The binding affinity and specificity are the key indicators of a successful antibody. The majority of existing antibodies were raised against either full-length protein antigens (>200 amino acids) or long-peptide (>20 amino acids) antigens. Full-length protein antigens often cause promiscuous bindings in antibody due to the large number of potential binding sites. Long peptides are very difficult to dissolve into water even in the presence of DMSO. They must be denatured prior to conjugation to carrier proteins, which is the major cause of non-specific or off-target binding.
BiCell Scientific® researchers have discovered that short-peptide antigens (13-19 amino acids) allow developing polyclonal and monoclonal antibodies with specific and bona fide binding to target proteins in their native conformation. Because the structural length of an antibody binding site spans 8 amino acids in a target protein, short-peptide antigens contain a limited number (often <10) of potential binding sites. This feature is particularly important for developing “precision” antibodies with improved binding specificity to suit high-resolution applications, such as immunohistochemistry studies that require undenatured proteins to be recognized in situ among millions of other proteins. Short antigen sequences also allow antibodies to be used in protein domain specific or amino acid site specific recognition.
Custom ordering polyclonal antibodies takes 7-12 weeks depending upon antigen sequence. Target proteins will be analyzed by advanced algorithms based upon DeepMind’s AlphaFold protein structures to facilitate the rational design of peptide antigens. Short peptide antigens (13-19 amino acid long) ensures high specificity of antibody binding and allows developing site-specific antibodies that can recognize point mutation, phosphorylation, acetylation, methylation and many other forms of modification in target proteins. Custom ordered antibodies will be manufactured, purified and validated against human or rodent tissues using ELISA and immunofluorescence labeling techniques. Successful binding signals are guaranteed. Projects that fail to produce meaningful binding signals will be fully refunded.
Custom ordering monoclonal antibodies takes 3-5 months depending upon antigen sequence. Target proteins will be analyzed by advanced algorithms to facilitate the rational design of peptide antigens. An antigen binding assay will be implemented to screen for the antibody secreting hybridoma cells. Both recombinant full-length protein antigen and short peptide antigen can be used to make monoclonal antibody. Short peptide antigens also allow making site-specific monoclonal antibodies. Custom ordered antibodies will be manufactured, purified and validated against human or rodent tissues using ELISA and immunofluorescence labeling techniques. One validated hybridoma cell line will be provided in addition to the purified antibody. Successful binding signals are guaranteed. Projects that fail to produce meaningful binding signals will be fully refunded.
Protein modifications that can be targeted for site specific polyclonal or monoclonal antibody production:
– Acetylation; Cleavage sites; Drug binding Isoforms; Glycosylation; Ligand binding; Myristolation;
– Phosphorylation; Prenylation; Splice variants; Sumoylation; Ubiquitination; Mutations/Polymorphisms.
All animal work is performed at St Louis University Department of Comparative Medicine and approved by IACUC under animal use protocol number AUP #2943.
BiCell Tech Team –
Claudin-2 has been a problem in terms of antibody detection for some time. We have tested most commercial monoclonal and polyclonal antibodies and had only occasionally found a polyclonal that works well across applications, including immunostaining of formalin-fixed paraffin-embedded tissues. Unfortunately, polyclonal antibodies purchased from can vendors very over time. We therefore tried to make our own monoclonal antibodies to claudin-2 and failed on 2 different attempts with an approach that had worked well for ZO-1 and occludin monoclonal antibodies. BiCell approached the problem differently in terms of antigen analysis, screening, and other unique aspects of their technique. As a result, we developed several outstanding monoclonal anti-claudin-2 antibodies. The one shown, clone E5, outperforms all previous antibodies, monoclonal and polyclonal, that we have used across all applications, including western blot, immunofluorescent staining of cultured cells, and immunofluorescent staining of tissues.
BiCell Tech Team posting review on behalf of Dr. Jerrold Turner, Dr. Shabnam Abtahi, Harvard Medical School
jhou23 –
Paraffin embedding and subsequent antigen retrieval protocols probably have caused minute changes in target protein folding structure, which make target protein different from that in cryosections or in live cells. Monoclonal antibodies raised against short peptide epitope likely will offer a wide range of binding affinities to suit different protein folding patterns in FFPE, cryosection or live cells, because short peptides in solution assume a wide range of folding possibilities, some of which will resemble FFPE. Full-length protein antigens likely have rigid structures. Antibodies raised against full-length proteins may only recognize a few protein folding possibilities.