Characterizing monoclonal antibodies to antigenic domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin

Figures & data

Figure 1.  Titer of anti-TCblR antibodies. Antibody titer in hybridoma supernatants was determined by ELISA; 100 ng of antigen was used to coat ELISA plates. Peroxidase conjugated goat anti-mouse secondary antibody and TMB substrate were used to detect the binding of anti-TCblR antibody.

Figure 2.  Specificity of antibody binding to cell surface receptors. K562 cells were incubated with Mab followed by FITC tagged anti-mouse IgG and analyzed by flow cytometric separation of antibody positive cells. (A) control mouse IgG, 4°C; (B) TCblR mAb 1-19, 4°C; (C) TCblR mAb 1-19, 37°C.

Table 1.  Binding of monoclonal antibodies to truncated TCblR proteins expressed in HEK293 cells.^*

mAb	AA sequence
	32-233	32-183	32-149	32-233-mycHis	32-94,142-233-mycHis	32-137,166-233-mycHis	32-52,64-233-mycHis
1-10	+++	++	++	++	−	++	−
1-18	++	++	++	+	−	−	−
1-19	++	−	−	+++	++	++	+++
1-23	++	−	−	++	++	++	++
1-25	++	+++	−	++	+	−	+++
2-2	++	+++	+++	+++	+	+	+++
2-3	++	+++	+++	+++	−	++	+++
2-4	++	+++	+++	+++	−	−	++
2-6	++	+++	+++	+++	−	−	+++

* Truncated TCblR proteins were captured in ELISA plates coated with anti-TCblR MAbs and were detected with an anti-TCblR polyclonal antibody. The extracellular TCblR was used as the positive control; normal HEK293 cell culture medium and normal mouse IgG served as negative controls.

OD 2–3:+++; OD 1–2:++; OD 0.5–1:+; OD <0.5: −.

Figure 3.  Diagrammatic representation of antibody binding regions within the TCblR protein. The peptide fragment reacting with specific antibody was identified from the ELISA data in Table 1.

Figure 4.  Effect of antibody in the culture medium on the cellular uptake of TC-Cbl. K562 cells were incubated with 120 pmoles mAb 1-19, 1-23, and 1-25, then 0.026 pmoles of recombinant TC saturated with ⁵⁷CoCbl was added to culture medium and incubated for 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, and 96 h. Cells were collected to measure uptake of TC-Cbl at each time point. MAb 1-25 inhibited uptake of TC-Cbl throughout the 96h culture period.