SITE-SPECIFIC IMMOBILIZATION OF MONOCLONAL-ANTIBODIES USING SPACER-MEDIATED ANTIBODY ATTACHMENT

Site-directed and random coupling of antibodies (Abs) was performed us ing aminated silica surfaces as substrates. The site-directed coupling linked the Ab, a monoclonal anti-fluorescein IgG1 (9-40), to the surf ace via 3400 Dalton (Da) poly(ethylene oxide) (PEO) spacers. The hydra zide end groups of these spacers were attached to aldehyde groups in t he hinge region of the oxidized Ab to yield surfaces with an Ab concen tration of 2.1 pmol/cm(2). The random coupling, in turn, linked the Ab via its available primary amines directly to the glutaraldehyde-activ ated surface with a yield of 3.8 pmol/cm(2). Despite a nearly twofold difference in Ab concentration, the two surfaces bound similar amounts of the FL-BSA antigen (0.56 vs 0.55 pmol/cm(2)), while their nonspeci fic uptake of protein was 0.11 vs. 0.21 pmol/cm(2), respectively, refl ecting the protein repellent quality of PEO-coated surfaces. Fab' frag ments of the 9-40 Ab were also Linked to the same tethers. Here, the s uccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) difu nctional coupling reagent was attached to the PEO-hydrazide via its su ccinimide end and to the carboxy-terminal thiol of the the Fab' via it s maleimide end. The concentration of reactive groups was varied by mi xing difunctionalized PEO (PEO - (HZ)(2)) with monofunctionalized poly mer (CH3O-PEO-HZ) prior to surface attachment. At 100% PEO-(HZ)(2) the FL-BSA (Ag) binding was 0.77 pmol/cm(2) while the nonspecific binding was 0.058 pmol/cm(2). Progressive dilution of the reactive PEO chains to 25% led to the remarkable binding of 0.57 pmol/cm(2) of Ag with 0. 037 pmol/cm(2) of nonspecific binding. Competitive release from these various surfaces showed more favorable kinetics for the Fab' surface w ith 25% active tethers.