Mouse Anti-NCAM1 Recombinant Antibody (clone 5A2) (CAT#: NS-054CN)

Figure 1 The specific binding of chimeric monoclonal ch.MK1 and monoclonal 5A2 antibodies to native NCAM.

NCAM molecules were precipitated from IMR-5 (NCAM+ neuroblastoma) cell lysates by ch.MK1 or 5A2 Abs plus Protein A sepharose beads. Precipitated NCAM was then analysed in a Western Blot and NCAM bands were detected using the murine reference antibody 123C3. Cell lysates were pretreated with neuraminidase to digest polysialic acid substitutes that could weaken the precipitation step. Abbreviations used: 5A2 contr., immunoprecipitation with 5A2 Abs without cell lysate as a control; MK1 contr., immunoprecipitation with ch.MK1 Abs without cell lysate as a control; 5A2, immunoprecipitation with 5A2 Abs with cell lysate; MK1, immunoprecipitation with ch.MK1 Abs with cell lysate; 123C3, immunoprecipitation with 123C3 Abs with IMR-5 cell lysate.

Figure 2 Murine monoclonal anti-NCAM antibody 5A2 specifically binds to NCAM on viable cells.

IMR5-75 (NCAM+ neuroblastoma) was incubated with 5A2 or ch.KLH Abs as a control. Abs reacting with cells were detected by goat anti-human IgG-biotin and streptavidin-PE. The results clearly demonstrate binding of 5A2 Abs to NCAM on cell surfaces.

Figure 3 The inhibitory effect of anti-NCAM Abs on subcutaneous IMR5-75 tumor growth in SCID mice.

Prior s.c. injection of IMR5-75 cells mice were treated i.v. with anti-ASGM1 (asialo-GM1) antiserum to deplete murine NK cells in order to synchronize tumour growth. Groups of 6 SCID mice were subcutaneously challenged with 2 x 10⁷ IMR5-75 NCAM expressing human neuroblastoma cells to induce subcutaneous growth of a tumor nodule. At day 14 mice were treated intravenously with 2 x 10⁷ freshly isolated human PBMC plus 100 µg 5A2 Abs (closed circles) or PBMC alone (open circles) as a control. Treatment was intensified on day 19, 21, and 24 by injecting 2 x 10⁷ freshly isolated human PBMC plus 100 µg 5A2 Abs intravenously and an additional intralesional injection of the same doses. Tumor growth was monitored by three-dimensional measurement by calliper and tumor volume was calculated (volume [mm³] =4/3*3.14159*x/2*y/2*z/2). From 6 mice per group 5 mice per group entered the analyses and one mouse per group was censored as an outlier. At day 25 and 26 tumor sizes differed significantly between experimental and control group using the students t-test to compare tumor sizes at each time point.