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Single-step colony assay for screening antibody libraries
Accepted Manuscript Title: Single-step colony assay for screening antibody libraries Authors: Mieko Kato, Yoshiro Hanyu PII: DOI: Reference:
S0168-1656(17)30291-2 http://dx.doi.org/doi:10.1016/j.jbiotec.2017.06.010 BIOTEC 7919
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
18-1-2017 19-4-2017 10-6-2017
Please cite this article as: Kato, Mieko, Hanyu, Yoshiro, Single-step colony assay for screening antibody libraries.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2017.06.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Single-step colony assay for screening antibody libraries
Mieko Kato,1,2 and Yoshiro Hanyu 2* 1
Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854 Japan. 2 Structural Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan.
*Corresponding author: Yoshiro Hanyu Structural Physiology Research Group, Biomedical Research Institute, National Institutes of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan Tel. +81-29-861-2716 Fax: +81-29-861-2706 E-mail: [email protected]
Highlights ・ An scFv selection method was developed based on colony-filter screening.
・The method does not involve transfer of filters with colonies before scFv expression.
・Controlled exposure to an expression-inducing agent causes enhanced scFv expression.
・Monoclonal scFvs were established using this single-step colony assay. ・Reactivity of scFv clones established using our method was evaluated.
Abstract We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3 × 103 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Abbreviations: scFv, single-chain Fv fragment; VL, light-chain variable region; VH, heavy-chain variable region; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-β-Dthiogalactopyranoside; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. Keywords: scFv, Escherichia coli, Colony assay, Screening, Antibody library, Expression
1. Introduction A critical step in the establishment of clones of antigen-specific monoclonal antibody fragments is the screening of recombinant antibody libraries (Hoogenboom, 2005; Hust et al., 2014). For rapid screening of large antibody libraries, one of the most extensively used methods is the phage-display method (Hoogenboom et al., 1998; McCafferty et al., 1990). The display of antibody repertories on the surface of bacteriophages and their selection through panning (Parmley and Smith, 1988) allow the isolation monoclonal antibodies (Haque and Tonks, 2012; Schofield et al., 2007). Moreover, in vitro screening performed using this method enables the establishment of monoclonal antibody fragments against various types of antigens, including toxic antigens (Geyer et al., 2012). Phage-display technology allows the handling of extremely large libraries, such as those including 1011 molecules (Bradbury and Sblattero, 2000; Schirrmann et al., 2011); by comparison, the number of hybridomas screened in hybridoma technology is in the order of 103. However, the specific antigen-binding activity is typically not the only driving force exploited during the panning process (Hammers and Stanley, 2014; Nelson, 2010). Multiple rounds of panning have been documented to frequently give rise to a strong bias for antibodies directed against immunodominant epitopes and abundant proteins (de Wildt et al., 2000), which results in the loss of diverse binding specificities and of valuable antibody clones. A common problem encountered is that several factors influence the selection of antigen-specific clones and produce undesired effects; these factors include high expression efficiency and high folding efficiency despite poor antigen-binding activity, hydrophobic binding properties of the phage particle itself, and enhanced tolerance with host cells. Thus, although phage-display screening is a powerful tool for enriching clones from diverse libraries, the method is inadequate for identifying single clones through several rounds of panning. Considerable effort is required to identify single clones exhibiting high specificity and affinity against an antigen after the bio-panning step (Rauth et al., 2010). As an antibody-screening tool, colony-lift immunoassay is superior to—and can
be used instead of—the phage-display method (Kato and Hanyu, 2015; Pini et al., 2002). The detection of antibody fragments released by bacteria was performed by a phage plaque assay in earlier experiments (Caton and Koprowski, 1990; Huse et al., 1989). Libraries of antibody fragments were expressed in Escherichia coli (E. coli) by using phage vectors (Mullinax et al., 1990; Persson et al., 1991). Then the active fragments secreted from viable E. coli colonies were detected by colony-lift immunoassay (Rodenburg et al., 1998). Colonies of E. coli engineered to express antibodies are grown on a plate, and these colonies are lifted onto nitrocellulose membranes to prepare replicas of the colonies (Radosević et al., 2003). The membranes are next transferred onto antigencoated membranes and then placed on plate containing an expression-inducing reagent, such as isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed antibodies diffuse out and react with the antigen and are detected, and thus the colonies that express an antibody that can react with the antigen are identified. The advantage of this method is that the antibody-antigen binding can be directly observed during the screening process, which reduces the selection of falsenegative clones. Dreher et al. improved the colony assay by developing the filter-sandwich colony-screening assay (hereafter the filter-sandwich assay) (Dreher et al., 1991; Skerra et al., 1991). Antibody-expressing E. coli colonies are grown directly on hydrophilic filters, which are then transferred onto antigen-coated membranes soaked with IPTG solution and placed on IPTG plates, to induce antibody production. The antibodies produced by the colonies diffuse out and bind to antigen on the membrane and are detected using immunological methods. The major advantage of this methodology is that it does not involve the lifting of colonies, which requires delicate handling, and Giovannoni et al. used this method to successfully establish several monoclonal antibodies (Giovannoni et al., 2001). However, the induction of expression in the filter-sandwich assay requires further improvement: expression induction is not tightly controlled because the induction reagent (IPTG) that is applied reaches the cells by diffusing through the filter from the antigen-coated membrane. Consequently, the IPTG concentration to which the cells are exposed is not precisely controlled. However, its concentration is critical for colony assay because exposure to excess IPTG induces cell death and prevents the isolation of genes from positive clones, whereas exposure to insufficient IPTG induces inadequate antibody expression for the detection of signals from positive clones.
