Urinary Assays

Albuwell M is an indirect competitive ELISA designed to monitor kidney function in the mouse by measurement of urinary albumin. To complete the assay, sample and rabbit anti-murine albumin antibody are added to albumin coated wells. The antibody interacts and binds with the albumin immobilized to the stationary phase or with albumin in the fluid phase, hence the notion of competitive binding.

A subsequent reaction with anti-rabbit -HRP conjugate labels the probe with enzyme. After washing, only the antibody-conjugate bound to the stationary phase remains in the well, and this is detected using a chromogenic reaction. Color intensity is inversely proportional to the logarithm of albumin in the fluid phase. The assay may be completed in less than 2.5 hours.

To complete the assay, sample and goat anti-collagen IV antibody are added to human collagen IV coated wells. The antibody interacts and binds with the collagen IV immobilized to the stationary phase or with the antigen in the fluid phase, hence the notion of competitive binding. After a suitable incubation period, the plates are washed, and a rabbit anti-goat -HRP conjugate is used to detect bound antibody. After washing, only the antibody-conjugate bound to the stationary phase remains in the well, and this is detected using a chromogenic reaction. Color intensity is inversely proportional to the logarithm of collagen IV in the fluid phase. The assay may be completed in less than 3 hours.

The “enhanced sensitivity” mode is completed by placing the samples and anti-collagen IV antibody into tubes and incubating 2-24 hours. Here, the antibody can only react with the collagen IV of the fluid phase. Subsequently, aliquots of the mixture are added to the plate where remaining free antibody can react with the antigen of the stationary phase. The bound antibody is detected as described above.

Normal Sensitivity: 0.156-10 ug/mL

Enhanced Sensitivity: 0.02-2.5 ug/mL

Collagen IV M is an indirect competitive ELISA designed to measure type IV collagen in biological and tissue culture specimens of murine origin. The kit also works with rat specimens. The assay may be completed in a "normal sensitivity" mode, with a useful assay range of 0.156-10 ug collagen IV/mL. A slightly different procedure, the "enhanced sensitivity" mode typically yields a useful assay range of 0.02-2.5 ug/mL, and is sensitive enough to detect collagen IV in urine specimens.

To complete the assay, sample and rabbit anti-collagen IV antibody are added to murine collagen IV coated wells. The antibody interacts and binds with the collagen IV immobilized to the stationary phase or with the antigen in the fluid phase, hence the notion of competitive binding. After a suitable incubation period, the plates are washed, and an anti-rabbit -HRP conjugate is used to detect bound rabbit antibody. After washing, only the antibody-conjugate bound to the stationary phase remains in the well, and this is detected using a chromogenic reaction. Color intensity is inversely proportional to the logarithm of collagen IV in the fluid phase. The assay may be completed in less than 3 hours.

The "enhanced sensitivity" mode is completed by placing the samples and anti-collagen IV antibody into tubes and incubating 2-24 hours. Here, the antibody can only react with the collagen IV of the fluid phase. Subsequently, aliquots of the mixture are added to the plate where remaining free antibody can react with the antigen of the stationary phase. The bound antibody is detected as described above.

Normal Sensitivity: 0.156-10 ug/mL

Enhanced Sensitivity: 0.02-2.5 ug/mL

The Creatinine Companion is a chemical assay designed to monitor urinary creatinine in a convenient microplate format. It is intended as a "companion" for Exocell urinary albumin assays.

The assay is based upon the Jaffe' reaction of alkaline picrate with creatinine. Standards or samples are added to a microplate, and alkaline picrate reagent is added. Absorbance at 500 nm is determined after a 10 minute incubation on the benchtop. Subsequently, an acid solution is added, and absorbance is again determined after a 5 minute incubation. The difference between these absorbance values is directly proportional to the the creatinine concentration, and a standard curve is generated from the response to the standards. Unknown samples are evaluated by comparing response to the standard curve. The assay may be completed in less than 30 minutes.

The assay serves as a normalization procedure for other analytes in urine samples, particularly for urinary albumin (1,2). It allows for a measure of kidney function based on a spot urine sample rather than the usual 24 hour pool.

1. Bennet, P. H. et. al. Screening and management of microalbuminuria in patients with diabetis mellitus. Am. J. Kidney Dis. 25:107, 1995.
2. Warram, J. H. et. al. Effect of duration of type 1 diabeties on the prevalence of stages of diabetic nephropathy defined by urinary albumin/creatinine ratio. J. Am. Soc. Nephrol. 7:930, 1996.

Nephrat II is a direct competitive ELISA for the quantitative determination of urinary albumin in rat specimens. To complete the assay, controls, standards and diluted samples are added to rat albumin coated plates. An anti-rat albumin antibody-HRP conjugate is added, and allowed to react. Specific antigen-antibody reactions occur either with the albumin of the fluid phase or with that of the stationary phase, hence the notion of competitive binding. Subsequently, the plate is washed, and unbound reactants are washed away. Only conjugate bound to the stationary phase remains. A chromogenic substrate, TMB, is added to the wells, and color development ensues. Color intensity is inversely proportional to the log of rat albumin concentration of the sample.

Nephrat II has a useful dynamic range of 1.56-100 ug rat albumin/ mL, and samples must be diluted appropriately to fall within that range. The kit includes 2 plates and sufficient reagents to complete 80 analyses in duplicate.

