Aim: To prepare humanized anti-digoxin(Dig) antibodies(single chain Fv, scFv) from a human phage antibody library and construct anti-Dig diabody vector.
Methods: A human phage antibody library was panned against immobilized Dig for four times, and the specificity of the selected scFv expression clones was identified by ELISA. Positive clones against Dig were analyzed by DNA fingerprint and sequencing. A clone which had high affinity to Dig was selected to construct a diabody vector. Diabody was secreted from E. coli by IPTG induction, and the specificity was also identified by ELISA.
Results: After four rounds of panning, four specific humanized anti-Dig antibodies(scFv) were obtained. DNA fingerprint and sequencing analysis proved that they had different Ab-encoding genes. The V(L) of the 4 expression clones belonged to lambda subgroup 1 and V(H) belonged to 3 and 4 subgroups, respectively. A scFv clone was picked out to construct diabody vector. The prepared humanized diabody reacted with Dig specifically.
Conclusion: Humanized anti-Dig antibodies(scFv) have been got by using phage display. A diabody has been obtained from scFv. These humanized anti-Dig antibodies may be used for the diagnosis and therapy of Dig toxication.