Validation of an ELISA for detection of neutrophil gelatinase‐associated lipocalin (NGAL) in equine serum

Abstract

Background: Neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be a useful marker of kidney injury in people and dogs, but has not been described in horses. Objectives:The aim of the study was to validate a commercially available porcinespecific ELISA to measure serum concentrations of equine NGAL.Methods: Intra-and interassay imprecisions were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by the linearity under dilution. Overlapping performance was assessed by measuring NGAL concentrations in horses with normal and elevated serum creatinine levels.Results: Intra-and interassay imprecision (coefficient of variation) ranged from 5.35% to 28.39%. The ELISA showed no signs of inaccuracy. Overlapping performance was acceptable, as the assay was able to detect the expected differences of NGAL levels in horses with normal and elevated serum creatinine concentrations. Conclusions:Equine serum NGAL concentrations could be quantified reliably using the porcine-specific ELISA.| 603 monitoring tool in patients with kidney injury. 10 Diagnostic use of NGAL has not been described in horses.The aim of this study was to validate a commercially available, porcine-specific ELISA for measurements of NGAL in equine serum by investigating (a) assay characteristics (imprecision, inaccuracy, and detection limit [DL]), and (b) the ability of the assay to detect the expected differences in NGAL levels between the two selected equine populations, one with elevated and one with normal serum creatinine concentrations. | MATERIALS AND METHODS | The NGAL assayThe assay, a commercially available ELISA (porcine NGAL ELISA kit, cat. no. KIT 044; Bioporto, Hellerup, Denmark) developed for measuring NGAL concentrations in porcine urine, plasma, serum, tissue extracts, and culture media was used for heterologous NGAL determination in equine serum samples.The analyses were performed using a Multiskan EX plate reader (Thermo Electron Corporation, Hvidovre, Denmark) supported with the Ascent software, Version 2.6 for the iEMS Reader MF according to the manufacturer's instructions. Absorbance was read at 450/ 620 nm. All samples were run in duplicate. The calibration curve was made using the calibrator supplied with the kit and fitted with a four-parameter logistic curve using the freely available software at www.myassays.com. When optical density values were outside the calibration curve, samples were further diluted and reanalyzed.

Influence of clinical and experimental intra‐articular inflammation on neutrophil gelatinase‐associated lipocalin concentrations in horses

“…Increased serum NGAL concentrations were found in horses with increased serum creatinine values by researchers investigating NGAL in horses suspected of kidney injury. 26 Similarly to in man, NGAL seems to be a marker of inflammation in horses. Serum NGAL concentrations increased after experimental intravenous injection of lipopolysaccharide (LPS) and were also elevated in clinical cases with systemic inflammation.…”

“…There are few reports on NGAL in equine medicine. Increased serum NGAL concentrations were found in horses with increased serum creatinine values by researchers investigating NGAL in horses suspected of kidney injury 26 . Similarly to in man, NGAL seems to be a marker of inflammation in horses.…”

“…The ELISA has been validated and found reliable for equine use by our research group. 26 Serum samples were diluted 1:1000 to 1:3000, and SF was diluted 1:2000 to 1:20000 to achieve absolute NGAL concentrations. The ELISA was performed according to the manufacturer's instructions.…”

Serum symmetric dimethylarginine concentration in healthy horses and horses with acute kidney injury

“…Due to the increase in biomarker demand, there is a growing interest in new AKI biomarkers in horses. However, the number of biomarker studies conducted in horses is limited [ 8 – 10 ].…”

Detecting acute kidney injury in horses by measuring the concentration of symmetric dimethylarginine in serum

“…Studies in horses have attempted to evaluate renal function by analysis of the fractional excretion of sodium (FENa), urine γ-glutamyl transpeptidase to creatinine ratio (GGT:Crea), as well as cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels, but with inconsistent results [8,[16][17][18]. Studies in human and companion animals, on the other hand, assessed multiple serum and urinary parameters as potential AKI biomarkers, symmetric dimethylarginine (SDMA) being the most common [19][20][21][22][23][24][25].…”