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Site-directed and random coupling of antibodies (Abs) was performed using aminated silica surfaces as substrates. The site-directed coupling linked the Ab, a monoclonal anti-fluorescein IgG1 (9-40), to the surface via 3400 Dalton (Da) poly(ethylene oxide) (PEO) spacers. The hydrazide end groups of these spacers were attached to aldehyde groups in the hinge region of the oxidized Ab to yield surfaces with an Ab concentration of 2.1 pmol/cm 2 . The random coupling, in turn, linked the Ab via its available primary amines directly to the glutaraldehyde-activated 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 nonspecific uptake of protein was 0.11 vs. 0.21 pmol/cm 2 , respectively, reflecting the protein repellent quality of PEO-coated surfaces. Fab′ fragments of the 9-40 Ab were also linked to the same tethers. Here, the succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) difunctional coupling reagent was attached to the PEO-hydrazide via its succinimide end and to the carboxy-terminal thiol of the Fab′ via its maleimide end. The concentration of reactive groups was varied by mixing difunctionalized PEO (PEO-(HZ) 2) with monofunctionalized polymer (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 0.87 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 with 25% active tethers.

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An antigen-binding fragment (Fab) from a murine monoclonal antibody (4-4-20) with high affinity for fluorescein was cocrystallized with ligand in polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) in forms suitable for X-ray analyses. In MPD the affinity of the intact antibody for fluorescein was 300 times lower than the value (3.4 x 10(10) M-1) obtained in aqueous buffers. This decreased affinity was manifested by the partial release of bound fluorescein when MPD was added to solutions of liganded Fab during crystallization trials. In PEG, the ligand remained firmly bound to the protein. The liganded Fab crystallized in the monoclinic space group P2(1) in PEG, with a = 58.6, b = 97.2, c = 44.5 A and beta = 95.2 degrees. In MPD the space group was triclinic P1, with a = 58.3, b = 43.4, c = 42.3 A, alpha = 83.9 degrees, beta = 87.6 degrees, and gamma = 84.5 degrees. X-ray diffraction data were collected for both forms to 2.5-A resolution. Surprisingly, the triclinic form of the liganed antifluorescyl Fab had the same space group, closely similar cell dimensions, and practically the same orientation in the unit cell as an unliganded Fab (BV04-01) with activity against single-stranded DNA.

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