**Colony Assay for Antibody Library Screening: Outlook and Comparison to Display Screening and Comparison to Display Screening**

**Colony Assay for Antibody Library Screening: Outlook** 

DOI: 10.5772/intechopen.72149

Mieko Kato and Yoshiro Hanyu Mieko Kato and Yoshiro Hanyu Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72149

#### **Abstract**

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102 Antibody Engineering

Recombinant monoclonal antibodies are established by screening the antibody libraries. To obtain antibodies with a high specificity and affinity, an efficient screening process with a highly diverse library including low background signals is necessary. One of the most extensively used methods is the phage display method. Although phage display screening is a powerful tool for enriching clones from vast libraries, it is not easy to identify single clones with an antigen recognition function only through several rounds of biopanning. The application of colony assays for screening antibody libraries can identify clones with a high reliability by a direct observation of the antibody-antigen binding during the screening process; however, the size of the library that can be dealt with is limited. This chapter describes the colony assay as a current screening technology used in recombinant monoclonal antibody production, the possible problems in this method, and discusses the outlook for this technology.

**Keywords:** colony assay, screening, antibody library, scFv, *E. coli*, phage display

#### **1. Introduction**

The use of recombinant technology for antibody selection offers several advantages over conventional antibody selection strategies, such as the selection of antibodies against toxic or non-immunogenic antigens unattainable using conventional methods [1, 2], the ability to select positive clones from vast libraries [3], the realization of in vitro screening [4], and the bypass of animal usage [5]. The selection and production of recombinant monoclonal antibodies require the creation of highly diverse libraries [6] and the subsequent identification of positive clones using a screening technology with low background signals [7]. In particular, the variable domains of the antibody heavy and light chain (VH and VL) are isolated

from the lymph tissue of immunized animals and linked together for creating a single-chain variable fragment (scFv) library, and Fab libraries are constructed too. In general, the antibody fragments used for screening are the scFvs. Currently, entirely synthetic libraries [8–11] and naïve libraries [12] are being used. These antibody gene libraries are incorporated into a phagemid or plasmid and expressed in phage or *Escherichia coli* (*E. coli*). Further, panning [13] or colony assays [14] are performed to isolate scFvs possessing affinity to the antigen, thereby establishing monoclonal antibodies. This step, the screening of antibody libraries, is critical for establishing monoclonal antibody fragments with a high affinity and specificity against the antigen. One of the most extensively used methods is the phage display method [15, 16]. The display of the antibody repertoires on the surface of bacteriophages and their selection through panning enables the isolation of monoclonal antibodies [17]. Phage display is also widely used for affinity maturation [18, 19], in which mutations are introduced into the variable domains of an antibody gene mainly into CDRs to produce antibodies with a higher affinity as the original clone [20]. In addition, cell surface panning techniques [1, 21, 22] are being developed to establish antibodies recognizing membrane proteins on living cells that are difficult to produce using the conventional methods. Technologies that enable liquid panning rather than immobilizing the antigens to a solid phase have also been proposed for phage display to establish antibodies that recognize protein conformation [23]. Screening with a colony assay induces the actual expression of the scFvs themselves and involves a direct confirmation of the antigen-antibody binding, lending it the advantage of a low falsepositive rate. In addition, the method can be easily used to screen libraries in the order of magnitude larger than those that can be screened with the hybridoma technology. However, this method poses several problems: it requires extensive and complex manipulation of assay steps, the expression of antibody fragments could be at times nonexistent or very low, and the extensive manipulation during the assay can lead to contamination and death of the *E. coli* cells, potentially preventing gene retrieval. Although this technique is not complete and not widely applied, further development and improvement can render it highly beneficial.
