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A key step for producing antibodies against a protein is selecting the right peptide fragment. This factor will fundamentally determine the quality of the antibody. One major drawback of antibodies is that not all antibodies are suitable for all techniques. This is because the structure of a protein and the accessibility of the epitope vary in line with experimental conditions. Biosiris has responded by developing a method that optimizes the choice of a well-suited peptide sequence, specifically designed to meet your precise needs.

Unique method

The Epiros™program has been carefully developed to improve the prediction of epitopes. This is achieved by focusing on the minimal epitope size, optimal selectivity and maximum sensitivity in your immunoassay.

Epiros™ consists of three steps:

Firstly, we characterize all potential linear epitopes in a protein sequence. The second step involves our calculating their structures and keeping those epitopes with the characteristics that will enable us to obtain high affinity antibodies. Finally, we select the fragment(s) that are most likely to be active in your immunoassay.

Epiros™ will select potential linear epitopes :
Regarding antigenicity, it is widely considered that all the solvent-accessible fragments of protein 3D structures represent potential antigen. This antigenic surface is effectively a 3D patchwork of conformational and linear epitopes. In a first instance, Epiros™ detects the solvent-accessible linear fragments - NB the linear ones are of importance in regard to the production of antibodies induced by peptides. Epiros™ prediction uses a wide range of the most modern techniques in epitope prediction.

Epiros™ will improve prediction through a structure study :
Antibodies have to be able to react successfully with the peptide used to produce this antibody as well as with the native protein. This cross-reaction demands a structural homology between the peptide and the equivalent fragment in the protein. Consequently, not all the selected linear epitopes will lead to epitopes that are of relevance. Through the advanced calculation and analysis of the structure of each putative epitope, Epiros™ is able to select the more suitable fragments. In addition, Epiros™ can efficiently study the type and number of residues of the epitope accessible to the antibody.

Epiros™ will choose the best epitope according to your immunoassay :
Last but not least, you should be aware that the structure of your protein may vary with your assay. Each immunoassay is performed under conditions that can alter the structure of the antigen and as a result influence antibody-antigen recognition. Significant factors contributing to changes in the epitope structure are denaturation by detergent and chemical modification by fixatives or other chemicals. We ascertain whether the antibody will be used on a native, a detergent- denaturated or a fixative-modified structure of your protein. In line with this, we will then select from either a flexible or a more rigid region of the protein, a turn or a helix.

Case studies

Using this theoretical strategy and an experimental strategy based on the production of anti-peptide antibodies, three monoclonal antibodies were produced against the H,K-ATPase membrane protein. The result of this was that all three reacted against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western Blots. This clearly demonstrated that not only they recognize the native and the SDS-denaturated ionic pump but also that the epitopes are located at the surface of the native ATPase. Antibody Kd were in the range 2-10 X 10-8M.

To study the distribution of the ClC-2 chloride channel in rat and human epithelial tissues, a polyclonal antibody was required. Using our strategy, we defined a fragment of rat ClC-2 chloride sequence and were able to successfully produce the corresponding antibody. The experimental studies cleary confirmed that this antibody was able to react with both the rat and the human ClC-2 isoforms. furthermore, it confirmed that it was not reactive against other member of the ClC family, whilst it was active in immunoprecipitation and immunocytochemistry. Therefore, the results were exactly as we anticipated.

Conclusion

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