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16 | AUGUST 2014 | GENengnews.com | Genetic Engineering & Biotechnology News Robert Durham, Ph.D, and Wei Zheng, Ph.D. The top reason for drug candidate attri- tion is not poor effcacy as one might ex- pect but rather unacceptable toxicity, high- lighting the need to identify drug-induced toxicity in the early stages of drug devel- opment. The challenge is to identify and validate sensitive and specifc biomarkers that can detect drug-induced organ dam- age early in exposure and also fulfll the requirements of regulatory authorities. In the pharmaceutical and chemical in- dustry, the kidney is routinely assessed dur- ing preclinical safety evaluations. The role of the kidney as a central detoxifcation or- gan leads to a high exposure of renal tissue to drugs, reactive metabolites, or environ- mental compounds. Traditional markers for assessing renal toxicity, such as blood urea nitrogen (BUN) and serum creatinine (SCr), have low sensitivity. Although both are direct measurements of renal function, serum concentrations of these biomarkers increase only after substantial renal injury, demonstrating a clear need for improved biomarker assays to detect damage earlier. The Predictive Safety Testing Consor- tium (PSTC) was formed in 2006 to support the development of new biomarkers. PSTC encourages pharmaceutical companies to share and validate innovative safety testing methods that can be submitted for formal regulatory qualifcation by the FDA (Food and Drug Administration), the EMA (Eu- ropean Medicines Agency), and the PMDA (Japanese Pharmaceutical and Medical De- vices Agency). This is a major step forward to establishing improved cross-industry methods that are applicable throughout the drug development spectrum and can be independently validated and accepted as proof of safety by regulatory authorities. KIM-1 and Clusterin Biomarkers The frst project of the PSTC identifed eight effective rat urinary biomarkers, in- cluding KIM-1 (Kidney Injury Molecule 1) DIRECT FROM SAMPLE MULTIPLEXED GENOMIC TESTING Quantitative genomics for under $50/sample www.dxterity.com For Research Use Only. Not for Use in Diagnostic Procedures. We see the future in a drop of blood. Nanoliter-Scale Immunoassays Can Be Used to Measure KIM-1 and Clusterin Analysis of Rat Kidney Injury Biomarkers DRUG DISCOVERY Tutorial Robert Durham, Ph.D. (rob.durham@ gyros.com), is director of feld application scientists, Gyros U.S. Wei Zheng, Ph.D. (wei.zheng@emdmillipore.com), is head of R&D; and licensing, research content and reagents, bioscience, EMD Millipore. and clusterin, that were endorsed by FDA and EMA for the detection of acute kidney toxic- ity during nonclinical drug development. Rat urinary KIM-1 and clusterin can be detected by ELISA, but this method has a number of disadvantages, including narrow dynamic range, large sample consumption (particular- ly problematic in preclinical work with small laboratory animals), and long assay times and hands-on time, especially when a large num- ber of samples need to be analyzed. EMD Millipore has overcome these weak- nesses with new kits for rat urinary KIM-1 and clusterin based on a nanoliter-scale im- munoassay platform developed by Gyros AB. This combination of ft-for-purpose assay kits and an automated high-throughput im- munoassay platform using small sample vol- umes offers the potential for a sensitive robust method with signifcant advantages. Nanoliter-Scale Immunoassays Gyrolab™ xP workstation performs auto- mated immunoassays within nanoliter-scale microfuidic structures in a compact disk (CD) format. Each structure on the Bioaffy CD comprises a 15-nanoliter affnity column prepacked with Streptavidin-coated particles, supporting a variety of assay types including sandwich and indirect antibody assays. Working at the nanoliter scale means that the consumption of sample and reagents is dramatically reduced compared with plate- based ELISA. Apart from obvious cost-saving advantages of reducing use of expensive re- agents, the availability of sample, often taken from small laboratory animals, can become a limiting factor with ELISA, especially as regu- latory demands drive the need to perform an increasing number of analyses on each sample. The microfuidics ensure controlled fow of capture reagents, sample, and detection reagents, so that all samples within a single CD are processed in parallel. Parallel process- Figure 1. The assays are linear over a broad dynamic range. Sensitivity can be increased by amplifying the signal using higher photo multiplier tube (PMT) levels while maintaining parallelism.