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Genetic Engineering & Biotechnology News | GENengnews.com | AUGUST 2014 | 17 ing eliminates time-dependent artifacts that may arise in a typical plate-based ELISA and ensures that samples are processed under uni- form conditions. Each individual microstruc- ture equates to one data point, thus eliminating cross talk. The control and analysis software is 21 CFR part 11 compliant and gives fex- ibility in run setup and data analysis, ensuring that assays can be developed and transferred through to GMP and GLP environments. Development of Fit-for-Purpose Assays Using Gyrolab xP workstation, EMD Mil- lipore developed and validated ft-for-purpose GyroMark™ HT Kits for rat urinary KIM-1 and clusterin that had the following properties: • High linearity over a broad dynamic range (Figure 1) • High sensitivity with LLOQ down to 0.02 ng/mL • Target detection of spiked control in rat urine samples = 80–120% • Minimal matrix effects, with minimum required dilutions as low as 1:2 • Dilutional linearity accuracy well within acceptance criteria over a broad dilution range • Inter- and intra-assay precision in the 3.6–8.6% range The results confrmed that the combina- tion could indeed be suitable for studying drug-induced nephrotoxicity. The results from GyroMark HT kits also correlated very well with those from an alternative method— MILLIPLEX ® MAP multiplex assays based on Luminex ® technology. Case Study: In Vivo Evaluation of Sub-acute Nephrotoxicity Biomarkers Researchers at Merck Serono decided to determine if rat urinary KIM-1 and clus- terin could be used to detect sub-acute drug- induced kidney damage. They measured the urinary protein biomarkers using GyroMark HT kits run on Gyrolab xP workstation and performed immunohistochemistry (IHC) in a 28-day rat study using vancomycin as the tox- icant. The study design involved 120 animals and 120 samples, run in duplicate or tripli- cate, thus generating hundreds of data points. Using Gyrolab xP workstation they were able to analyze all samples in one day. The baseline study involved histopathol- ogy studies and IHC staining for KIM-1 on all animals to correlate and confrm kidney dam- age. Levels of the traditional biomarkers BUN and SCr also increased signifcantly after eight days at the high vancomycin dose, includ- ing a dip in the recovery phase (Figure 2). No changes were seen in low-dose animals. The results from assays using GyroMark HT KIM-1 and clusterin kits correlated well with levels of the traditional biomarkers for both male and female (results not shown) animals, with signifcant changes being detected after only four days of exposure. Receiver-operating characteristic (ROC) analysis of urinary proteins confrmed that KIM-1 and clusterin indeed have great diag- nostic value. The area under the curve (AUC) was used as a measure of the overall ability of the biomarker to discriminate animals with- out histopathological fndings in kidneys from those with signs of tubular damage. KIM-1 had an AUC of 0.90–0.95 and clusterin 0.94– 0.98 for males and females, respectively. Conclusions The study confrmed the high accuracy and predictivity of rat urinary KIM-1 and clusterin as markers to detect sub-acute neph- rotoxicity when treated with vancomycin. The combination of GyroMark HT kits and Gyrolab xP workstation generated reliable and sensitive measurements of the markers from a large number of 1 μL samples per day, thus promising to be a useful tool for drug de- velopment and basic research when studying kidney damage in rat models. 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DRUG DISCOVERY Tutorial Figure 2. There was an excellent correlation between results for GyroMark HT Kits (Clusterin and KIM-1) and traditional biomarkers (SCr and BUN). The data show re s u l t s fo r m a l e rat s ex p o s e d t o l ow- dose (LD) and high-dose (HD) levels of vancomycin for 4–29 days, compared with controls (C). The animals were allowed to recover on day 15. Statistical signifcance was determined by ANOVA + Dunnett-Test; *p < .05, **p < .01, ***p < .001.