Antibody Engineering for Pursuing a Healthier Future

Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

Principally, specific antibody is immobilized on high binding ELISA plates by incubation overnight at 4 • C or for 1-2 h at 37 • C, and then followed by 3-5 washes with PBST (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , and 0.5% Tween 20; Murayama et al., 1996) . Then, plates are blocked with irrelevant protein e.g., albumin (for example, PBS containing 4-5% skimmed milk), and incubate overnight at 4 • C or for 1-2 h at 37 • C. After washes, samples and standard dilutions are added to the wells to be captured by bound protein, incubate for 1-2 h at 37 • C and wash properly. Next, specific peroxidase-conjugated enzyme labeled detection antibody is added to the wells to enable detection of the captured protein and incubate for 1 h at 37 • C. After appropriate wash, colorimetric substrate is added to the wells and incubated for 15-20 min at 37 • C for the color development as catalyzed by the enzyme. For instance, addition of 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) substrate develops blue color that can be read by microplate plate reader at the wavelength of 562 nm. Additionally, addition of 0.16-2 M sulfuric acid (H 2 SO 4 ) as a stop reaction solution develops yellow color that can be read at 405-450 nm correspondingly. In addition, the incubation conditions and reagent formulations should be preferably optimized based on the type of ELISA (Grange et al., 2014; Thiha and Ibrahim, 2015) .