Albustix

Serum was collected from treated and untreated mice at different time points, and IgM- and IgG- anti-dsDNA antibodies were measured by ELISA as described previously using biotin-labeled (detection) goat anti-mouse IgG (γ chain specific) and IgM (μ chain specific) antibodies (Southern Biotech) [6 (link)]. Proteinuria was monitored monthly using Albustix (Bayer).

C57BL/6 J mice were inoculated once with 0.5 ml pristane (2,6,10,14-tetramethyl-pentadecane; Sigma-Aldrich). The mice were divided into four groups: B7-1 antibody, IgG isotype, B7-1 shRNA and wild-type group. The B7-1 antibody mice were treated with 200 μg B7-1 antibody through an intravenous injection in the tail on day 1, 3, 5, 8 and 15 every month for three months after the pristane injection. The IgG isotype mice were treated with IgG control in parallel to the B7-1 antibody mice. The B7-1 shRNA mice were treated with 0.4×10⁸ TU LV-B7-1 shRNA on days 1 and 60 through an intravenous injection in the tail following pristane injection. The mice were bled every month following pristane inoculation and the sera were frozen for analysis of the autoantibodies. All mice were monitored for proteinuria once a month and were sacrificed via cervical dislocation at 8 or 10 months to harvest their kidneys. Kidney disease was assessed in mice treated with B7-1 antibody, B7-1 shRNA and in wild-type littermates. Proteinuria was measured on a 0–4 scale using a colorimetric assay strip for albumin (Albustix; Bayer, Elkhart, IN, USA), with scoring performed as follows: 0, (absent); 1, 30 mg/dl (mild); 2, 100 mg/dl (moderate); 3, 300 mg/dl (moderate to severe); and 4, 2000 mg/dl (severe). The experiment included 60 mice, which were allocated into six groups (10 mice per group).

In a separate cohort of eight NZB/W mice (six 13-week-old mice and two 21-week-old mice), systolic blood pressure (BP) was measured weekly using a tail cuff attached to a sphygmomanometer (Perfect Aneroid with automatic valve, ERKA, Bad Tölz, Germany) and the TSE Blood Pressure Monitoring System Software (TSE-Systems, Bad Homburg, Germany). In these mice, proteinuria was also measured weekly in spontaneous urine using Albustix® (Bayer AG). Before the start of the recordings, the mice were trained to this procedure for 4 weeks to prevent false results due to extraordinary stress. To get accustomed to this procedure, we began fixing the mice in a metal restrainer 2–3 times a week following another 3 weeks where we also used the tail cuff and performed test measurements. The weekly results obtained prior to the onset of proteinuria were averaged to a mean BP for each animal; the mean BP at proteinuria was calculated from the mean of the BP at the onset of proteinuria and the BP values of the following week. The final measurement was performed when the mice exhibited advanced LN, and the experiment was terminated at the age of 27–29 weeks.

Proteinuria was assessed semiquantitatively using dip sticks (Albustix, Bayer) at
weekly intervals (0=none;
1=30–100 mg dl⁻¹;
2=100–300 mg dl⁻¹;
3=300–1,000 mg dl⁻¹;
4≥1,000 mg dl⁻¹).