Meny

Javascript is not activated in your browser. This website needs javascript activated to work properly.
Du är här

Two-step in vitro antibody affinity maturation enables estradiol-17β assays with more than 10-fold higher sensitivity.

Författare:
Publiceringsår: 2010
Språk: Engelska
Sidor: 1027-1038
Publikation/Tidskrift/Serie: Analytical Chemistry
Volym: 82
Nummer: 3
Dokumenttyp: Artikel
Förlag: American Chemical Society

Sammanfattning

Immunoassays for haptens depend on competitive hapten-anti-hapten reactions, and consequently their sensitivities are significantly influenced by the affinities of anti-hapten antibodies. Thus, genetically engineered antibodies, which have much higher affinities than native antibodies, should increase assay sensitivities. Here, we created a mutated single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) that allowed immunoassays with a much improved sensitivity. Two steps of affinity maturation were performed on a "wild-type" scFv (scFv#E4-4) composed of V(H) and V(L) domains from a mouse anti-E(2) antibody (Ab#E4-4). First, we conducted complementarity-determining region (CDR)-targeted mutagenesis by "CDR-shuffling". Gene fragments encoding CDRs H2, H3, L1, and L3, each of which contained random point mutations, were combined by "shuffling" into the gene encoding the scFv#E4-4 scaffold. After phage display and repeated panning, we isolated a mutated scFv clone [scFv#m1-e7; Ile(L29)Val] that had 5-fold higher affinity (K(a) = 2.6 x 10(8) M(-1)) compared to the Ab#E4-4 Fab fragment (Fab#E4-4). Next, the entire V(H) and V(L) of this clone were randomly mutated by error-prone polymerase chain reaction (PCR). From this library, we found an improved clone, scFv#m2-c4 (K(a) = 6.3 x 10(8) M(-1); Lys(H19)Arg, Tyr(H56)Phe, Ser(H84)Pro, Glu(H85)Gly, Gln(L27)Arg, Leu(L36)Met, Ser(L63)Gly, and Ser(L77)Gly). ScFv#m2-c4 had more than 10-fold higher sensitivity (the midpoint of its dose-response curve was 0.56 ng) than Fab#E4-4 (midpoint 9.0 ng/assay) in a competitive E(2) radioimmunoassay, and even higher sensitivity [midpoint 21 pg/assay, and a limit of detection of 0.47 pg (1.7 fmol)/assay] in a competitive enzyme-linked immunosorbent assay. Cross-reactivity with selected E(2)-related endogenous steroids strongly suggested that scFv#m2-c4 has improved specificity compared to conventional antibodies.

Disputation

Nyckelord

  • Biology and Life Sciences

Övriga

Published
Yes
  • ISSN: 0003-2700

Box 117, 221 00 LUND
Telefon 046-222 00 00 (växel)
Telefax 046-222 47 20
lu [at] lu [dot] se

Fakturaadress: Box 188, 221 00 LUND
Organisationsnummer: 202100-3211
Om webbplatsen