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.
Nanda
Sarita
S
Department of Biochemistry, Daulat Ram College, Delhi, India. saritananda@hotmail.com
Muralidhar
K
K
Kar
S K
SK
D000818:Animals; D000947:Antigens, Helminth / Q000032:analysis; D001407:Bacillus / Q000201:enzymology; D002020:Buffaloes; D003432:Cross-Linking Reagents / Q000737:chemistry; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005370:Filarioidea / Q000276:immunology; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry; D007986:Luteinizing Hormone / Q000032:analysis / Q000276:immunology; D015335:Molecular Probes / Q000737:chemistry; D008970:Molecular Weight; D011817:Rabbits; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry
D000947:Antigens, Helminth; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D007074:Immunoglobulin G; D015335:Molecular Probes; D007986:Luteinizing Hormone; D000516:alpha-Amylases; D005976:Glutaral