Purification of RBC Agglutinating Antibodies by Affinity Chromatography on Peptides Representing Specific Variant MNS Antigens

Aim  Variant MNS (vMNS) phenotypes are characterised by the presence of multiple antigens that provoke complex antibody responses. The hypothesis tested was that red cell‐agglutinating antibodies of a single specificity could be purified by affinity chromatography on peptides representing vMNS antigens. The Vw and Hut/Mut antigens vary only at glycophorin residue 28, Vw methionine‐28, Hut and Mut lysine‐28. The Mur peptide TYPAHTANEV was aa 33 to 42 of GpMur. For each experiment a 5 ml pool of 4 and 10 sera that agglutinated Mi‐positive cells of known MNS‐peptide‐ELISA activity, was subjected to affinity chromatography on a single peptide. Result  For a serum pool that was strong peptide‐ELISA positive with the lysine‐28 peptide but was methionine‐28 peptide negative, the antibody eluate was lysine‐28 pos by ELISA and agglutinated all MUT and HUT pos cells tested but not Vw cells or Mi(a) negative cells. For the MUT antigen the hypothesis that a rbc‐agglutinating antibody of a defined specificity could be purified by affinity chromatography on a linear peptide representing the antigen was confirmed. In a similar fashion a specific anti‐Mur was prepared. However, for one serum pool the antibody eluted from the Mur‐column reacted with Hut (MiII) as well as Mur‐positive cells, suggesting that Mut.Mur was recognised as a single antigen. Conclusion  Purified monospecific polyclonal antibodies that define vMNS phenotypes may be useful in resolving difficult serological investigations. The complexity of responses to vMNS glycophorins is confirmed by the recovery of an antibody reacting with sequentially adjacent antigens as a single entity.