Integrated Biomarkers for the Management of Indeterminate Pulmonary Nodules.

Rationale: Patients with indeterminate pulmonary nodules (IPNs) at risk of cancer undergo high rates of invasive, costly, and morbid procedures. Objectives: To train and externally validate a risk prediction model that combined clinical, blood, and imaging biomarkers to improve the noninvasive management of IPNs. Methods: In this prospectively collected, retrospective blinded evaluation study, probability of cancer was calculated for 456 patient nodules using the Mayo Clinic model, and patients were categorized into low-, intermediate-, and high-risk groups. A combined biomarker model (CBM) including clinical variables, serum high sensitivity CYFRA 21-1 level, and a radiomic signature was trained in cohort 1 (n = 170) and validated in cohorts 2-4 (total n = 286). All patients were pooled to recalibrate the model for clinical implementation. The clinical utility of the CBM compared with current clinical care was evaluated in 2 cohorts. Measurements and Main Results: The CBM provided improved diagnostic accuracy over the Mayo Clinic model with an improvement in area under the curve of 0.124 (95% bootstrap confidence interval, 0.091-0.156; P < 2 × 10-16). Applying 10% and 70% risk thresholds resulted in a bias-corrected clinical reclassification index for cases and control subjects of 0.15 and 0.12, respectively. A clinical utility analysis of patient medical records estimated that a CBM-guided strategy would have reduced invasive procedures from 62.9% to 50.6% in the intermediate-risk benign population and shortened the median time to diagnosis of cancer from 60 to 21 days in intermediate-risk cancers. Conclusions: Integration of clinical, blood, and image biomarkers improves noninvasive diagnosis of patients with IPNs, potentially reducing the rate of unnecessary invasive procedures while shortening the time to diagnosis.

Lysophosphatidic acid (LPA)-antibody (504B3) engagement detected by interferometry identifies off-target binding.

BACKGROUND: Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that acts through its six cognate G protein-coupled receptors. As a family, lysophospholipids have already produced medicines (e.g., sphingosine 1-phosphate) as is being pursued for LPA through the use of specific antibodies that reduce ligand availability. METHODS: The binding properties of a commercially available, reportedly specific, monoclonal LPA antibody named 504B3 that is related to the clinical candidate Lpathomab/LT3015 were reexamined using a free solution assay (FSA) measured in a compensated interferometric reader (CIR). RESULTS: Measurement of 504B3 binding properties with an FSA-CIR approach revealed similar binding affinities for 504B3 against LPA as well as the non-LPA lipids, phosphatidic acid (PA) and lysophosphatidylcholine (LPC). CONCLUSIONS: Antibody binding specificity and sensitivity, particularly involving lipid ligands, can be assessed in solution and without labels using FSA-CIR. These findings could affect interpretations of both current and past basic and clinical studies employing 504B3 and related anti-LPA antibodies.

Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader.

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

Lysophospholipid G protein-coupled receptor binding parameters as determined by backscattering interferometry.

Lysophosphatidic acid (LPA) activates cognate G protein-coupled receptors (GPCRs) to initiate biological signaling cascades. Lysophospholipid (LP) receptor binding properties remain incompletely assessed because of difficulties with ligand lipophilicity and lipid "stickiness." These inherent attributes produce high levels of nonspecific binding within cell-membrane preparations used to assess GPCRs, as has been shown in classical binding assays using radiolabeled ligands, making accurate measurements of lipid binding kinetics difficult to achieve. Backscattering interferometry (BSI) is an optical technology that measures molecular binding interactions by reporting changes in the refractive index of a solution after binding events. Here, we report the use of BSI to assess LPA1 for its ability to bind to naturally occurring lipids and a synthetic LPA1 antagonist (ONO-9780307), under both primary- and competition-binding conditions. Assessment of 12 different lipids demonstrated that the known LP ligand, 1-oleoyl-LPA, as well as an endocannabinoid metabolite, anandamide phosphate, are specific ligands for LPA1, whereas other LPs tested were not. Newly determined dissociation constants (Kd values) for orthosteric lipid ligands approximated 10-9 M, substantially lower (i.e., with higher affinity) than measured Kd values in classical binding or cell-based assays. These results demonstrate that BSI may have particular utility in assessing binding interactions between lipid receptors and their lipid ligands and could provide new screening approaches for lipid receptor identification and drug discovery.

Quantification of Opioids in Urine Using an Aptamer-Based Free-Solution Assay.

