Mouse Anti-RPRD1B Recombinant Antibody (clone 3E10) (CAT#: FAMAB-0671WJ)

Figure 1 Examination of specificity and sensitivity of antibodies.

(A) ELISA assays were performed to detect the titer and specificity of ascites from different CREPT hybridoma clones. (B) ELISA assays were performed to detect the difference of ascites from individuals injected with the same CREPT hybridoma. (C) Sandwich ELISA assay was performed, coating with different dilutions of CREPT multiple clonal antibodies. Different concentrations of GST-CREPT proteins were added to detect the sensitivity of the antibody. Minimal concentration was determined as the concentration with no significant difference to base level of BSA. (D) Capture ELISA assay was performed, coating with CREPT MAb 3E10. Different concentrations of GST-CREPT were used to detect the minimal concentration of CREPT protein that the antibody recognized.

Ren, F., Wang, R., Zhang, Y., Liu, C., Wang, Y., Hu, J., ... & Chang, Z. (2014). Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 401-408.

Figure 2 Epitope mapping of CREPT MAb 3E10.

(A) HEK293T cells were transfected with Flag-tagged CREPT or control vector. Cell lysates were blotted with MAb 3E10 and then, after stripping the membrane, with an anti-Flag antibody. (B) HEK293T cells were transfected with Myc-tagged CREPT and Myc-tagged p15RS. Western blot was performed using the indicated antibody. No cross-reaction was observed. (C) Random clones from the whole library were aligned to full-length CREPT sequence. (D) Positive clones binding 3E10 were enriched by a sorting process. (E) Alignment of positive clones. Green lines represent the forward sequences and red lines represent reverse ones. The overlapping nucleotide sequences of positive yeast clones were selected and aligned to full-length CREPT sequence (marked in red box). The below sequence of 160-168 amino acids, corresponding to the red box, was shown for the comparison with the sequence of p15RS. The sequences of the epitope in different animals are shown. (F) HEK293T cells were transfected with Flag-tagged full-length RPR domain and CCT domain of CREPT. Cell lysates were detected with either an anti-Flag antibody or monoclonal antibody 3E10.

Ren, F., Wang, R., Zhang, Y., Liu, C., Wang, Y., Hu, J., ... & Chang, Z. (2014). Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 401-408.

Figure 3 Application of monoclonal antibody 3E10.

(A) Protein levels of CREPT in different cell lines. Western blot was performed with a CREPT MAb 3E10. (B) mRNA levels of human and mouse homologous sequence in different cell lines. RT-PCR assay was performed using the indicated cells.

Ren, F., Wang, R., Zhang, Y., Liu, C., Wang, Y., Hu, J., ... & Chang, Z. (2014). Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 401-408.

Figure 4 ELISA assays were performed using different dilutions of CREPT monoclonal antibody 3E10 ascites from mouse hybridoma cells as a primary antibody.

An irrelevant antibody was used as negative control.

Ren, F., Wang, R., Zhang, Y., Liu, C., Wang, Y., Hu, J., ... & Chang, Z. (2014). Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 401-408.

Figure 5 ELISA assays were performed with different dilutions of CREPT monoclonal antibody 3E10 produced in supernatant from HEK293T cells expressing the chimeric antibody.

The supernatant from HEK293T cells transfected vector was used as negative control.

Ren, F., Wang, R., Zhang, Y., Liu, C., Wang, Y., Hu, J., ... & Chang, Z. (2014). Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 401-408.

Figure 6 Western blot analysis was performed in colon cancer samples.

CREPT monoclonal antibody 3E10 produced in mouse ascites and supernatant produced by 293T cells were used as primary antibodies to detect colon tumor tissues. P, paired non-tumor tissue; T, tumor tissue from same patient.

Ren, F., Wang, R., Zhang, Y., Liu, C., Wang, Y., Hu, J., ... & Chang, Z. (2014). Characterization of a monoclonal antibody against CREPT, a novel protein highly expressed in tumors. Monoclonal antibodies in immunodiagnosis and immunotherapy, 33(6), 401-408.