**3. Colony assay for antibody library screening**

As an alternative antibody-screening tool, the colony assay can be used which is sometimes superior to the phage display method [42]. The advantage of this method is that the antibodyantigen binding can be directly observed during the screening process, reducing the selection of false-negative clones [24]. Thus, the colony assay presents notable advantages over the phage display and biopanning method.

#### **3.1. Principle of the colony assay**

repertoires with high clone numbers (10<sup>9</sup>

the positive ratio is low.

106 Antibody Engineering

**2.3. Screening with a phage display**

the antibody fragment affinity.

–1012 clones). During the assay, a clone from an

*E. coli* library is cultured, and its expression is induced. The reactivity of the expressed scFv against the antigen is measured. Clones exhibiting high reactivity are selected as the positive clones. In this method, only a few thousands of clones can be examined simultaneously, even if a multi-well microtiter plate is used. Although this number is higher than the clones obtained by the hybridoma technique, positive clones cannot be efficiently obtained, when

Colony assay in which periplasmic expression and *E. coli* colony formation lifted onto filters are used provides a method for handling large libraries (**Figure 1C**). In colony assay, the clones do not need to be picked up individually before screening; all the colonies on the plate can be assayed simultaneously. Thus, numerous clones can be assayed from a single plate. Antibody fragments released by bacteria were detected by a phage plaque assay in earlier experiments [32, 33]. Libraries of the antibody fragments were expressed in *E. coli* using phage λ vectors [34, 35]. Then, the active fragments secreted from the viable *E. coli* colonies were detected by colony-lift immunoassay [36]. With the colony assay, considerably larger libraries can be dealt with because the number of colonies screened can be easily increased.

In a phage display system, panning is used to isolate phages that display the antibody fragments exhibiting affinity to the antigen (**Figure 1A**). Positive clones are established by selecting only the phages that displays antibody fragments (primarily scFvs) fused to the g3p coat proteins on the surface of the filamentous phage, which have affinity to the antigen. This method has the advantage of processing large libraries (~1011) [3, 37]. The antigen is immobilized, and the recombinant phage bound to the antigen is left intact; weakly bound recombinant phages are washed away. The remaining recombinant phages, which possess a binding capacity are detached from the antigen by acid treatment and infected into *E. coli*. Further, *E. coli* cells are cultured to propagate positive clones. The *E. coli* clones expressing the phagemids are then infected with a helper phage, and the phages displaying scFvs with binding capacities are collected. Panning is performed repeatedly for the selected group of phages. The repeated selection and propagation of positive clones enrich clones with antibodies comprising binding capacities to the antigen. Then, single clones are isolated at the final step with

high binding capacities [38]. This method renders it possible to handle large libraries.

One limitation of this method is that the high background during panning selection often results in false-positive clones. A specific antigen-binding activity is typically not the only driving force exploited during the panning process [39, 40]. Multiple rounds of panning have been documented to frequently cause a strong bias for antibodies directed against immunodominant epitopes and abundant proteins [41], resulting in the loss of the library's diversity and of valuable antibody clones. Several factors influence the selection of the antigen-specific clones and produce undesired effects; these factors include a high efficiency of expression and folding despite poor antigen-binding activity, the nonspecific hydrophobic binding properties of the phage particle itself, and a superior compatibility with the host cells, not related to In the colony assay (**Figure 2**), antibody libraries are expressed in *E. coli* for the selection of clones with a favorable affinity to the antigen. An scFv library is transformed into *E. coli* cells, and afterward transformed *E. coli* cells are plated on appropriate agar plates. After growing of the colonies, they are lifted onto a filter. Further, an expression-inducing reagent such as isopropyl-β-D-thiogalactopyranoside (IPTG) is applied, inducing the expression and secretion of scFvs from the *E. coli* cells (**Figure 2A**). scFvs with the desired affinity will diffuse out and bind the antigen coated on the membrane beneath the colonies. However, scFvs without affinity will not bind the antigen (**Figure 2B**), and the unbound scFvs are washed away. Then, the bound scFvs with an affinity against the antigen are detected using an enzymatic method. The His-tags attached to the scFvs are detected with anti-His antibodies (**Figure 2C**). Positive clones are identified as the colonies corresponding to positive signals (**Figure 2D**).

#### **3.2. Filter-sandwich assay**

Dreher et al. [43, 44] improved the colony assay by developing the filter-sandwich colonyscreening assay (hereafter, the filter-sandwich assay) for selecting positive clones; *E. coli* colonies are grown directly on a hydrophilic filter, which is then transferred to an antigen-coated membrane soaked with IPTG solution and placed on an agar plate containing IPTG to induce antibody fragment production. The antibody fragments produced by the colonies diffuse out and bind to the antigen on the membrane. The presence of antibody fragments bound to the membrane is then detected, and the spot is superimposed on the colony. This method circumvents the difficult technique of lifting the colony [14, 36]. In addition, the filter-sandwich assay was further optimized. The procedure can now be performed by a single step [45] under tightly controlled IPTG concentration for expression of the scFvs.

