Peptides for Immunoenzymatic Assays Immobilized on Microplates

A simple, fast, and inexpensive protocol has been described that shows how immobilized synthetic peptides can be bound to plastic surfaces for standard ELISA, which could be used as an alternative to the common technique of conjugating peptide antigens to carrier proteins.

Scientists at the University of Sassari (Italy) devised the technique, which is based on the use of maleimide-activated bovine serum albumin or keyhole limpet hemocyanin as a protein anchor adsorbed on the polystyrene surface of the microtiter plate. Following adsorption of the carrier protein, sulfhydryl-containing peptides are cross-linked with an in-well reaction, allowing their correct orientation and availability to antibody binding, avoiding the time consuming steps needed to purify the hapten–carrier complexes.

Conventional in-solution maleimide-mediated coupling reaction (ISC) was performed by using the Imject Maleimide Activated Immunogen Conjugation kit (Pierce; Rockford, IL, USA). Nine synthetic peptides (Nurex; Sassari, Italy) with length ranging from 10 to 15 amino acids were designed to display different hydrophobic/hydrophilic characteristics and to carry either an N or C terminal cysteine, to allow cross-linking with maleimide-activated carrier proteins. The immunoreactivity of peptides was tested by using both monoclonal and polyclonal antibodies in standard ELISA assays, and compared with established coating methods.

The efficiency of direct cross-linking method (DCLM) was tested with four different peptides and subsequent standard indirect ELISA with polyclonal antibodies was performed. All the sera specifically recognized the corresponding specific antigens when directly cross-linked onto the multiwell plate. Results in terms of signal strength were comparable to that obtained with ELISAs performed by using plates coated with ISC conjugates.

The authors concluded that DCLM proved to be simple, reproducible, cost-effective, and suitable with peptides of different lengths and hydrophobic/hydrophilic characteristics. The DCLM can be considered a straightforward, faster, and much more convenient alternative to standard coupling protocols, allowing saving time and cut costs in ELISA applications. When considering its time- and cost-effectiveness, DCLM may prove to be applicable also to automated systems for high-throughput peptide screenings. The study was published in the August 2012 edition of the Journal of Immunological Methods.

Related Links:
The University of Sassari
Pierce
Nurex