High-affinity antibodies

Table I describes several of the fluorescent assays that have been used in our lab to study neutrophil activation. Fluorescein-labeled W-formylhexapeptide (FLPEP) has been used to characterize the ki- netics of ligand binding, dissociation, and internalization at 37°C (7,8). FLPEP is added to a suspension of cells, then receptor-bound and free FLPEP in solution are distinguished by adding antibody to fluorescein. This is a high-affinity antibody which binds free FLPEP within 1 s hut does not bind cell-bound FLPEP. When it binds the FLPEP, it quenches the fluorescein fluorescence. Hence the residual fluorescence after antibody addition represents FLPEP that is bound to the cell. Nonspecific binding is determined in cell suspensions that contain an excess of nonfluorescent peptide.

Complexity of LR Dynamics in Intact Neutrophils at 37°. At present, our most versatile assay to analyze receptor binding uses fluorescent ligand and a high-affinity antibody to fluorescein which discriminates free from receptor-bound hexapeptide ligand (FLPEP, N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-Fl). The assay has a time resolution of 1... 

The occurrence of mutation only after exposure to antigen probably accounts for early reports that high affinity antibodies were observed only late during immune... 

Affinity purification may be performed using gravity flow through a disposable column (e.g., a Bio-Rad Poly-Prep chromatography column). In this case, flow reversal is not possible and recovery of high affinity antibodies may be compromised. Nevertheless, acceptable results are often achieved. 

De Haardt, H. J., van Neer, N., Reurst, A., Hufton, S. E., Roovers, R. C., Henderikx, P., et al. (1999) A large non-immunized human fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274, 18,218-18,230. 

Schmidt, A., Muller, D., Mersmann, M., Wtiest, T., Gerlach, E., Garin-Chesa, P., et al. (2001) Generation of human high-affinity antibodies specific for the fibroblast activation protein by guided selection. Eur. J. Biochem. 268, 1730-1738. 

The first step in developing an ELISA to detect the stem peptide product of the coupled enzyme reaction was to determine if antibodies to the stem peptide could be raised in animals. Only small quantities of stem peptide could be isolated or prepared enzymatically, so we used the commercially available pentapeptide portion of the stem peptide as the hapten in the hopes of generating high-affinity antibodies that would cross-react with the stem peptide. 

Hanes, J.,Jermutus, L., Weber-Bornhauser, S., Bosshard, H. R., and Pluckthun, A. (1998). Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. USA, 95(24), 14130—14135. 

Isolation of high affinity antibodies against any antigen... 

Hoogenboom HR, Designing and optimizing library selection strategies for generating high-affinity antibodies, Trends Biotechnol., 15 62-70, 1997. 

An optimal antibody dilution is also governed by the intrinsic affinity of an antibody. If the titer is held constant, a high-affinity antibody is likely to react faster with the tissue antigen and give more intense staining within the same incubation period than an antibody of low affinity. 

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