sigma metrics have become a useful tool for all parts of the quality control (QC) design process. Through the allowable total error model of laboratory testing, analytical assay performance can be judged on the Six Sigma scale. This not only allows benchmarking the performance of methods and instruments on a universal scale, it allows laboratories to easily visualize performance, optimize the QC rules and numbers of control measurements they implement, and now even schedule the frequency of running those controls.

Abstract 728: Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay

Abstract Introduction: Colorectal cancer is a highly preventable disease as early detection increases rates of patient survival to near 100%. Herein we report the development and validation of a novel multigene mutation biomarker real-time PCR based assay for qualitative detection of colorectal cancer associated biomarkers that comprise tumor specific mutations in the following genes: APC (Exon 15), KRAS (Exon 2), BRAF (Exon 15) and CTNNB1 (Exon 3) called ColoScapeTM . The assay allows the sensitive detection of the presence or absence of mutations in the targeted regions of the genes interrogated. Methodology: The high sensitivity of this multigene biomarker assay is achieved due to xenonucleic acid (XNA) probe technology. XNA probes are novel backbone modified oligomers having natural nucleoside bases (A,T,C and G) that hybridize by Watson-Crick base pairing to natural DNA and RNA with much higher binding affinity than natural deoxyribonucleic acid oligomers of the same sequence. XNA probes are designed that bind to the selected wild-type sequences at the respective genetic loci in the target genes. These XNA probes have a much higher Tm than the primer annealing temperature and suppress amplification of WT DNA templates and only allow amplification of the target mutant DNA templates in the sample. Both single nucleotide polymorphisms (SNP’s) and insertion/deletions (indels) mutations can be detected. For each of the selected mutation sites primers and FAM-labeled TaqMan hydrolysis probes were designed and tested together with the selected XNA oligomers and analytical assay performance confirmed. An internal PCR control amplicon selected in the Human β-Actin (ACTB) gene was employed utilizing a HEX-labeled TaqMan probe. Performance parameters of the assay were established on DNA of colorectal cancer patients extracted from FFPE as well as reference DNA materials (synthetic and cell line derived DNA). The assay demonstrates high sensitivity and specificity in detection of colon cancer and adenoma samples based on the set of biomarkers involved in colorectal cancer neogenesis and disease progression. Conclusion: The ColoScapeTM Colorectal Cancer Mutation Detection assay is shown to be a sensitive tool intended to facilitate research in colon cancer development, early detection, disease monitoring and therapeutic interventions. Citation Format: Michael J. Powell, Elena Peletskaya, Anne Vallerga, Qing Sun, Larry Pastor, Aiguo Zhang. Development and validation of ColoScape™ - a new colorectal cancer mutation detection assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2017-728

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.

What Criteria Should Be Used to Assess Troponin Assays?

Since the introduction of cardiac troponin testing in the 1990s, troponin measurement has become the main diagnostic criterion for the diagnosis of myocardial infarction. Multiple improvements have been made in the available commercial assays, with lower detection limits, improved imprecision at low concentrations, and improvements in standardization. Because troponin has been shown to be a powerful predictor of death and requirement for revascularization in patients presenting with the acute coronary syndrome, the analytical performance of assays is very important. Frequently, clinical decisions are based on increased troponin concentrations, and the performance of different assays is difficult to assess objectively. To allow better evaluation for the clinical and laboratory community, Apple suggested a scorecard for cardiac troponin assays (1), with the value of the troponin assay being based on 2 criteria. The main criterion is the imprecision of the assay at the 99th percentile of a reference population. A good assay was defined as one with a 10% CV at the population’s 99th percentile, a clinically usable assay was defined as one with a CV between 10% and 20% at that percentile, and a CV >20% at this …

Adapting Homogeneous Enzyme-Linked Competitive Binding Assays to Microtiter Plates

Recently devised homogeneous enzyme-linked binding assays useful for the rapid detection of carbohydrate structure/content of intact glycoproteins (via use of lectins as binders) and for quantitating given vitamins (e.g., biotin; using soluble binding proteins) are adapted successfully to a microtiter plate reader format. The problem of nonspecific adsorption of the binders and enzyme-saccharide/vitamin conjugates is solved via the addition of Tween 20 to the assay buffer. More convenient and reliable photometric detection of the preferred labeling enzyme, glucose-6-phosphate dehydrogenase (G6PDH), is accomplished by monitoring the rate of generation of reduced thio-NAD (from thio-NAD) at 405 nm instead of NADH (from NAD) at 340 nm. By employing these modifications it is shown that homogeneous enzyme-linked binding assays can be readily adapted to microtiter plates without loss in analytical assay performance. Results further suggest that other homogeneous assays based on G6PDH, including commercial EMIT assays used routinely in clinical chemistry laboratories for detecting drugs of abuse, could, in principle, be run on microtiter plates to significantly enhance sample throughput.