We Select the Best Immunogen for the Desired Antibody
R&D Systems has long recognized the need for reliable anti-GPCR antibodies and the difficulties in developing them. We believe that generating anti-GPCR antibodies that display the maximum immunogenicity with minimal antibody cross-reactivity begins with using the right antigenic epitope.
When generating our antibodies for GPCRs, we utilize a combination of immunization techniques to get the best selection of antibodies. We use short and long synthetic peptides to generate antibodies against specific domains and extracellular loops, respectively. Using immunogenic peptides, though, is not ideal for generating antibodies for applications that require the GPCR’s native structure, such as flow cytometry, as the peptides often lack the conformation and relevant post-translational modifications. Additionally, these antibodies tend to not be suitable for receptor mapping due to their lack of specificity and functional activity. To produce antibodies against the full, native GPCR, we pioneered the use of whole cell transfectants as immunogens (Figure 3).
Many commercial GPCR antibodies are made only using peptides; however, we have been using whole cell transfectants to generate anti-GPCR antibodies for decades. This commitment is illustrated by a 1999 paper published in The Journal of Biological Chemistry and through ongoing efforts in a 2017 Nature paper. Authors of the papers partnered with R&D Systems to develop antibodies to intricately map the epitopes of the CCR5 chemokine receptor and PAR2, respectively, to study receptor structure and function.8,9
We Screen Using Native GPCR-Expressing Cells
Antibody screening is performed to identify which animals or hybridoma clones produce high levels of antigen-specific antibodies. To screen for anti-GPCR antibodies, we use samples that naturally express GPCRs to ensure that our antibodies recognize the native protein (Figure 4). Antibody specificity is then verified using transfected cells and irrelevant transfectants. (Figure 5).
Our Validation Protocol is Unparalleled
R&D Systems has always taken rigorous steps to test our antibodies. By developing and manufacturing over 90% of our antibodies, we can not only ensure we use optimized products during product development, but we also can also execute quality tests to ensure lot-to-lot consistency and outstanding performance, which most antibody suppliers are unable to do since they don't manufacture their own product. Additionally, we continuously confirm the specificity of our antibodies by testing using a variety of applications such as true and irrelevant transfectants (Figure 5) and knockout cell lines (Figure 6). We also perform additional tests for select GPCR antibodies, such as live cell staining, which confirms detection of low expressing GPCRs (Figure 7). These tests ensure that we provide the highest quality and most consistent anti-GPCR antibodies.
Figure 7. R&D Systems CCR7 Antibody is Tested at Different Temperatures to Ensure Optimal Staining at Physiological Conditions. CCR7, like all GPCRs, are fluid structures. These receptors move within the lipid membrane as well as undergo internalization and endocytic recycling. These factors can mask the antibody binding site, resulting in no staining by flow cytometry. We examined the effect of temperature on CCR7 staining to determine if our antibody exhibits optimal staining at physiological temperatures (37 °C). Human whole blood was stained with a Mouse anti-Human CCR7 APC-conjugated Monoclonal Antibody (Catalog #AB197A) and a Mouse anti-Human CD4 PE-conjugated Monoclonal Antibody (Catalog #FAB3791P) at 4 °C, room temperature (RT), or 37 °C. Cells were washed and red blood cells were lysed with Flow Cytometry Human Lyse Buffer (10X) (Catalog #FC002) prior to the final wash and flow analysis. All products listed are from R&D Systems.