Characterization of 11-dehydro-thromboxane B2 recombinant antibody obtained by phage display technology.
Recombinant monoclonal antibodies specific for 11-dehydro-thromboxane B(2) (11D-TX) were isolated from the combinatorial libraries on a pComb3 phage-display vector using a magnetic cell sorting (MACS) system. The libraries were constructed from repertories of light and heavy-chains derived from the total RNA of 11D-TX conjugated keyhole limpet haemocyanin-immunized mice. Biotinylation of 11D-TX conjugated bovine serum albumin (BSA) was performed through free thiol groups on BSA using 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC). Affinity bio-panning was performed to enrich the phage display libraries against biotinylated 11D-TX conjugated BSA with the MACS system. Results indicated that the selected anti-11D-TX Fab fragments expressed by E. coli exhibited a five-fold higher affinity for BSA conjugated 11D-TX compared to BSA alone and little specificity to other related compounds as determined by the binding assay and inhibition enzyme-linked immunosorbent assay (ELISA). This is the first report of an antibody against prostaglandin produced by phage display technology and also determination of the DNA sequence of this antibody. The MACS system was shown to be a simpler and more efficient method of panning than the conventional ELISA procedure. According to our results, we concluded that the phage display technique combined with the MACS system allowed the selection of the antibody with high affinity and some specificity.
Tsuruta
Lilian Rumi
LR
Division of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.
Tomioka
Yoshihisa
Y
Hishinuma
Takanori
T
Kato
Yoshinori
Y
Itoh
Kunihiko
K
Suzuki
Toshio
T
Oguri
Hiroki
H
Hirama
Masahiro
M
Goto
Junichi
J
Mizugaki
Michinao
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001710:Biotin / Q000031:analogs & derivatives / Q000494:pharmacology; D019921:Biotinylation; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005822:Genetic Vectors; D007136:Immunoglobulins / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D010957:Plasmids / Q000378:metabolism; D011453:Prostaglandins / Q000737:chemistry / Q000276:immunology; D012680:Sensitivity and Specificity; D013929:Thromboxane B2 / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*
C479840:1-biotinamido-4-(4'-(maleimidomethyl)cyclohexanecarboxamido)butane; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007136:Immunoglobulins; D019151:Peptide Library; D011453:Prostaglandins; D013929:Thromboxane B2; C049235:11-dehydro-thromboxane B2; D001710:Biotin