**Figure 2.** Scheme of the colony assay principle. (A) scFvs are expressed and secreted from *E. coli*. (B) scFvs with the desired affinity bind the antigen beneath the colonies. (C) Bound scFvs with an affinity against the antigen are detected using an enzymatic method. (D) Positive clones are identified as the colonies corresponding to positive signals.

Then, the filter harboring the colonies is removed, placed on a fresh plate, and stored for the later recovery of the bacteria. Subsequently, antigen-bound scFvs on the nitrocellulose membrane are detected with chemiluminescence from a horseradish peroxidase (HRP)-conjugated anti-His antibody. The filter harboring the colonies and the image presenting the chemiluminescence data are superimposed, and positive colonies corresponding to the chemiluminescence signals are identified. These positive clones are transferred to a medium and incubated. The plasmid encoding the scFv gene with an affinity against the antigen is purified, and the

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A colony assay is used for screening the antibody fragments against a variety of antigens, with optimizations for each specific purpose. The recombinant antibody fragments against EspA and the intimin of *E. coli* O157:H7 were established by colony filter screening [47]. Colony-lift assay was combined with phage display, using cell-coated filters to screen the phage libraries for cell-binding clones [48]. Robert et al. developed subtractive colony filter screening to select scFvs that recognize atherosclerotic but not the normal aorta [49]. Giovannoni et al.

antibody coding sequence is determined.

**Figure 3.** Procedure for filter-sandwich colony assay.

**3.4. Establishing monoclonal antibody fragments by colony assay**

#### **3.3. Procedure for filter-sandwich assay**

The procedure used in the filter-sandwich colony assay is depicted schematically in **Figure 3**.

In particular, the RNAs are isolated from the lymph tissue of immunized animals, and the corresponding cDNA is synthesized; this cDNA is used as the template for the polymerase chain reaction (PCR) amplification of the VL and VH domains. Further, the variable domains are assembled to an scFv and cloned into an expression vector to create the scFv libraries [46]. As expression vector, for example, pET22b (+), containing a *pelB* signal sequence for periplasm expression and a His-tag sequence for the detection of the scFv expression driven by the T7 promoter, is used. The antibody repertoire is transformed into *E. coli*, and the filter sandwich assay is performed as described in **Figure 3**.

The hydrophilic PVDF filter is placed on an agar plate. Transformed *E. coli* with the scFv libraries is spread onto the filter and incubated. After the formation of the bacterial colonies on the filter surface, the filter harboring the colonies is transferred to an antigen-coated nitrocellulose membrane on the agar plate containing IPTG and incubated to induce scFv expression. Colony Assay for Antibody Library Screening: Outlook and Comparison to Display Screening http://dx.doi.org/10.5772/intechopen.72149 109

**Figure 3.** Procedure for filter-sandwich colony assay.

**3.3. Procedure for filter-sandwich assay**

108 Antibody Engineering

sandwich assay is performed as described in **Figure 3**.

The procedure used in the filter-sandwich colony assay is depicted schematically in **Figure 3**. In particular, the RNAs are isolated from the lymph tissue of immunized animals, and the corresponding cDNA is synthesized; this cDNA is used as the template for the polymerase chain reaction (PCR) amplification of the VL and VH domains. Further, the variable domains are assembled to an scFv and cloned into an expression vector to create the scFv libraries [46]. As expression vector, for example, pET22b (+), containing a *pelB* signal sequence for periplasm expression and a His-tag sequence for the detection of the scFv expression driven by the T7 promoter, is used. The antibody repertoire is transformed into *E. coli*, and the filter

**Figure 2.** Scheme of the colony assay principle. (A) scFvs are expressed and secreted from *E. coli*. (B) scFvs with the desired affinity bind the antigen beneath the colonies. (C) Bound scFvs with an affinity against the antigen are detected using an enzymatic method. (D) Positive clones are identified as the colonies corresponding to positive signals.

The hydrophilic PVDF filter is placed on an agar plate. Transformed *E. coli* with the scFv libraries is spread onto the filter and incubated. After the formation of the bacterial colonies on the filter surface, the filter harboring the colonies is transferred to an antigen-coated nitrocellulose membrane on the agar plate containing IPTG and incubated to induce scFv expression. Then, the filter harboring the colonies is removed, placed on a fresh plate, and stored for the later recovery of the bacteria. Subsequently, antigen-bound scFvs on the nitrocellulose membrane are detected with chemiluminescence from a horseradish peroxidase (HRP)-conjugated anti-His antibody. The filter harboring the colonies and the image presenting the chemiluminescence data are superimposed, and positive colonies corresponding to the chemiluminescence signals are identified. These positive clones are transferred to a medium and incubated. The plasmid encoding the scFv gene with an affinity against the antigen is purified, and the antibody coding sequence is determined.

