PMC

(A) BLG-coated ELISA plates were incubated with indicated concentrations of chimeric huIgE (closed symbols) or chimeric huIgG1 (open symbols) anti-BLG antibody clone 8F7.1. Binding of these chimeric anti-BLG antibodies was measured at OD A450 nm. Results (n = 1) are representative of 2 independent experiments. (B) In addition, BLG-coated plates were pre-incubated with indicated concentrations of chimeric huIgG1 anti-BLG antibody clone 8F7.1 followed by adding saturating chimeric huIgE anti-BLG antibody clone 8F7.1. Binding of chimeric huIgE anti-BLG antibody clone 8F7.1 was measured using HRP-labelled huIgE-specific antibodies. Percentage of inhibition of this chimeric huIgE antibody clone 8F7.1 binding by pre-incubated chimeric huIgG1 antibody clone 8F7.1 is shown. Results are expressed as mean ± SD (n = 4); *P<0.01 and **P<0.001 when compared with binding of chimeric IgE anti-BLG antibody clone 8F7.1 without chimeric huIgG1 anti-BLG antibody clone 8F7.1 pre-incubation.

A) Staining of CEA-expressing frozen tumor tissue samples with the chimeric antibody conjugate gave a brighter fluorescence signal compared to staining with the mouse antibody conjugate (p=0.046). Despite the higher dye:mole ratio obtained with the chimeric antibody conjugate, a lower fluorescence signal intensity was consistently observed in the chimeric antibody-stained normal human tissue samples compared to mouse antibody-stained tissue. However, given the significantly brighter signal of chimeric antibody-labeled tumor, the contrast between normal tissue and tumor tissue achieved with the chimeric antibody was greater. N, normal. B) The human colon cancer tumor used to generate the PDOX nude-mouse models was stained with either mouse or chimeric anti-CEA-Alexa 488 antibody and compared to a negative control IgG-488 antibody. The chimeric antibody (b) demonstrated improved binding to the CEA-expressing colon cancer and thus yielded a significantly brighter fluorescence signal (p<0.001) compared to the sample labeled with the mouse antibody conjugate (a). As expected, there was no binding with the IgG-488 antibody.

(A) BLG-coated ELISA plates were incubated with indicated concentrations of chimeric huIgE or chimeric huIgG1 anti-BLG antibody clones 8F7.1, 5D6.1, 11B6.2, 13A5.2, 7D5.1, and 8C3.3. Binding of these chimeric anti-BLG antibodies was measured at OD A450 nm. Results (n = 1) are representative of 2 independent experiments. (B) Western blotting of non-reduced and denatured bovine BLG (18 kDa), and subsequent detection with chimeric huIgE anti-BLG antibody clones 5D6.1 (lane 1), 7D5.1 (lane 2), 8F7.1 (lane 3), 8C3.3 (lane 4), 11B6.2 (lane 5), and 13A5.2 (lane 6). (C) Human FcεRI α chain expressing RBL cells were incubated with saturating chimeric huIgE anti-BLG antibody clones 8F7.1, 5D6.1, 11B6.2, 13A5.2, 7D5.1, and 8C3.3. Binding of these chimeric huIgE anti-BLG antibodies was flow cytometrically measured after detection with FITC-labelled goat anti-human IgE-specific antibodies. Expression level of human FcεRI α chains on RBL cells was examined with a PE-labelled mouse anti-human FcεRI α chain-specific antibody. White histograms represent background staining and grey histograms represent human FcεRI α chain expression or chimeric human IgE anti-BLG antibody binding on RBL cells.

The chimeric antibody bound to the colon tumor thereby fluorescently labeling the tumor. As indicated by the white arrow, small microscopic, sub-millimeter tumor deposits were easily visualized by labeling with the chimeric antibody.