Human α- and β-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-β-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (β-enolase). Selective anti-α- and anti-β-enolase antibodies were obtained by affinity chromatography on either α- or β-enolase-Sepharose columns. On Western blots, antibodies directed against human β-enolase, did not react with human α-isoenzyme, but recognized pig and rat β-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both α- and β-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing α-enolase epitope (the C-terminal portion of the epitope 3 - S262PDDPSRYISPDQ273) reacted with anti-α-enolase, while the other also derived from the α-enolase sequence (epitope 2 - N193VIKEKYGKDATN205) was recognized by anti-β-enolase antibodies. Interestingly, neither anti-α- nor anti-β-antibody reacted with a peptide corresponding to the epitope 2 in β-enolase (G194VIKAKYGKDATN206). Further analysis showed that substitution of E197 with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native β-enolase.
Keywords: AP, alkaline phosphatase; BSA, bovine serum albumin; Cross-reactivity; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray injection; Enolase purification; Epitope prediction; HRP, horse radish peroxidase; IgG, immunoglobulin G; LC, liquid chromatography; MS, mass spectrometry; Mass spectrometry; MeOH, methanol; OPD, ortho-phenylenediamine; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecylsulfate; Specific antibodies; TBST, 20 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween-20; UPLC-Q-TOF-MS, ultrapressure liquid chromatography, quadrupole-time-of-flight mass spectrometer; WB, western blotting; pNPP, para-nitrophenyl phosphate.