Application Note: Novel Rat Kidney Injury Biomarker Assays Using a Gyros Nanotechnology Platform

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A leading cause of failure in any drug development program is organ toxicity, such as in the kidney. Traditional nephrotoxicity biomarkers may show insufficient tissue specificity and often cannot be detected until damage is irreversible. New markers, such as KIM-1 and Clusterin, help detect damage to the kidneys and offer clues to specific tissue injury in a timelier manner.

We developed rat KIM-1 and clusterin GyroMark™ HT biomarker immunoassays, the first two commercially available toxicity assay kits for the Gyrolab® xP Workstation platform. This automated compact disc-based microfluidic nanotechnology for sandwich ELISA analysis uses a biotinylated capture antibody and a fluorescent-labeled detection antibody (Figure 1).

The capture antibody is conjugated to particles, which are packed into 15-nL columns inside the microfluidic structures of spinning Bioaffy™ CDs inside the workstation. Compared to traditional ELISAs, GyroMark™ assays consume less reagents and require only 200 –1000 nL of sample. Because samples are assayed in parallel using microfluidics, conditions are uniform across samples, without time-dependent or plate location artifacts. Gyros technology also provides high sensitivity, broad dynamic range, and short assay time (1 hour per assay).

Merck:/Freestyle/BI-Bioscience/Protein-Detection/elisas/gyrolab-workstation.jpg
Figure 1. Gyrolab® XP Workstation (left) houses spinning Bioaffy® CDs, which contain prepacked columns within microstructures (right). The columns are packed with capture antibody-conjugated particles, which bind the analyte and detection antibody as they flow past.


In this study, we determined assay performance characteristics for the GyroMark™ HT rat KIM-1 and Clusterin assays, and then measured these biomarkers from urine samples of rats treated with gentamicin. We compared the results to those obtained using an electrochemiluminescence assay on the Mesoscale Discovery (MSD) platform, to a traditional ELISA, and to a MILLIPLEX® MAP assay for the Luminex® platform.

Materials and Methods

We evaluated rats treated with no, low, and high doses of gentamicin, a known nephrotoxicant. Urine samples (n=36) were collected on days 0, 3, 7, and 14. Urine samples were centrifuged briefly to pellet debris. Samples were then diluted 1:10 in Diluent GM1. 10 µL sample was added 90 µL of Diluent GM1 in polypropylene microfuge tubes and mixed well.

The GyroMark™ HT Rat Clusterin Assay (Cat. No. GYRCLU-37K) and Rat KIM-1 Assay (Cat. No. GYRKIM1-20K) were developed and used for biomarker quantitation, according to the protocols supplied with the kits. Also used were the MILLIPLEX® MAP Rat Kidney Toxicity Magnetic Bead Panel 1 - Toxicity Multiplex Assay (Cat. No. RKTX1MAG-37K), and a traditional ELISA kit from R&D Systems.

Results and Conclusions

For the KIM-1 assay, we determined dynamic range, sensitivity, spike recovery, dilution linearity, intra-, and inter-assay variations of 0.015-60 ng/mL, 0.015 ng/mL, 100%, 107%, < 10% and < 15%, respectively for 1:2 diluted rat urine samples (Table 1).

The clusterin assay had a dynamic range, sensitivity, spike recovery, dilution linearity, intra-, and inter-assay variations of 0.7-3000 ng/mL, 0.36 ng/mL, 93%, 108%, < 15% and <15% respectively for 1:10 diluted rat urine samples (Table 1).

KIM-1 Clusterin (uses 1:10 diluted sample)

Standard Curve Range (ng/mL)

LLOQ (ng/mL)

Spiked Recovery

Dilutional Linearity

Intra-Assay Precision(16 replicates)

Inter-Assay Precision (8 runs)

0.015-60

0.015

100%

107%

<10%

<15%

0.7-3000

0.36

93%

108%

<10%

<15%


Table 1. Assay performance standards for the GyroMark™ HT Rat KIM-1 and Clusterin Assay Kits

The standard curves for the GyroMark™ HT KIM-1 and clusterin assays were compared to the curves obtained for the same standards but using the MSD platform. The KIM-1 standard curves showed a high degree of correlation between the two platforms (Figure 2A). GyroMark™ HT Standard was observed to be less potent than MSD standard by 26-fold (Figure 2B).

2A 2B
KIM-1 Standard Curve GryoMark™ vs. MSD Graph Clusterin Standard Curve GyroMark™ vs. MSD Chart

Figure 2. Comparing quantitation of serially diluted KIM-1 (A) and clusterin (B) standards using GyroMark™ HT and MSD assays.


These GyroMark™ HT assays were also compared to the MSD platform in the analysis of vancomycin-treated rat urine samples (n=36); correlations were r = 0.97 (KIM-1, Figure 3A) and 0.87 (clusterin, Figure 3B).

3A 3B
Merck:/Freestyle/BI-Bioscience/Protein-Detection/elisas/rat-kim-1_Fig3A.jpg Merck:/Freestyle/BI-Bioscience/Protein-Detection/elisas/rat-clusterin_Fig3B.jpg

Figure 3. Comparing quantitation of KIM-1 (A) and clusterin (B) in urine from gentamicin-treated rats, using GyroMark™ HT and MSD assays.


The rat urine samples were also tested for clusterin using a MILLIPLEX® MAP bead-based assay for the Luminex® platform (Figure 4). A high degree of correlation (R=0.9825) was observed between the two assay platforms.

Figure 4
Merck:/Freestyle/BI-Bioscience/Protein-Detection/elisas/rat-clusterin-vs-map_Fig4.jpg

Figure 4. Comparing quantitation of clusterin in urine from gentamicin-treated rats, using GyroMark™ HT and MILLIPLEX® MAP assays.


Quantitation of the GyroMark™ HT Clusterin Standard was also compared to a clusterin standard from Prospec (Table 2A) and to measurements using a traditional clusterin ELISA (R&D Systems, Table 2B). In Table 2A, comparing the second and third columns reveals that the two clusterin standards evoked similar assay response in the GyroMark™ HT assay. Table 2B shows that the traditional Clusterin ELISA yielded data that were quite similar to the results of the GyroMark™ HT assay.

Table 2A
Std. Conc. (ng/mL)Control ReponseProspec Response
0 0.042 0.171
0.36 0.059 0.179
1.46 0.142 0.191
5.86 0.409 0.310
22.43 1.570 0.859
93.75 5.940 2.860
375 18.800 11.000
1500 48.800 42.100


Table 2B
GyroMark in R&D (ng/mL)AbsorbanceCal Conc.
0.5 0.05
1 0.07
5 0.255 3.22
25 1.435 19.75

Table 2. Quantitation of the GyroMark™ HT clusterin standard closely matched the Prospec standard (2A) and the R&D Systems ELISA standard (2B)

These data demonstrate that our GyroMark™ HT toxicity assays for rat KIM-1 and clusterin, are robust and reproducible, using 1000 nL samples, and can be useful for detecting kidney damage in rat urine samples for drug development and basic research.