1. Potent enzyme inhibitors derived from dromedary heavy‐chain antibodies
Analysis of antigen‐specific antibodies. (A) Presence of antigen‐specific antibodies in total serum as a function of time. Antigens were immobilized in microtitre plates and incubated with total serum (at 8000‐fold dilution) from different blood samples (days 0,7,14,21,28,35,42,54). α‐Amylase‐ (▪) and carbonic anhydrase‐ (▴) specific immunoglobulins were subsequently detected with a rabbit anti‐dromedary IgG antiserum and anti‐rabbit IgG‐alkaline phosphatase conjugate. Optical densities were measured after 10 min. (B, C) ELISA experiment with individual immunoglobulin subclasses isolated from serum collected at day 54. Bound IgG1 (▪), IgG2a (▴), IgG2b (▾) and IgG3 (●) to solid‐phase coated α‐amylase (B) and carbonic anhydrase (C) was detected as described above. (D) Analysis of amylase‐binding compounds in serum collected at days 0, 28 and 54. Serum samples (200 μl) were incubated with immobilized α‐amylase (50 μl wet gel). After washing, beads were resuspended in 100 μl non‐reducing (lanes 1–3) or reducing (lanes 4 and 5) sample buffer and 10 μl aliquots were loaded on 10% SDS–PAGE. Lane 1, day 0; lanes 2 and 4, day 28; lanes 3 and 5, day 54. SDS–PAGE was stained with Coomassie Blue.
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EMBO J, Volume: 17, Issue: 13, Pages: , First published: 01 July 1998, DOI: ( /emboj/ )