Table 1.
Specificity of endostatin ELISA for mouse and rat samples.
Table 2.
No cross-reactivity with or interference of other proteins.
Table 3.
Intra- and inter-assay precision of mouse/rat endostatin ELISA.
Table 4.
Limit of detection.
Table 5.
Lower limit of quantification.
Fig 1.
Robustness of the endostatin ELISA.
(A) Influence of assay incubation temperature (23°C vs. 30°C) on recombinant endostatin (STD 2–5) and sample OD. (B) Influence of assay buffer and detection antibody solution temperature (23°C and 8°C) as well as 20 minutes delayed pipetting of samples (= drift over plate) on delta OD of recombinant endostatin (STD 1–7).
Fig 2.
Dilution linearity of recombinant and endogenous endostatin.
Dilution recovery in % (y-axis) of recombinant (rec.) mouse endostatin spiked in seven mouse serum and plasma samples and afterwards diluted 1:2, 1:4 and 1:8 with assay buffer and of endogenous endostatin of seven mouse serum and plasma samples diluted 1:2 and 1:4 with assay buffer. Dilution recovery (%) of each sample was calculated and should be within ± 25% (grey area).
Table 6.
Accuracy (spike recovery) of mouse/rat endostatin ELISA.
Fig 3.
Freeze/thaw stability of recombinant and endogenous endostatin.
(A) Delta OD of recombinant endostatin (STD 2–7) and (B) concentration (nmol/L) of endogenous endostatin (sample 1–7) subjected to up to four freeze/thaw (F/T) cycles.
Fig 4.
BCL2tg and ETV6/RUNX1tg;BCL2tg mice develop glomerulonephritis.
Representative pictures of kidney sections of wild type (wt), BCL2tg and E/Rtg;BCL2tg mice (n = 2 for each genotype) are shown. Pictures in lower panels (middle and right) correspond to further progressed glomerulonephritis than in upper panels (middle and right). Periodic Acid Schiff (PAS) staining of the kidney sections indicates enlarged glomeruli and crescents in BCL2tg and E/Rtg;BCL2tg mice.
Fig 5.
BUN and endostatin levels in young and older E/Rtg, BCL2tg, double transgenic and control mice seem to display the kidney disease phenotype.
Tukey’s boxplots indicate (A) blood urea nitrogen (BUN) levels (mg/dL) and (B) endostatin levels (nmol/L) in serum of 9–13 weeks and more than 24 weeks old wild type (wt), E/Rtg, BCL2tg and E/Rtg;BCL2tg mice. For BUN, n = 6, n = 8, n = 20, n = 5 for younger and n = 7, n = 11, n = 7 and n = 5 for older mice. For endostatin n = 8, n = 8, n = 19, n = 5 for younger and n = 10, n = 10, n = 8 and n = 6 for older mice. + indicates significance to wild type control (+ p<0.05) and * to young animals of same genotype with ** p<0.01 and * p<0.05.
Fig 6.
Increased BUN and endostatin levels of transplanted mice with impaired kidney function.
(A) BUN (mg/dL) and (B) endostatin (nmol/L) in serum of immunocompromised NSG mice receiving cells from either control (ctrls) animals, or terminally diseased BCL2tg or E/Rtg;BCL2tg mice, n = 4, n = 5 and n = 7 for BUN, n = 5, n = 13 and n = 8 for endostatin, respectively, measured 40 weeks after transplantation. Significance to controls is stated with **** p<0.0001, ** p<0.01 and * p<0.05.