Mouse Anti-NPrPSA Recombinant Antibody (clone 13D9) (CAT#: PABL-288)

Figure 1 Binding characteristics of mAbs 735 and 13D9.

(A) and (B) The specificities of mAbs 735 (A) and 13D9 (B) were determined by indirect ELISA against HSA conjugates of NAcPSA (B), NPrPSA (O), and NBuPSA (P). (C) and (D) Graded amounts of pure NAcPSA, NPrPSA, and NBuPSA polysaccharides were used to competitively inhibit binding of mAb 735 to NAcPSA-HSA (C) or mAb 13D9 (D) to NPrPSAand NBuPSA-HSA antigens in ELISA experiments. Bars represent the inverse amount of inhibitor required for 50% inhibition of mAb binding to each coating antigen without the presence of polysaccharide inhibitor.

Pon, R. A., Biggs, N. J., & Jennings, H. J. (2006). Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment. Glycobiology, 17(3), 249-260.

Figure 2 Effects of ManPr and ManBu on PSA expression in RMA-s tumor cells.

RMA-s cells in log-phase growth were subjected to a 3-day course of 10 mM ManPr (þManPr), ManBu (þManBu), or medium alone (untreated), followed by surface staining with mAbs 735 (A) and 13D9 (B) and analysis by flow cytometry. Gates were set on viable cells only, as determined through propidium iodide exclusion, and 10 000 events were acquired. RMA-s autofluorescence appears within the first decade of each histogram. (C)–(E) Mixed polysaccharide inhibitors of defined length and composition (NPr:NAcPSA; NBu:NAcPSA) were prepared, as described in the Materials and methods section and used in competitive inhibition studies. RMA-s tumor cells were cultured for 3 days with media alone (C), 10 mM ManPr (D), or ManBu (E), stained with either mAbs 735 [(C)– (E)] or 13D9 [(D) and (E)], which were pretreated with 1 mg of the indicated mixed inhibitor, and evaluated by flow cytometry. The percentage of inhibition was calculated relative to the mean channel fluorescence values obtained from each respective mAb without polysaccharide inhibitors.

Pon, R. A., Biggs, N. J., & Jennings, H. J. (2006). Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment. Glycobiology, 17(3), 249-260.

Figure 3 The effects of ManPr and ManBu sialic acid precursors on PSA expression on NT2 neurons at various stages of maturation.

NT2 neurons at different maturation stages were prepared. Cells were then treated with 10 mM ManPr (þManPr), ManBu (þManBu), and medium alone (untreated) for 3 days, followed by surface staining with mAbs 735 and 13D9 and evaluation by flow cytometry. Histogram profiles are based on viable cells found within the respective gates defined within the FS/SS scattergrams. Autofluorescence was adjusted to appear within the first logarithmic decade of each histogram. (A) Relatively immature neurospheres; (B) moderately mature neurite-expressing neurospheres; and (C) fully mature classical neurons. Note the homogeneity of either mAb 735 or 13D9 surface staining within the neurosphere fraction (A) as opposed to both neurite-expressing neurospheres and classically derived neurons [(B) and (C)].

Pon, R. A., Biggs, N. J., & Jennings, H. J. (2006). Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment. Glycobiology, 17(3), 249-260.

Figure 4 Effects of ManPr and ManBu sialic acid precursors on polysialylation of NCAM in NT2 neurons by western blot analysis.

(A) NT2 neurospheres (N), (B) neurite-expressing neurons (NENs), or classical neurons (CNs) were derived as previously described, followed by a 3-day treatment with either 10 mM ManPr or ManBu. Proteins from whole-cell lysates were resolved by SDS–PAGE (7.0%), transferred onto PVDF membranes, and probed with mAbs 735 (PSA), 13D9 (NPr- or NBuPSA), or OB11 (NCAM). (Neuron-f, untreated; neuron-Pr, ManPr treated; and neuron-Bu, ManBu treated.)

Pon, R. A., Biggs, N. J., & Jennings, H. J. (2006). Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment. Glycobiology, 17(3), 249-260.

Figure 5 Complement-dependent cytotoxicity of ManBu precursor-treated NT2 neurospheres with mAbs 735 and 13D9.

NT2 neurospheres labeled for 1 h with Na2[ 51Cr]O4 were combined with graded amounts of mAbs 735 or 13D9 in the presence of 5% baby rabbit complement for 4 h at 378C. Released 51Cr was measured by scintillation counting and the percentage of specific cytotoxicity calculated, as described in the Materials and methods section. Maximum release was determined in the presence of cetrimide and spontaneous release in the presence of medium alone and was consistently,20%.

Pon, R. A., Biggs, N. J., & Jennings, H. J. (2006). Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment. Glycobiology, 17(3), 249-260.