The sensitivity of different renal regions to xenobiotics requires the development of a multiplex immunoassay for the simultaneous analysis of kidney biomarkers. Calbindin D28K is a distal tubule-specific protein that can be detected in urine under pathological conditions. In this study, a pair of anti-calbindin D28K antibodies was used in an immunoassay for the detection of calbindin D28K expression in rat and human kidney and urine. Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms. Analysis on both platforms detected calbindin D28K concentrations between 100 ng/mL and 100 pg/mL. Luminex detected 10-fold the amount of calbindin D28K in samples analyzed as compared to MSD, whereas calbindin D28K level in rat and human urine was below detection limit in both platforms. The application of the immunoassays described herein may be useful in toxicological and pathological studies of distal tubular damage in rats and human.
The high renal blood flow, renal biotransformation of chemicals to reactive metabolites, and the nephrons ability to concentrate tubular fluid render the kidney sensitive to xenobiotics. Due to the kidneys regional sensitivity to xenobiotics, it is important to colocalize sites of biomarkers release with pathological lesions [
Calbindin D28K is an intracellular, vitamin D-dependent, calcium-binding protein that is expressed in the epithelial cells of renal distal tubules [
There is an emerging trend in pharmaceutical industry to evaluate biomarkers to determine the safety of drugs early in clinical development. These biomarkers should predict specific damage before functional loss. Limited sample volume and excess costs have led to the development of multiplex immunoassays for the simultaneous analysis of multiple biomarkers.
The aim of this study was to compare an immunoassay for calbindin D28K analysis on two multiplex platforms. The same antibody pair was used for the single-plex analysis of calbindin D28K in Luminex as compared to Meso Scale Development (MSD). Analyses were performed using rat recombinant calbindin D28K protein as a standard. Kidney homogenates were used for validating the immunoassay on both platforms. Results from this work may provide clues to the further development of a multiplex immunoassay for the simultaneous analysis of multiple kidney biomarkers.
Rat and human homogenates were kindly provided by AstraZeneca (UK) and Cardiff university (UK), respectively. Briefly, frozen tissue samples were chopped into small cubes (approximately
Anti-mouse immunoglobulin plates (MSD, Gaithersburg, Md, USA) were used to immobilize anti-calbindin D28K monoclonal antibody from murine ascetic fluid (Sigma, Saint Louis, Mo, USA). The monoclonal antibody was diluted in Tris-Base Saline (TBS) buffer to 5
Anti-mouse IgG1 beads (Upstate, Temecula, Calif, USA) were used to capture anti-calbindin D28K murine antibody. The beads were vortexed vigorously for one minute and diluted
The need for multiple biomarker analysis in limited sample volumes as well as the demand of decreasing costs has led to the development of multiplex immunoassay techniques. Several studies have previously compared the analysis of different biomarkers on different commercially available multiplex platforms [
Recombinant calbindin D28K was used as a standard and was diluted to produce 8- versus 7-standard concentrations for the MSD and Luminex immunoassay, respectively. Figure
(a) Calbindin D28K standard curve analysis on MSD. (b) Luminex analysis of calbindin D28K; with standard curve indicating the fluorescence intensity (FI) at the different concentrations.
The earliest report on a calbindin D28K immunoassay, made use of anti-calbindin D28K-coupled Sepharose beads and the standard used was native calbindin D28K purified from human kidney tissue [
In an attempt to assess the immunoassays ability in measuring native calbindin D28K, kidney homogenates were analyzed. Since calbindin D28K is expressed in distal tubuli, rat cortical kidney homogenate was used in validating the immunoassays sensitivity and linearity. Whereas MSD had a higher sensitivity for recombinant calbindin D28K, Luminex detected calbindin D28K in kidney homogenate at the highest dilution factor tested (Table
Linearity analysis of rat cortical kidney homgenate dilutions.
MSD | Luminex | |||||
---|---|---|---|---|---|---|
Dilution factor | Measured pg/mL | % | Measured pg/mL | % | ||
100 | 5554 | 1.2 | 131 | 49762 | 0.1 | 128 |
200 | 2410 | 1.9 | 114 | 22802 | 5.1 | 117 |
400 | 1233 | 11 | 116 | 11568 | 1.2 | 119 |
800 | 424 | 12 | 80 | 5421 | 8.0 | 112 |
1600 | 247 | 6.7 | 93 | 2709 | 5.2 | 111 |
3200 | 110 | 16 | 83 | 1120 | 3.7 | 92 |
6400 | 55 | 14 | 83 | 422 | 12 | 69 |
12800 | — | 155 | 6.2 | 51 |
To investigate the immunoassays measurement of high versus low concentrations of calbindin D28K, two different dilutions of rat cortical kidney homogenate were analyzed. Precision analysis of the two different platforms indicated that Luminex had lower cv values as compared to MSD, at both high and low concentrations of calbindin D28K (Table
Precision analysis using rat cortical kidney homogenate.
MSD | Luminex | |||
---|---|---|---|---|
Dilution | ||||
Mean | 945 | 87 | 14167 | 1041 |
97 | 17 | 723 | 103 | |
10% | 20% | 5% | 10% |
Interassay variation analysis of rat cortical kidney homogenate.
Assay | MSD (ng/mL) | Luminex ( |
---|---|---|
1 | 424 | 3.9 |
2 | 407 | 3.1 |
3 | 283 | 3.3 |
76 | 0.4 |
Whereas the recombinant calbindin D28K is cloned from rat and the analyses presented so far was with rat kidney homogenate, the antibodies used in this immunoassay cross-reacts with human calbind D28K. To test the specificty of the immunoassay, homogenates from different regions of the human kidney were analyzed with MSD. The result demonstrated the immunoassays specifcity to the predominant expression of the distal tubule calbindin D28K in the cortical region (Table
MSD specificity analysis of human kidney homogenate.
Region | Concentration (ng/mL) |
---|---|
Cortex | 64 |
Medulla | 3 |
Papilla | — |
The methods described herein will enable future multiplex analysis of calbindin D28K in combination with other kidney-damage biomarkers, which may then be used in investigating distal tubule damage in rat and human toxicological studies. Whereas MSD had a wider dynamic range, analysis of calbindin D28K indicated that Luminex has higher sensitivity and precision. The selection of the platform most suitable for kidney biomarker analysis will depend on the other analytes available and other practical factors such as cost and ease of analysis.
This work was supported by the Technology Strategy Board in the United Kingdom.