Hrp conjugated goat anti human fab specific antibody

The expression of hTLR4-Fab01 in E. coli were performed by Western blot as described previously [21 (link)]. Typically, bacteria lysate was prepared supplemented with a proteinase inhibitor cocktail (Roche, IN, USA). Protein concentration was examined using a bicinchoninic acid (BCA) Protein Assay kit according to the manufacturer’s instruction (Pierce, IL, USA). The protein from whole-cell lysate were separated by 10% SDS-PAGE and transferred to Nitrocellulose membrane (Bio-Rad, CA, USA). To determine the antigenicity of the purified Fab fragment, the membrane was incubated with HRP-conjugated goat anti-human Fab specific antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. The bands were visualized using DAB Chromogenic Reagent (Boster company, Wuhan, China) according to the manufacturer’s instructions.

Non-competitive ELISA was employed to identify the affinity of ROR1-cFab. Briefly, serial dilutions of ROR1-cFab were added into 96-well plates that were pre-coated with 50 ng recombinant human ROR1 protein(Sino Biological Inc., Beijing, China) per well. After incubation and washing with PBST, HRP-conjugated goat anti-human Fab specific antibody(Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added into each well of the plates, while a commercial anti-human ROR1 antibody (Abcam, Cambridge, MA, USA) was used as a positive control. The absorbance value at 450 nm was then detected using a spectrophotometer (Thermo Electron Corporation).