Storage
1. Store the kit with all reagents at 2-8°C. Do not freeze.
2. Unopened materials/reagents remain stable until the expiry date on the kit box label.
3. The stability of materials/reagents after opening is indicated on the kit box label.
4. Diluted Wash Solution Conc. remains stable for 24 hours at 2-8°C. If not using all wells, dilute only the portion of Wash Solution Conc. required.
5. Diluted Sample Diluent Conc. remains stable for 24 hours at 2-8°C. If not using all wells, dilute only the portion of Sample Diluent Conc. required.
6. For subsequent use, store unused wells in the foil pouch with the desiccant provided and reseal. Always allow the foil pouch to equilibrate to room temperature before opening to avoid condensation in/on the coated microwells.
Detection Limit
The lowest concentration of monkey NGAL giving an absorbance reading greater than 2 SD above the mean zero (calibrator 1) reading was 1.5 pg/mL, which is significantly lower than the value of the lowest calibrator concentration above zero (10 pg/mL).
General Description
NGAL (neutrophil gelatinase-associated lipocalin) belongs to the lipocalin family of proteins. These are secreted proteins characterized by their ability i) to bind small hydrophobic molecules in a structurally conserved pocket formed by β-pleated sheet, ii) to bind to specific cell-surface receptors, and iii) to form macromolecular complexes. NGAL has many synonyms: perhaps the most widely used is lipocalin 2 (LCN 2); more recently the name siderocalin has been used to express NGAL' s ability to bind certain bacterial siderophores.
The NCBI reference sequence XP_001083008.1 from genomic sequencing of Macaca mulatta (chromo-some 15) suggests that Rhesus monkey NGAL consists of a single disulfidebridged polypeptide chain of 178 amino-acid residues with a calculated molecular mass of 20.4 kDa. By analogy with NGAL from other mam-malian species, glycosylation would be expected to increase its apparent molecular mass on SDS-PAGE to about 24-25 kDa. The N-glycosylation site at residue 65 is intact and is in fact conserved in all known NGAL sequences from placental mammals.
Because Rhesus monkey NGAL has not yet been isolated and no antibodies or immunochemical detec-tion methods have hitherto been developed to detect or measure it, there are no published data on the physi-ological or pathological functions of this protein. How-ever, it is likely that some features of the biosynthesis, location and release of Rhesus monkey NGAL will be similar to those of its homologues in other species, particularly man. The present kit has in fact been de-veloped to extend NGAL research and the use of NGAL as a marker molecule, especially for kidney injury, to the Rhesus and closely related Cynomolgus monkey. In the following, some pathophysiological features of NGAL in humans and other mammalian species will be mentioned, because these two monkey species may show similar features.
In some situations, NGAL may be co-expressed with matrix metalloproteinase-9 (MMP-9), but whether Rhesus monkey NGAL forms a complex with this enzyme is unknown, given that, unlike its human homologue, it does not possess a third cysteinyl residue capable of forming an intermolecular disulffde bridge with the enzyme.
In other mammals, NGAL is expressed in neutrophil polymorphonuclear leucocytes1 and at lower basal levels in various other cell types, tissues or organs. These include epithelial cells of the uterus, mammary gland, lung, spleen, vagina and epididymis. It may be expressed in additional cell types during embryonic development and in response to various stimuli. This applies to certain kidney cells (see below), LPS-stimulated macrophages, dexamethasone-stimulated Lcells,
and ffbroblasts stimulated with basic ffbroblast growth factor. In the mouse, NGAL (often called 24p3 or uterocalin) is a type-1 acute-phase protein, being secreted by hepatocytes in the in-vivo turpentine model or in response to TNF-α or dexamethasone in vitro.
In disease, NGAL may be moderately raised in bacterial infections because of release from activated neutrophils11 or from epithelial cells. It may also be moderately raised in atheroma and the ailing myocardium, and in certain epithelial cancers such as those of the mammary gland, gastrointestinal tract or urothelium.
NGAL and acute kidney injury
Apart from the expression of mammalian NGAL that occurs in the above situations, NGAL was found to undergo an early and dramatic upregulation in mouse kidney cells after infection with SV40 or polyoma virus. This was the ffrst indication of NGAL's early response to kidney cell injury.
Years later this ffnding was extended to post- ischemic and nephrotoxic injury. The marked up regulation of NGAL mRNA and protein levels in the early post-ischemic rat kidney was detected predominantly in proximal tubule cells18 and NGAL was easily detected by Western blotting of urine after ischemia and cisplatin-induced nephrotoxicity.
Urinary NGAL has also been found to be raised in mouse models of diabetic and obstructive nephropathy. However, there is a paucity of quantitative data on NGAL levels in non-human urine, plasma or serum, whether in the basal state or after kidney injury, and there are as yet no published data from any monkey species. It is the purpose of the present kit to allow such data to be obtained.
In man, it is now widely recognized that NGAL, whether measured in urine or plasma, is the earliest responding marker of acute kidney injury known, with the further advantage that its response is high and easy to measure while retaining proportionality with the severity of injury. It is therefore to be hoped that the determination of urinary or plasma NGAL may become a convenient end-point in Rhesus and Cynomolgus monkey models of renal injury, nephrotoxicity and other nephropathies. In the following instructions, the unqualiffed term "monkey" refers to Rhesus and/or Cynomolgus monkeys.
COA
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