Immunoassays are sensitive, quantitative, rapid and economic tools, widely used in environmental analytical measurements. Synthesis of one of its components, haptens, small molecules that are used both for immunizing animals and to develop the competition assay with the analite measured, is a trial and error process that involves a great amount of chemical synthesis. One potential approach that could be more systematic and would help to diminish the extent of chemical synthesis in the generation of haptens is the use of analyte peptidomimetics.

A vast repertory of candidate peptides can be found in phage displayed peptide libraries, where randomly generated amino acid sequences are fused to coat proteins of the filamentous phage of the fd family Phage libraries are enriched for specific clones by repetitive rounds of affinity selection (biopanning), which includes binding to the desire selector molecule, washing and elution, reinfection of bacteria and growth to amplify the selected phages. After some rounds of selection, phages obtained as supernatans of bacterial cultures are tested for binding to the target by an enzyme linked immunosorbent assay (ELISA). Positive supernatants are titulated and employed in a competitve assay with the molecule under study to test the specificity of that union. DNA from phages is extracted and sequenced to deduce the aminoacid composition of the displayed peptide.

Using monoclonal and polyclonal antibodies against the pesticides molinate and atrazine, it was possible to isolate peptide-bearing phages that can be used to set up competitive ELISAs for this pesticide. This strategy allowed to develop assays with similar or better sensitivities than that of the conventional ones. As phage particles are very robust and can be easily purified, we have found a simple and rapid method for the development of immunoassays. Phage particles can be produced in large amounts and constitute highly standardize reagents that substitute the conventional competing antigens, diminishing the extent of chemical synthesis and lowering the amount of organic solvents waste. In addition the free peptide can be use as surrogate standard substituting for highly toxic components, such as fungal toxins, dioxins, etc.

Soledad Cardozo

Phage: virus that attacks bacteria.