**Author details**

isolated antiangiogenesis antibodies from combinatorial libraries by iterative colony filter screening: colonies located around the positive signals were selected, and the screening step was repeated; monoclonal scFvs were established after several rounds of the assay [50]. Neumann-Schaal et al. developed a colony-screening method in which *E. coli* colonies producing the required scFv were selected in the presence of ampicillin conjugated to the antigen of interest; this method relies on the neutralization of the conjugate by the produced scFv. The scFvs were identified against biotin by the growth of the scFv library-expressing *E. coli* in the presence of a biotin-ampicillin conjugate [51]. Kumada et al. improved the sensitivity of the colony assay

whereas in the hybridoma method, dozens of 96-well microtiter plates are required for screening these clones. Further, the filter-sandwich assay can be readily upscaled by increasing the number of plates. Therefore, the number of positive clones from the filter-sandwich assay can be higher than that from the hybridoma method. This would increase the chance of obtaining

However, the filter-sandwich assay needs to be improved further for the selection of positive clones, particularly with respect to the reliability of the antibody fragment expression and the handling of the colonies during the assay. For the colony assay, the control of the expression level is critical. Because the scFv expression by itself is considerably toxic to *E. coli*, an excess induction of expression, namely, exposure to an excess of the expression-inducing reagent (IPTG), leads to cell death and prevents the selection of antigen-specific scFvs. Conversely, exposure to insufficient IPTG induces inadequate antibody expression for the detection of signals from positive clones. In the filter-sandwich assay, expression induction is not stringently controlled because the concentration of the IPTG added to the cells cannot be precisely controlled. IPTG reaches the colonies by diffusing through the filter from the antigen-coated membrane and the agar plate. Quantitative control of the expression level is required for superior screening. This uncertainty in the IPTG concentration in the filter-sandwich assay also causes a problem in the induction timing. For appropriate induction, the colony size is a critical factor [14, 44]; however, the colony continues to grow during the assay. Hence, the timing of the expression induction is crucial for proper expression. If the ITPG diffusion is delayed, an initially small colony would grow too large for proper induction to occur; however, if the colonies are too small, the signal from each colony is inadequate for detecting the antigen binding. The induction strength cannot be accurately determined, particularly during the step, when the filter is transferred to the IPTG-containing plate to initiate the induction of expression. These induction-related uncertainties in the filter-sandwich assay lead to unstable expression and failure in isolating the antibody-encoding genes. Stringent control of the expression level is critical. Various factors related to the expression vector, such as the promoter, strength of the ribosomal binding site, fusion tags, and the copy number, must be optimized [53–57]. The incubation temperature is also an important factor in controlling the

monoclonal antibody fragments with the desired affinity, specificity, and function.

clones on a 10-cm diameter plate in a filter-sandwich assay,

utilizing antibody-coupled liposome encapsulating HRP [52].

**4. Summary**

110 Antibody Engineering

It is possible to screen 3–5 × 103

Mieko Kato1 and Yoshiro Hanyu2 \*

\*Address all correspondence to: y.hanyu@aist.go.jp

1 Bio-Peak Co., Ltd., Takasaki, Japan

2 Structural Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
