Synthetic Peptide Mimotopes of blood Group Antigens for Red Cell Antibody Screening

Background: Antibody screening is performed both routinely in the blood group typing procedures for donors and patients and in more detail as part of special investigations for transfusion-dependent patients such as those suffering from Sickle Cell Disease and Thalassaemia. However, despite the care taken, intrinsic limitations of traditional serological diagnostic tests mean that alloimmunisation of pregnant women and multiply transfused patient may still go undetected, resulting in Hemolytic Disease of the Newborn or Hemolytic Transfusion Reactions, respectively. Furthermore, although the genes encoding the majority of blood group antigens have been characterised, the expression of recombinant gene products and the subsequent determination of protein structure that might lead to novel diagnostic reagents have proved more difficult to achieve.

Methods: Phage Display libraries that express random peptide sequences (~10¹⁵) on the virion surface were screened using a series of monoclonal antibodies and an anti-RhD polyclonal preparation to identify peptides that mimic epitopes of clinically important blood group antigens. The peptides thus identified, were then synthesised in macroarrays and evaluated using SPOTs (Simple Precise Optimal Test system) in a step towards development of a novel diagnostic antibody-screening assay.

Results: The combined approach of phagepeptide display and SPOTs proved powerful. From 490 phage-peptides selected by biopanning, 86 mimotopes bound their cognate antibody in SPOTs assays and represented the clinically important blood group antigens RhD (including epitopes 1.1, 3.1 and 6.3), RhE, Rhe, Fy^a and Fy^b. These peptides ranged in size from 7 to 15 residues and included 7-mers that were constrained at their termini by a di-sulphide bridge. Further SPOTs analyses showed 26 of these phage-peptides (12 RhD, 3 RhE, 1 Rhe, 2 Fy^a and 8 Fy^b) have the appropriate strength of signal and binding specificity for inclusion in any future diagnostic antibody-screening assay. A subset of these peptides has been further tested. These peptides were immobilised on polystyrene microspheres and shown to specifically bind their cognate antibodies in both (1) monoplex gel agglutination immunoassays and (2) microsphere-based, multiplex suspension arrays.

Conclusions: We have shown that, regardless of whether or not the mimotopes resemble the original antigen sequence, they bind their cognate antibodies specifically and are therefore genuine mimics of the natural antigenic epitopes. It has also been demonstrated that the context in which a peptide is presented is fundamentally important for antibody recognition. The value of the phage-peptide approach in identifying mimotopes to clinically significant blood group antigens has also been established. Moreover, these peptides could be used in a single, comprehensive screening assay and eliminate many of the problems associated with agglutination assays and may herald the possibility of a synthetic, diagnostic array for routine antibody screening for all patients and donors and patients in the near future.