Nephrin ELISA is an indirect competitive ELISA designed to measure nephrin in urine specimens of rodent or human origin. Nephrin, a transmembrane protein expressed in epithelial cells (podocytes) of the renal glomerulus, regulates passage of plasma proteins across the glomerular filtration barrier and may be shed into the urine in diseases affecting the glomerulus such as diabetes mellitus.

To complete the assay, standard or sample containing nephrin in the soluble phase is added to microtiter wells that contain nephrin antigen immobilized onto the solid phase, and to which rabbit anti-nephrin antibody is added in the fluid phase. The antibody binds to either the immobilized nephrin or the soluble nephrin, which compete for this binding. After a one hour incubation at room temperature, the plates are washed and a goat anti-rabbit – HRP conjugate is used to detect bound antibody. Plates are washed and only the antibody conjugate bound to the stationary phase antigen remains in the well, which is detected with a chromogenic reaction. Color intensity is inversely proportional to the concentration of nephrin in the fluid phase. Concentrations in experimental samples are determined from a standard curve.

Catalogue Numbers:

1020

Methodology:

Chemical Assay

Summary of procedure:

The TBARs assay kit measures malondialdehyde (MDA), a reactive compound formed from lipid peroxides that are generated under conditions of oxidative stress. Oxidative modification of lipids occurs with aging and various diseases, and increased oxidative stress is associated with diabetes and its complications. Cellular exposure to oxidative stress gives rise to highly reactive and unstable lipid hydroperoxides. Decomposition of unstable peroxides derived from polyunsaturated fatty acids leads to the MDA formation. MDA reacts with thiobarbituric acid (TBA) to form an adduct that can be measured spectrophotometrically or fluorometrically, the latter providing greater sensitivity. Determination of TBARs in biological samples has become the preferred method for assessing lipid peroxidation and oxidative stress. Exocell's TBARs assay can be used with a spectrum of biological samples including body fluids, tissue and cell specimens.
To complete the assay, standard or sample is reacted with thiobarbituric acid and heated in the presence of acetic acid. After cooling and clarification by centrifugation, aliquots are placed into microtiter wells of a black plate and read in a fluorometer at excitation 484 nm, emission 530 nm, or into a plastic plate for reading at 530-540 nm in a spectrophotometer. MDA concentration in samples is determined from the standard curve.

Specimen Required:

100uL

Assay Range:

0-30  nmole fluorometrically
0-300 nmole spectrophotometrically

Precision:

Intra- and inter-assay precision <8%

Catalogue Numbers:

1024 

Methodology:

Quantitative ELISA

Summary of Procedure:

Adiponectin-ELISA is designed to measure the concentration of adiponectin in samples of rodent or human origin. Decreased levels of adiponectin, an adipose-derived protein with anti-inflammatory, anti-artherogenic and insulin-sensitizing properties, are linked to obesity, insulin resistance and Type 2 diabetes. Serum adiponectin levels are elevated in patients with overt diabetic nephropathy and may be due to either an increase in synthesis of adiponectin in adipose tissue secreted into the circulation or to a reduction in adiponectin clearance. Quantification of adiponectin in serum or urine may be used as a marker for renal insufficiency and tubular damage especially in overt diabetic nephropathy.

Specimen Required:

Serum, Urine, Culture Medium or Cell extracts (Mouse, Rat, or Human)

Assay Range:

0.156 ng/ml - 10.0 ng/ml

Precision:

Intra- and inter-assay precision for samples within the assay range have a C.V. o f <7%

Catalogue Numbers:

1025 & 1026

Methodology:

Quantitative Sandwich ELISA

Summary of Procedure:

ßIG-H3 ELISA is designed to measure ßig-h3 (beta-induced H3) in urine, serum or cell culture supernatant of mouse origin. ßig-h3 is a 68 kD protein induced in diverse cell types by the growth factor Transforming Growth Factor-ß (TGF-ß) that is secreted into the extracellular matrix and is involved in cell growth, differentiation, adhesion, and wound healing. ßig-h3 expression is altered in various conditions including tumorigenesis, breast cancer, corneal dystrophy, osteogenesis, and atherothrombosis. ßig-h3 is elevated in the urine of rodents with experimental diabetes and in patients with cyclosporine nephrotoxicity and with type 2 diabetes, in whom it shows positive correlation with the albumin excretion rate, making it a useful biomarker of renal dysfunction due to diabetes in its earliest phases and in monitoring clinical progression of diabetic nephropathy.
To complete the assay, standard or sample containing ßig-h3 in the soluble phase is added microtiter wells coated with anti-mouse ßig-h3 antibody. The ßig-h3 in standard or sample is captured by this antibody on the solid phase, and unbound materials are washed away. Biotinylated anti-mouse ßig-h3 antibody is then added, followed by a reaction with Streptavidin –HRP conjugate to label the biotinylated probe with enzyme. After washing, ßig-h3 bound to the capture antibody on the stationary phase is detected after addition of a chromogenic substrate. Samples with absorbance values above the standard curve should be diluted before assay.

Specimen Required:

50 ul urine, serum or tissue extract

Assay Range:

1025 = 50 - 4000 pg/ml : 1026 = 150 – 10,000 pg/ml

Precision:

Intra- and inter-assay precision for samples within the assay range have a C.V. o f <7%.