Developing a Custom Polyclonal Antibody for Detection of a Phosphorylated Protein

Peter was focused on studying phosphorylation regulation for a group of proteins involved in signaling pathways. To study this, he needed highly specific antibodies that would only bind with the protein in its phosphorylated state.

He initially thought that it would be necessary to develop a monoclonal antibody against the full-length phosphorylated protein and then screen to find antibodies that wouldn’t recognize the non-activated protein. But, we explained that he could save a significant amount of time and money by immunizing with a synthetic peptide containing the phosphorylated amino acid and using a quick two-step affinity purification process to isolate highly specific antibodies that would recognize only an epitope where the serine residue was phosphorylated.

We began by designing a peptide that offered maximum epitope variability surrounding the phosphorylated serine residue. From there, we synthesized both a phosphorylated form of the peptide and a non-phosphorylated form of the peptide. We immunized with only the phosphorylated peptide, saving the non-phosphorylated peptide for the later ELISA and affinity purification steps.

At the first production bleed, we ran the ELISA against both peptides to measure antibody titers and see if there would be a stronger response against the phospho peptide. The ELISA did confirm a strong response in both animals and showed slightly higher titers against the phosphorylated peptide.

When the time came to affinity purify the serum, we first affinity purified against the non-phosphorylated peptide to capture antibodies that recognized common epitopes that existed on both the phosphorylated and non-phosphorylated peptides (and, by extension, both forms of the native protein). We then took the flow-thru from this column and affinity purified it against the phosphorylated peptide to isolate only those antibodies that would be able to recognize any epitopes containing the modified phosphorylated serine.

To confirm everything, we ran a series of six ELISA assays to measure each stage of the purification and confirm that antibody from the phospho column did not recognize the non-phospho peptide. The ELISA tests confirmed specificity to the phosphorylated epitope and showed a promising yield of 1.2 mg purified antibody.

Peter was anxious to see if the purified antibody would be able to distinguish between the phosphorylated and non-phosphorylated proteins. The following week, he sent us a congratulatory email describing how the antibody was able to perfectly detect the activated phosphorylated protein and showed no reactivity against the non-phosphorylated protein.

For a fraction of the cost and time associated with developing a monoclonal antibody, Peter was amazed with the results and moved ahead with a full study to develop antibodies against the rest of the phosphorylated proteins involved in the signaling pathway. We also began a large-scale affinity purification of the serum from the original project so that he would have plenty of working antibody for the foreseeable future.

Around a year after the final batch of antibodies was developed and purified, he again expressed his gratitude and sent us a copy of the paper that his lab had just published using the collection of custom phosphospecific antibodies.