The opioid epidemic continues in the United States. Many have been impacted by this epidemic, including neonates who exhibit Neonatal Abstinence Syndrome (NAS). Opioid diagnosis and NAS can be negatively impacted by limited testing options outside the hospital, due to poor assay performance, false-negatives, rapid drug clearance rates, and difficulty in obtaining enough specimen for testing. Here we report a small volume urine assay for oxycodone, hydrocodone, fentanyl, noroxycodone, norhydrocodone, and norfentanyl with excellent LODs and LOQs. The free-solution assay (FSA), coupled with high affinity DNA aptamer probes and a compensated interferometric reader (CIR), represents a potential solution for quantifying opioids rapidly, at high sensitivity, and noninvasively on small sample volumes. The mix-and-read test is 5- to 275-fold and 50- to 1250-fold more sensitive than LC-MS/MS and immunoassays, respectively. Using FSA, oxycodone, hydrocodone, fentanyl, and their urinary metabolites were quantified using 10 µL of urine at 28-81 pg/mL, with >95% specificity and excellent accuracy in ∼1 h. The assay sensitivity, small sample size requirement, and speed could enable opioid screening, particularly for neonates, and points to the potential for pharmacokinetic tracking.

Origin and prediction of free-solution interaction studies performed label-free.

Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for free-solution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R(2)> 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.

Longitudinal pixel averaging for improved compensation in backscattering interferometry.

Longitudinal averaging of the interference pattern in a compensated backscattering interferometer provides improved compensation for temperature induced refractive index perturbations. Fringe pattern likeness between two discrete detection regions of an off-the-shelf microfluidic chip illuminated by an inexpensive diode laser scales with interrogation length. Averaging the intensity distribution along a 2.75 mm length of the channel results in a 750-fold reduction in sensitivity to temperature and a baseline noise level of 3×10-8 refractive index units (RIU). These observations enable nanoliter-volume interferometric measurements at a level of 10-7 RIU in the presence of a 2°C temperature variation without the need for temperature control.

Taylor Dispersion Analysis of Polysaccharides Using Backscattering Interferometry.

Taylor dispersion analysis (TDA) allows the determination of the molecular diffusion coefficient (D) or the hydrodynamic radius (Rh) of a solute from the peak broadening of a plug of solute in a laminar Poiseuille flow. The main limitation plaguing the broader applicability of TDA is the lack of a sensitive detection modality. UV absorption is typically used with TDA but is only suitable for UV-absorbing or derivatized compounds. In this work, we present a development of the TDA method for non-UV absorbing compounds by using a universal detector based on refractive index (RI) sensing with backscattering interferometry (BSI). BSI was interfaced to a capillary electrophoresis-UV instrument using a polyimide coated fused silica capillary and an in-house designed flow-cell assembly. Polysaccharides were selected to demonstrate the application of TDA-BSI for size characterization. Under the conditions of validity of TDA, D and Rh average values and the entire Rh distributions were obtained from the (poly)saccharide taylorgrams, including non-UV absorbing polymers.

Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry.

BACKGROUND: Invasion of host erythrocytes by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion involves recognition events between erythrocyte receptors and ligands on the merozoite, the invasive blood form of the parasite. Identifying and characterizing host-parasite interactions is impeded by the biochemical challenges of working with membrane-embedded glycoprotein receptors. For example, the interaction between P. falciparum erythrocyte binding antigen 175 (PfEBA175) and glycophorin A (GYPA) depends on post-translational modifications that are not easily added in recombinant expression systems, and the use of native GYPA is limited by the hydrophobic transmembrane region, making it difficult to biochemically manipulate. It would, therefore, be desirable to perform quantitative binding assays with receptors embedded within the membranes of intact human erythrocytes. METHODS: The extracellular region of GYPA was over-expressed as a soluble protein in HEK293E cells. This protein was characterized, sialylated and evaluated for binding to the PfEBA175 protein. The label-free and free-solution assay, backscattering interferometry (BSI), was used to perform binding assays of two well-characterized P. falciparum invasion ligands to intact unmodified human erythrocytes. RESULTS: Findings indicate that the post-translational modifications present on native GYPA are required for it to bind recombinant PfEBA175 and that these modifications cannot be recapitulated in vitro using mammalian overexpression methods. Here, BSI was used to obtain quantitative, high fidelity interaction determinations on intact, unmodified erythrocytes. Using BSI and purified recombinant proteins constituting the entire ectodomains of the P. falciparum merozoite ligands PfEBA175 and PfRH5, K Ds of 1.1 µM and 50 nM were measured for the PfRH5-BSG and PfEBA175-GYPA interactions, respectively, in good agreement with previous biophysical measurements of these interactions. CONCLUSIONS: These results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.

