Antibody Combinatorial Library

To study the role of lysine residues in susceptibility to formalin fixation, the amino acid composition of immunoreactive peptides (to various monoclonal antibodies) was studied. Each peptide was evaluated to determine if immu-noreactivity was lost after formalin fixation. Formalin sensitivity was correlated with the peptides amino acid composition. The first step in the method is biopanning from a peptide combinatorial library with a monoclonal antibody. The peptides that bind to the antibody were tested for their sensitivity to formalin fixation. Some peptides remain immunoreactive whereas others do not. The peptides were then sequenced to look for differences between those that were sensitive to formaldehyde versus those that were not. The goal was to find whether there is a particular amino acid that is present in formalin-sensitive epitopes but absent in formalin-resistant epitopes, or vice versa. An advantage of this approach is that it is open-ended, without excluding any amino acids. 

Hoet, R.M.A., Raats, J.M.H., de Wildt, R.M.T., van Venrooij, W.J. (1995). Isolation and characterization of antibody fragments directed to disease markers isolated from patient derived combinatorial libraries. Immunology, 86(suppl 1), 96. 

Catalytic antibodies, like enzymes, must be isolated and purified to homogeneity before they can be studied. Initially this was done by using the hybridoma technique for isolation of monoclonal antibodies (Box 31-A). After induction of antibody formation by injecting a selected hapten into a mouse, large numbers of monoclonal antibodies had to be tested for catalytic activity. Even if several thousand different monoclonal antibodies were tested, only a few with catalytic properties could be found.1 Newer methods have incorporated recombinant DNA techniques (Box 31-A) and use of combinatorial libraries and phage display.) Incorporation of acidic or basic groups into the haptens used to induce antibody formation may yield antibodies capable of mimicking the acid-base catalysis employed by natural enzymes. 0... 

Most of the affinities of Fabs or scFvs isolated from combinatorial libraries have affinities similar to those of antibodies found in the primary immune response in vivo (Kd 10-6—10 7M), but in vivo, these affinities are improved during affinity... 

In 1991, we first introduced the one-bead one-compound (OBOC ) combinatorial library method.1 Since then, it has been successfully applied to the identification of ligands for a large number of biological targets.2,3 Using well-established on-bead binding or functional assays, the OBOC method is highly efficient and practical. A random library of millions of beads can be rapidly screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The amount of acceptor needed is minute compared to solution phase assay in microtiter plates. The positive beads with active compounds are easily isolated and subjected to structural determination. For peptides that contain natural amino acids and have a free N-terminus, we routinely use an automatic protein sequencer with Edman chemistry, which converts each a-amino acid sequentially to its phenylthiohydantoin (PTH) derivatives, to determine the structure of peptide on the positive beads. 

Barbas, C.F, J.S. Rosenblum, and R.A. Lemer. 1993. Direct selection of antibodies that coordinate metals from semisynthetic combinatorial libraries. Proceedings of the National Academy of Sciences, Vol. 90, pp. 6385-6389. 

Kang AS, Barbas CF, Janda KD, Benkovic SJ, Lerner RA, Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces, Proc. Natl. Acad. Sci. USA, 88 4363-4366, 1991. 

Bender E, Pilkington GR, Burton DR, Human monoclonal Fab fragments from a combinatorial library prepared from an individual with a low serum titer to a virus, Hum. Antibodies Hybridomas, 5 3-8, 1994. 

This strategy has been applied to select catalytic antibodies from phage-displayed libraries. Two catalytic single-chain antibodies catalyzing the hydrolysis of ampicillin with rate accelerations kcit/kunc lt of 5200 and 320 (kcat = 0.29 and 0.018min-1) were isolated from combinatorial libraries prepared from mice immunized with penam sulfone conjugates and selected with a biotinylated penam sulfone [61]. 

---