[Preparation of humanized anti-digoxin scFv and diabody].
OBJECTIVE
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.


CONCLUSIONS
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.
Chen
Yu-ping
YP
Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. Yupingch@sohu.com
Zhang
Guo-min
GM
Qiao
Yuan-yuan
YY
Wang
Gang
G
Liu
Yu-feng
YF
Hua
Bing
B
Wang
Yan
Y
D000906:Antibodies / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics; D016172:DNA Fingerprinting; D004077:Digoxin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D019151:Peptide Library
D000906:Antibodies; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004077:Digoxin