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发表于 2021-4-2 13:09:33
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3.6. Affinity determinationAffinity of nanobodies determined according to the procedureof Beatty et al. (1987). The kaffof Nb42, Nb35, nb23 and Nb22 was60, 45, 10 and 0.1 nM respectively (Table 4). The affinity graphs ofnanobodies are shown in Fig. 7. The graph for negative control (BSA)was not show2.12. Affinity measurementThe affinity of purified VEGF specific nanobodies were deter-mined according Beatty et al. method (Beatty et al., 1987). Briefly,n.96-well plate was coated with 100 l of two different concen-trations (10 and 1 g/ml) of VEGF121antigen or BSA (as negativecontrol) at 4◦C overnight. The next day, plate was blocked for 1 hat room temperature. After washing with PBST, the nanobody atincreasing concentration (0–10 nM/ml) were added to the wellsand incubated for 1 h at room temperature. The wells were washedwith PBST and incubated with HRP conjugated anti-His antibody(1:500). The peroxides activity of enzyme was detected by TMB.The signal intensity was measured at 450 nm. The affinity (kaff) ofVEGF specific nanobodies were obtained using following equation(Beatty et al., 1987):
[Ag]/[Ag]
=
N
kaff =
N
−
1/2(N[Nb]
−
[Nb])
[Ag] and [Ag] refer to the concentration of 10 and 1 g/ml anti-gen, and [Nb] and [Nb] are the concentrations of nanobody at 50%binding in the Ag and Agcurves.
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发表于 2023-10-6 20:38:18