Non-Radiologic Assessment of Kidney Stones by KIT, a Spot Urine Assay.
To evaluate the utility of the KIT Assay urinary biomarkers to detect kidney stones and quantify stone burden.</AbstractText><AbstractText>136 spot urine samples from 98 individuals, with and without kidney stone disease, were processed for measuring a pre-defined assay consisting of 6 DNA and protein markers, to generate a risk score for non-invasive detection of nephrolithiasis. From this cohort, 56 individuals had spot, non-timed, urine samples collected at the time of radiographically confirmed kidney stones and 54 demographically matched, healthy controls, without kidney stone disease, also provided spot, non-timed urine samples. Sixteen individuals with persistent stone disease had more than one urine sample.
Using a proprietary microwell based kidney injury test (KIT) assay, we measured cell-free DNA, methylated cell-free DNA, clusterin, creatinine, protein, and CXCL10. A KIT Stone-Score was computed across all markers, using the prior locked KIT algorithm. The KIT Stone-Score, scaled from 0 – 100, was then correlated with demographic variables, kidney stone burden, obstructive kidney stone disease, and urine solutes by 24-hour urine collections.
The scaled KIT stone-score (KITstone), as a composite of all 6 biomarkers, readily discriminated individuals with current or prior radiographically confirmed kidney stones, from healthy controls without kidney stone disease (P < 0.0001). KITstone also correlated in individuals with nephrolithiasis, with radiologically measured stone size (P = 0.0174) and also differentiated patients with a clinical radiological diagnosis of obstructive nephrolithiasis, associated with upper renal tract dilatation (P = 0.0010).
On the other hand, stone burden as assessed by KITstone, did not correlate with the any of the traditional measures of 24-hour urine solutes or the 24-hour urine supersaturation levels. In patients with persistent stone disease, where multiple urine samples were collected over time and different interventions, KITstone could non-invasively track stone burden over time by a spot urine, non-timed urine sample.
A random, spot urine-based assay, KITstone, can non-invasively detect, quantify, and monitor current stone burden, and may thus minimize radiographic exposure for kidney stone detection. The KITstone assay may also help monitor for stone recurrence risk for patients with nephrolithiasis, without the requirement of 24 hour urine collections.
Comparison of Commercial ELISA Kits, a Prototype Multiplex Electrochemoluminescent Assay, and a Multiplex Bead-Based Immunoassay for Detecting a Urine-Based Bladder-Cancer-Associated Diagnostic Signature.
The ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of clinical tests composed of multiple biomarkers. We investigated the diagnostic accuracy of the two multiplex protein array platforms for detecting a bladder-cancer-associated diagnostic signature in samples from a cohort of 80 subjects (40 with bladder cancer). Banked urine samples collected from Kyoto and Nara Universities were compared to histologically determined bladder cancer. The concentrations of the 10 proteins (A1AT; apolipoprotein E-APOE; angiogenin-ANG; carbonic anhydrase 9-CA9; interleukin 8-IL-8; matrix metalloproteinase 9-MMP-9; matrix metalloproteinase 10-MMP10; plasminogen activator inhibitor 1-PAI-1; syndecan-SDC1; and vascular endothelial growth factor-VEGF) were monitored using two prototype multiplex array platforms and an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s technical specifications.
The range for detecting each biomarker was improved in the multiplex assays, even though the lower limit of quantification (LLOQ) was typically lower in the commercial ELISA kits. The area under the receiver operating characteristics (AUROC) of the prototype multiplex assays was reported to be 0.97 for the multiplex bead-based immunoassay (MBA) and 0.86 for the multiplex electrochemoluminescent assay (MEA). The sensitivities and specificities for MBA were 0.93 and 0.95, respectively, and for MEA were 0.85 and 0.80, respectively. Accuracy, positive predictive values (PPV), and negative predictive values (NPV) for MBA were 0.94, 0.95, and 0.93, respectively, and for MEA were 0.83, 0.81, and 0.84, respectively. Based on these encouraging preliminary data, we believe that a multiplex protein array is a viable platform that can be utilized as an efficient and highly accurate tool to quantitate multiple proteins within biologic specimens.
A nanocellulose-based colorimetric assaykit for smartphone sensing of iron and iron-chelating deferoxamine drug in biofluids.
The current work describes the development of a “nanopaper-based analytical device (NAD)”, through the embedding of curcumin in transparent bacterial cellulose (BC) nanopaper, as a colorimetric assay kit for monitoring of iron and deferoxamine (DFO) as iron-chelating drug in biological fluids such as serum blood, urine and saliva. The iron sensing strategy using the developed assay kit is based on the decrease of the absorbance/color intensity of curcumin-embedded in BC nanopaper (CEBC) in the presence of Fe(III), due to the formation of Fe(III)-curcumin complex. On the other hand, releasing of Fe(III) from Fe(III)-CEBC upon addition of DFO as an iron-chelating drug, due to the high affinity of this drug to Fe(III) in competition with curcumin, which leads to recovery of the decreased absorption/color intensity of Fe(III)-CEBC, is utilized for selective colorimetric monitoring of this drug.
The absorption/color changes of the fabricated assay kit as output signal can be monitored by smartphone camera or by using a spectrophotometer. The results of our developed sensor agreed well with the results from a clinical reference method for determination of Fe(III) concentration in human serum blood samples, which revealed the clinical applicability of our developed assay kit. Taken together, regarding the advantageous features of the developed sensor as an easy-to-use, non-toxic, disposable, cost-effective and portable assay kit, along with those of smartphone-based sensing, it is anticipated that this sensing bioplatform, which we name lab-on-nanopaper, will find utility for sensitive, selective and easy diagnosis of iron-related diseases (iron deficiency and iron overload) and therapeutic drug monitoring (TDM) of iron-chelating drugs in clinical analysis as well.
GABA enzymatic assaykit.
We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder’s reagent enabled accurate quantification of 0.2 – 150 mg/L GABA.
A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.