The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

Baclofen and other GABAB receptor agents are allosteric modulators of the CXCL12 chemokine receptor CXCR4.

CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca(2+) channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K(+) (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.

Toward rapid, high-sensitivity, volume-constrained biomarker quantification and validation using backscattering interferometry.

Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was <15% for BSI and <10% for ELISA. The two techniques correlated well, ranging from 3-29% difference for Cyfra 21-1 in a blinded patient sample analysis. The label-free and free-solution operation of BSI allowed for a significant improvement in analysis speed, with greater ease, improved LOQ values, and excellent day-to-day reproducibility. In this unoptimized format, BSI required 5.5-fold less sample quantity needed for ELISA (a 10 point calibration curve measured in triplicate required 36 µL of serum for BSI vs 200 µL for ELISA). The results indicate that the BSI platform can enable rapid, sensitive analytical validation of serum biomarkers and should significantly impact the validation bottleneck of biomarkers.

Characterizing aptamer small molecule interactions with backscattering interferometry.

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule-aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM-aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R(2) values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.

Synthesis and in vitro efficacy of MMP9-activated NanoDendrons.

Chemotherapeutics such as doxorubicin (DOX) and paclitaxel (PXL) have dose-limiting systemic toxicities, including cardiotoxicity and peripheral neuropathy. Delivery strategies to minimize these undesirable effects are needed and could improve efficacy, while reducing patient morbidity. Here, DOX and PXL were conjugated to a nanodendron (ND) through an MMP9-cleavable peptide linker, producing two new therapies, ND2(DOX) and ND2(PXL), designed to improve delivery specificity to the tumor microenvironment and reduce systemic toxicity. Comparative cytotoxicity assays were performed between intact ND-drug conjugates and the MMP9 released drug in cell lines with and without MMP9 expression. While ND2(DOX) was found to lose cytotoxicity due to the modification of DOX for conjugation to the ND; ND2(PXL) was determined to have the desired properties for a prodrug delivery system. ND2(PXL) was found to be cytotoxic in MMP9-expressing mouse mammary carcinoma (R221A-luc) (53%) and human breast carcinoma (MDA-MB-231) (66%) at a concentration of 50 nM (in PXL) after 48 h. Treating ND2(PXL) with MMP9 prior to the cytotoxicity assay resulted in a faster response; however, both cleaved and intact versions of the drug reached the same efficacy as the unmodified drug by 96 h in the R221A-luc and MDA-MB-231 cell lines. Further studies in modified Lewis lung carcinoma cells that either do (LLC(MMP9)) or do not (LLC(RSV)) express MMP9 demonstrate the selectivity of ND2(PXL) for MMP9. LLC(MMP9) cells were only 20% viable after 48 h of treatment, while LLC(RSV) were not affected. Inclusion of an MMP inhibitor, GM6001, when treating the LLC(MMP9) cells with ND2(PXL) eliminated the response of the MMP9 expressing cells (LLC(MMP9)). The data presented here suggests that these NDs, specifically ND2(PXL), are nontoxic until activated by MMP9, a protease common in the microenvironment of tumors, indicating that incorporation of chemotherapeutic or cytostatic agents onto the ND platform have potential for tumor-targeted efficacy with reduced in vivo systemic toxicities.

Assessing the clinical utility of biomarkers using the intervention probability curve (IPC).

BACKGROUND: Assessing the clinical utility of biomarkers is a critical step before clinical implementation. The reclassification of patients across clinically relevant subgroups is considered one of the best methods to estimate clinical utility. However, there are important limitations with this methodology. We recently proposed the intervention probability curve (IPC) which models the likelihood that a provider will choose an intervention as a continuous function of the probability, or risk, of disease. OBJECTIVE: To assess the potential impact of a new biomarker for lung cancer using the IPC. METHODS: The IPC derived from the National Lung Screening Trial was used to assess the potential clinical utility of a biomarker for suspected lung cancer. The summary statistics of the change in likelihood of intervention over the population can be interpreted as the expected clinical impact of the added biomarker. RESULTS: The IPC analysis of the novel biomarker estimated that 8% of the benign nodules could avoid an invasive procedure while the cancer nodules would largely remain unchanged (0.1%). We showed the benefits of this approach compared to traditional reclassification methods based on thresholds. CONCLUSIONS: The IPC methodology can be a valuable tool for assessing biomarkers prior to clinical implementation.

Improving lung cancer diagnosis with cancer, fungal, and imaging biomarkers.

