Figure 6. Enzyme linked immunosorbent assay to determine the substrate specificity of MtBPL.

(a) Incorporation of biotin / desthiobiotin into BCCP in BPL catalyzed reaction. The wells were coated with 15 µM BCCP and incubated overnight at 4°C. The non-specific sites were blocked with 1% BSA and biotinylation reaction carried out with biotin/desthiobiotin as the substrate. The incorporated substrate was detected streptavidin-HRP. All experiments were done in triplicates and on three different occasions. (b) Competitive ELISA to determine the affinity of streptavidin for biotin and desthiobiotin. The wells were immobilized with biotinylated BCCP. Streptavidin HRP (1∶2500) was incubated with equal volume and different concentration of biotin or desthiobiotin for 1 h at 37°C. The reaction mixture was then added to biotinylated BCCP. The unreacted streptavidin bound to immobilized BCCP which was detected by TMB/H₂O₂. All experiments were done in triplicates and at three different occasions.