Technical Notes: Antibodies to Complex Antigens

Because of their complexity, some of the initial work on chemokine receptors has been performed with polyclonal antibodies derived from peptide immunogens. While these anti-peptide reagents are often useful in the detection of denatured proteins on western blots, they have limited usefulness in the detection of membrane-anchored native proteins.

R&D Systems now uses an alternative approach to monoclonal antibody development: use of overexpressing cells as immunogens. The benefits of antibody products generated by this method is exemplified by our anti-human CCR8 monoclonal antibody (Clone 191704; Catalog # FAB1429P). This antibody can easily detect varying levels of CCR8 expression on peripheral blood monocytes and on the monocytic leukemic cell line THP-1 (Figure 1). These same cells were stained with a commercially available anti-human CCR8 antibody that was generated using a peptide immunogen with a sequence similar to part of CCR8. This polyclonal antibody was unable to detect CCR8 on these cells (Figure 2). Further, R&D Systems’ anti-human CCR8 (Clone 191704; Catalog # MAB1429) antibody can also neutralize the chemotactic action of I-309/CCL1, a CCR8 ligand, with an ED50 of approximately 30 ng/mL (Figure 3).

Although the process of using whole cells as immunogens may be more labor intensive, the resulting antibodies can be superior to those generated from linear peptide immunogens.

Figure 1 Human peripheral blood monocytes
(yellow) or THP-1 cells (green) were stained with 10 µL of R&D Systems’ PE-conjugated anti-human CCR8 monoclonal antibody (Catalog # FAB1429P) or an isotype control reagent (black). When required, erythrocyte lysis following antibody staining was performed using R&D Systems’ RBC Lysis kit (Catalog # WL1000) and staining was quantified using flow cytometric analysis.
Figure 2 The same human peripheral blood monocytes (yellow) or THP-1 cells (green) as used in Figure 1 were stained with 10 µL of another commercially available anti-human CCR8 polyclonal antibody followed by a PE-conjugated porcine anti-goat secondary antibody. Isotype control staining is also shown (black). When required, erythrocyte lysis following antibody staining was performed using R&D Systems’ RBC Lysis kit (Catalog # WL1000) and staining was quantified using flow cytometric analysis. Figure 3 R&D Systems’ recombinant human I-309/CCL1 (Catalog # 272-I) was added to the lower compartment of a 5 µm pore size chemotaxis chamber at varying concentrations. Human CCR8-transfected BaF/3 cells (2.5 x 105 cells in 100 µL) were added to the upper compartment of the chamber. After incubation, cells in the lower chamber were counted using R&D Systems’ Resazurin (Catalog # AR002) as a measure of chemotaxis (orange). To neutralize the I-309/CCL1 (20 ng/mL) effect R&D Systems’ anti-human CCR8 monoclonal antibody (Clone 191704; Catalog # MAB1429) was added to the cells at varying concentrations and chemotaxis was measured as above (blue).