OBJECTIVE: Indeterminate pulmonary nodules (IPNs) represent a significant diagnostic burden in health care. We aimed to compare a combination clinical prediction model (Mayo Clinic model), fungal (histoplasmosis serology), imaging (computed tomography [CT] radiomics), and cancer (high-sensitivity cytokeratin fraction 21; hsCYFRA 21-1) biomarker approach to a validated prediction model in diagnosing lung cancer. METHODS: A prospective specimen collection, retrospective blinded evaluation study was performed in 3 independent cohorts with 6- to 30-mm IPNs (n = 281). Serum histoplasmosis immunoglobulin G and immunoglobulin M antibodies and hsCYFRA 21-1 levels were measured and a validated CT radiomic score was calculated. Multivariable logistic regression models were estimated with Mayo Clinic model variables, histoplasmosis antibody levels, CT radiomic score, and hsCYFRA 21-1. Diagnostic performance of the combination model was compared with that of the Mayo Clinic model. Bias-corrected clinical net reclassification index (cNRI) was used to estimate the clinical utility of a combination biomarker approach. RESULTS: A total of 281 patients were included (111 from a histoplasmosis-endemic region). The combination biomarker model including the Mayo Clinic model score, histoplasmosis antibody levels, radiomics, and hsCYFRA 21-1 level showed improved diagnostic accuracy for IPNs compared with the Mayo Clinic model alone with an area under the receiver operating characteristics curve of 0.80 (95% CI, 0.76-0.84) versus 0.72 (95% CI, 0.66-0.78). Use of this combination model correctly reclassified intermediate risk IPNs into low- or high-risk category (cNRI benign = 0.11 and cNRI malignant = 0.16). CONCLUSIONS: The addition of cancer, fungal, and imaging biomarkers improves the diagnostic accuracy for IPNs. Integrating a combination biomarker approach into the diagnostic algorithm of IPNs might decrease unnecessary invasive testing of benign nodules and reduce time to diagnosis for cancer.

Comparison of free-solution and surface-immobilized molecular interactions using a single platform.

While it is generally accepted that surface immobilization affects the binding properties of proteins, it has been difficult to quantify these effects due to the lack of technology capable of making affinity measurements with species tethered and in free solution on a single platform. Further, quantifying the interaction of binding pairs with widely differing masses has also been challenging, particularly when it is desirable to tether the high molecular weight protein. Here we describe the use of backscattering interferometry (BSI) to quantify the binding affinity of mannose and glucose to concanavalin A (ConA), a 106 KDa homotetramer protein, in free solution using picomoles of the protein. Using the same platform, BSI, we then studied the effect on the binding constants of the ConA-carbohydrate interactions upon chemically immobilizing ConA on the sensor surface. By varying the distances (0, 7.17, and 20.35 nm) of the ConA tether and comparing these results to the free-solution measurements, it has been possible to quantify the effect that protein immobilization has on binding. Our results indicate that the apparent binding affinity of the sugar-lectin pair increases as the distance between ConA and the surface decreases. These observations could lend insight as to why the affinity values reported in the literature sometimes vary significantly from one measurement technique to another.

Back-scattering interferometry: an ultrasensitive method for the unperturbed detection of acetylcholinesterase-inhibitor interactions.

A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22,000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer's disease, by detecting dual-binding interactions. E = enzyme, I = inhibitor, S = substrate.

Assay Performance of a Label-Free, Solution-Phase CYFRA 21-1 Determination.

CYFRA 21.1, a cytokeratin fragment of epithelial origin, has long been a valuable blood-based biomarker. As with most biomarkers, the clinical diagnostic value of CYFRA 21.1 is dependent on the quantitative performance of the assay. Looking toward translation, it is shown here that a free-solution assay (FSA) coupled with a compensated interferometric reader (CIR) can be used to provide excellent analytical performance in quantifying CYFRA 21.1 in patient serum samples. This report focuses on the analytical performance of the high-sensitivity (hs)-CYFRA 21.1 assay in the context of quantifying the biomarker in two indeterminate pulmonary nodule (IPN) patient cohorts totaling 179 patients. Each of the ten assay calibrations consisted of 6 concentrations, each run as 7 replicates (e.g., 10 × 6 × 7 data points) and were performed on two different instruments by two different operators. Coefficients of variation (CVs) for the hs-CYFRA 21.1 analytical figures of merit, limit of quantification (LOQ) of ca. 60 pg/mL, B max, initial slope, probe-target binding affinity, and reproducibility of quantifying an unknown were found to range from 2.5 to 8.3%. Our results demonstrate the excellent performance of our FSA-CIR hs-CYFRA 21-1 assay and a proof of concept for potentially redefining the performance characteristics of this existing important candidate biomarker.