Maximizing Specificity and Affinity with a Custom Monospecific Peptide Antibody

Mary contacted us to find out how much it would cost to develop a monoclonal antibody against a novel Alzheimer’s protein that her lab had recently sequenced. After discussing the high costs associated with developing monoclonal antibodies and the disadvantages that they present in comparison to polyclonal antibodies, we recommended that she consider having us generate polyclonal antibodies against three peptide sequences corresponding to unique regions of this novel protein.

We explained that for less than the cost of a single monoclonal antibody project, she could have antibodies against three distinct regions of her protein with specificity that approaches monoclonal antibodies, but with the superior affinity and robustness of polyclonal antibodies.

We ran the protein sequence through our innovative antigen prediction algorithms and offered five peptide sequences that we felt would correspond to exposed regions of the native protein. After analyzing homology and known binding patterns, we narrowed this down to three sequences.

After synthesizing the three peptides, we conjugated each peptide individually to a KLH carrier protein and immunized each conjugated peptide into a separate pair of animals.

At the first production bleed, we ran an ELISA for each project against the immunizing peptide to compare antibody titers against the peptide sequence in the first production bleed against the pre-immune serum. Each animal showed exceptional titers of greater than 1:500,000 against the peptide sequence and confirmed that a strong and specific immune response had occurred.

Four weeks later, after the 3rd production bleed was taken, we prepared to affinity purify the serum against the peptide by creating an affinity column with the peptide as the capture antigen. Because both animals showed a strong response, we combined serum from both animals to maximize epitope variability against the peptide sequence.

After affinity purifying the serum against the peptide sequence, we ran a series of ELISA tests to measure antibody activity against the peptide in the starting serum, the flow-thru from the column and in the final purified antibody eluted from the column. The ELISA tests confirmed that no antibody against the peptide remained in the serum and that each project had yielded between 2.5 mg and 4 mg of antibody specific to the peptide.

Excited, Mary began assaying the native protein in Western Blot and IHC assays to see if the purified antibodies would recognize the native protein. Three weeks later, she sent a glowing email explaining that two of the antibodies had worked great for Western Blots and that one of the antibodies had worked beautifully for IHC.

Six months later, Mary contacted us again to discuss two more novel proteins that her lab had recently discovered. She also couldn’t resist mentioning that the paper her lab had written using the previous antibody had just been accepted by a prestigious research journal.