Mouse albumin elisa kit

Blood glucose was measured weekly for the first 4 weeks postsurgery and from there on every third week by collecting blood into heparinized glass capillary tubes and immediate suspension in glucose/lactate system solution buffer (EKF‐diagnostics). Blood glucose was measured using a BIOSEN c‐Line glucose meter (EKF‐diagnostics) according to the manufacturer's instructions. Glycated haemoglobin A1c (HbA1c) levels were measured at termination (16 weeks after surgery) using commercial kits (Roche Diagnostics) on a Cobas^TM C‐501 autoanalyzer (Roche Diagnostics). At study week 15, spot urine samples were collected directly from the penis/vulva to determine the urinary albumin‐to‐creatinine ratio (ACR). Urine creatinine was measured using the CREP2 kit (Roche Diagnostics) on a Cobas^TM C‐501 autoanalyzer. Urinary albumin was measured using a mouse albumin ELISA kit (Bethyl Laboratories, Inc.). Plasma blood urea nitrogen (BUN) levels were measured at termination (16 weeks after surgery) using commercial kits (Roche Diagnostics) on the Cobas^™ C‐501 autoanalyzer.

Mice were sacrificed 7 and 35 days after the rAd injections. Urine, blood and kidney samples were harvested and kept frozen at −80°C. Blood urea nitrogen was assessed using a QuantiChrom Urea Assay kit (BioAssay Systems, DIUR-500) as described previously (Mattison et al., 2012 (link)). The urinary creatinine levels were measured using a colorimetric assay (R&D Systems, KGE005). Albuminuria was measured with a mouse albumin ELISA kit (Bethyl Laboratories, E99-134) and expressed as ratio of the urinary creatinine. In addition, SDS-PAGE (4-12%) was carried out with 5 ml of urine that was then stained with Coomassie blue stain solution (Bio-Rad) to detect changes in high and low molecular weight urinary proteins as described previously (Das et al., 2016 (link)).

Quantification of BUN in serum was performed using a Quantichrom Urea Assay Kit (Bioassay Systems), according to the manufacturer's protocol. Urinary albumin and creatinine were assayed using a mouse albumin ELISA kit (Bethyl Laboratories) and urinary creatinine assay kit (Cell Biolabs). Results were expressed as albumin-to-creatinine ratio. To get rid of any floating cells in urine, the urine was centrifuged and then 2 µl was loaded onto 10% SDS-polyacrylamide gel for electrophoresis followed by Coomassie Blue staining. Bovine serum albumin (BSA) (2 µl; 10 mg/ml) was used as a positive control.

Total urinary protein concentrations were determined spectrophotometrically using Bradford assay (Sigma, United States). Urinary albumin concentration was measured with mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX, United States) according to manufacturer’s instructions.

Blood glucose was measured throughout the study by collecting blood from the tail vein of nonfasted mice into heparinized glass capillary tubes and immediately suspending in glucose/lactate system solution buffer (EKF Diagnostics, Barleben, Germany). Blood glucose was measured using a BIOSEN c-Line glucose meter (EKF Diagnostics) according to the manufacturer's instructions. Spot urine samples were collected 6 and 12 weeks after the injection of viral vector directly from the vulva to determine the urine ACR. Urine creatinine was measured using the CREP2 kit (Roche Diagnostics, Mannheim, Germany) on a Cobas C-501 autoanalyzer. Urine albumin was measured using a Mouse Albumin ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA).