Thermostable alpha-amylase conjugated antibodies as probes for immunodetection in ELISA

Thermostable alpha-amylase from B. licheniformis has been conjugated with high efficiency to goat antibodies against human, mouse, and rabbit immunoglobulins to prepare second-step reagents which can be used in Enzyme Linked Immunosorbent Assays (ELISA). Various conjugation methods, such as one- and two-step glutaraldehyde coupling and cross-linking, using heterobifunctional reagents such as sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carbonate (sulfo-SMCC) and N-succinimidyl-S-acetylthioacetate (SATA), yielded active alpha-amylase labeled second antibodies. Such conjugates had molecular sizes ranging between 200-300 kDa. Filter sterilized solutions of conjugates, when stored at 37 degrees C for two weeks, retained 32% of their biological activity and were thermostable even after keeping for 1 h at 90 degrees C.