#### **3.4. Establishing monoclonal antibody fragments by colony assay**

A colony assay is used for screening the antibody fragments against a variety of antigens, with optimizations for each specific purpose. The recombinant antibody fragments against EspA and the intimin of *E. coli* O157:H7 were established by colony filter screening [47]. Colony-lift assay was combined with phage display, using cell-coated filters to screen the phage libraries for cell-binding clones [48]. Robert et al. developed subtractive colony filter screening to select scFvs that recognize atherosclerotic but not the normal aorta [49]. Giovannoni et al. 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 utilizing antibody-coupled liposome encapsulating HRP [52].

expression strength [58]. For inducing expression, additional methods such as the cold-shock system [59] should be examined. Expression-inducing reagents that are less toxic than IPTG

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In the filter-sandwich assay, before the induction of antibody expression, the filter harboring the colonies must be transferred without disturbance. This transfer requires delicate manipulation of the filter and frequently produces unwanted stress on the filter, occasionally disturbing the colonies themselves. A method that does not require the transfer of the filter should be developed for more efficient antibody establishment. Recently, a single-step colony assay was established by us using a tightly controlled IPTG concentration for scFv expression [45]. One advantage is also that no transfer of the filter on which the colonies are grown to the antigen-

The establishment of a high-quality antibody library and efficient screening are the most important factors for successful recombinant antibody selection and production. Improvements in the screening technology are critical for quickly and reliably establishment of high-performance antibodies. Phage display screening is a powerful tool for this purpose; however, it has certain disadvantages such as the frequent selection of false-positive clones, but it can easily deal with a vast library. On the other hand, screening with a colony assay could identify the positive clones reliably; however, it cannot deal with a large complex library. Thus, screening methods using a display panning system and a colony assay have certain advantages and disadvantages, respectively. They should be utilized cooperatively, depending on the purpose of the experiments. Hence, condensing the library by phage display and then cloning the positive clones by colony assay would be advantageous. To efficiently establish high-quality antibodies, the adequate

2 Structural Physiology Research Group, Biomedical Research Institute, National Institute of

[1] Jones ML, Alfaleh MA, Kumble S, Zhang S, Osborne GW, Yeh M, et al. Targeting membrane proteins for antibody discovery using phage display. Scientific Reports. [Internet]. May 18, 2016;**6**:26240. Available from: http://www.nature.com/articles/srep26240

to *E. coli*, such as rhamnose [60], should also be tested.

choice of these technologies and their combination would be crucial.

\*

Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

and Yoshiro Hanyu2

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

[Accessed: Jan 28, 2017]

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

coated membrane is necessary.

**Author details**

Mieko Kato1

**References**

#### **4. Summary**

It is possible to screen 3–5 × 103 clones on a 10-cm diameter plate in a filter-sandwich 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 monoclonal antibody fragments with the desired affinity, specificity, and function.

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 expression strength [58]. For inducing expression, additional methods such as the cold-shock system [59] should be examined. Expression-inducing reagents that are less toxic than IPTG to *E. coli*, such as rhamnose [60], should also be tested.

In the filter-sandwich assay, before the induction of antibody expression, the filter harboring the colonies must be transferred without disturbance. This transfer requires delicate manipulation of the filter and frequently produces unwanted stress on the filter, occasionally disturbing the colonies themselves. A method that does not require the transfer of the filter should be developed for more efficient antibody establishment. Recently, a single-step colony assay was established by us using a tightly controlled IPTG concentration for scFv expression [45]. One advantage is also that no transfer of the filter on which the colonies are grown to the antigencoated membrane is necessary.

The establishment of a high-quality antibody library and efficient screening are the most important factors for successful recombinant antibody selection and production. Improvements in the screening technology are critical for quickly and reliably establishment of high-performance antibodies. Phage display screening is a powerful tool for this purpose; however, it has certain disadvantages such as the frequent selection of false-positive clones, but it can easily deal with a vast library. On the other hand, screening with a colony assay could identify the positive clones reliably; however, it cannot deal with a large complex library. Thus, screening methods using a display panning system and a colony assay have certain advantages and disadvantages, respectively. They should be utilized cooperatively, depending on the purpose of the experiments. Hence, condensing the library by phage display and then cloning the positive clones by colony assay would be advantageous. To efficiently establish high-quality antibodies, the adequate choice of these technologies and their combination would be crucial.