Here, we describe a simple and fast colony-assay method for screening singlechain Fv fragment (scFv) libraries. In this assay, E. coli colonies produce antibody fragments as soluble molecules that are secreted into the periplasm. Bacterial colonies are formed on a hydrophilic filter that is positioned atop the antigencoated membrane in an agar plate, and the expression-inducing reagent is applied to the filter in this configuration. Thus, this method does not involve the transfer of filters harboring colonies before scFv expression. The method ensures high scFv expression by E. coli colonies and enables the isolation of positive clones from libraries.
2. Materials and Methods 2.1. Immunization Approximately 6-week-old female Wister rats, obtained from SLC (Tokyo, Japan), were intraperitoneally (i.p.) injected with 100 μg of the antigen, 3 times once every 2 weeks; the first injection was in complete Freund’s adjuvant (SigmaAldrich, St. Louis, MO) and the second and third were in incomplete Freund’s adjuvant (Sigma-Aldrich) in a volume of 0.2 mL. The rats were intravenously (i.v.) boosted with 100 μg of the antigen in normal phosphate-buffered saline (PBS) one week after the third i.p. injection and then sacrificed 3 days after the i.v. boost, following which their spleens were aseptically removed. All animals were cared for and maintained in accordance with the guidelines of the National Institute of Advanced Industrial Science and Technology. 2.2. Amplification of VH and VL fragments RNA from the collected rat spleens was purified using NucleoSpin RNA II (Macherey-Nagel, Germany), and the corresponding cDNA was synthesized using PrimeScript Reverse Transcriptase (Takara Bio, Japan) and used as the template for PCR amplification of VL and VH sequences; the amplification protocol was the following: initial denaturation at 94 °C for 2 min, followed by 25 cycles of 98 °C for 3 s, 56 °C for 15 s, and 68 °C for 2 min, and a final elongation at 68 °C for 2 min. PCR was performed by using KOD FX polymerase (Toyobo, Japan) with 10 pM of the primers reported by Sepulveda and Shoemaker (Sepulveda and Shoemaker, 2008). NcoI and XhoI restriction sites were added to the 5ʹ end of the forward and reverse VH primers, and NheI and NotI sites were added to the
5ʹ end of the forward and reverse VL primers, respectively. 2.3. Preparation of expression vector A schematic of the DNA construct that we prepared is shown in Fig. 1 together with the restriction sites. The vector used for the single-step assay, pET-NXNN, was designed and constructed based on pET22b(+) (Merck Millipore, Darmstadt, Germany) for periplasmic protein expression. DNA fragments (Fig. 1A) used for inserting the linker and the restriction sites for cloning VH and VL fragments were synthesized by GenScript (Piscataway, NJ). The generated fragment was digested with NcoI and Bpu1102I (New England Biolabs, MA) and inserted into pET22b(+) (Fig. 1B). 2.4. Construction of scFv library VL and VH sequences PCR-amplified from spleen cDNA of immunized rats were purified and cloned into pET-NXNN. First, pET-NXNN and VL fragments were digested with NheI and NotI (New England Biolabs) and purified using NucleoSpin Gel and PCR clean-up (Macherey-Nagel). The VL library was constructed by ligating VL fragments and the vector, and E. coli DH5 chemically competent cells (Nippon Gene, Japan) were transformed with the resulting vectors. The transformed bacteria were incubated in 6 mL of SOC medium for 1 h at 37 °C, after which the cells were plated on 2YT plates supplemented with 1% glucose and 0.1 mg/mL carbenicillin (Wako, Japan) and cultured overnight. The cells were then collected and suspended in 10 mL of 2YT medium, and the vectors containing the VL fragments (termed pET-NX-VL) were purified using NucleoSpin Plasmid EasyPure (Macherey-Nagel). Lastly, pET-NX-VL and VH fragments were digested with NcoI and XhoI (New England Biolabs), and the scFv library was constructed by ligating the VH fragments to the vector. E. coli DH5 cells were transformed with these vectors and cultured as described above, and then the cells were collected and suspended in 10 mL of 2YT medium, supplemented with 0.1 mg/mL carbenicillin, and used for the colony assay. 2.5. Construction of expression vector for anti-rabbit IgG scFv (scFv(A10B)) For the colony assay, we used scFv(A10B), which recognizes the constant region of rabbit IgG; scFv(A10B) consists of VH and VL sequences joined by the linker sequence GGGGSGGGGSGGGGS (Shen et al., 2005a). The DNA for scFv(A10B), which was synthesized by GenScript, was digested with NcoI and
NotI (New England Biolabs) and inserted into the pET-NXNN vector for the colony assay. E. coli DH5 cells were transformed with this vector. The transformed bacteria were incubated in 6 mL of SOC medium for 1 h at 37 °C and then plated on 2YT plates supplemented with 1% glucose and 0.1 mg/mL carbenicillin and cultured overnight. Cells were collected and suspended in 10 mL of 2YT medium, supplemented with 0.1 mg/mL carbenicillin, and used for the colony assay. 2.6. Colony growth and scFv expression The procedure used in the single-step colony assay is depicted schematically in Fig. 2. The antigen-coated membrane was prepared as follows. A 9-cmdiameter nitrocellulose membrane was coated with the antigen by incubating it for 6 h in PBS containing rabbit IgG (Sigma-Aldrich; 10 μg/mL). Subsequently, the membrane was blocked for 2 h in PBS containing 5% nonfat dry milk, washed twice in PBS, and placed on a 2YT plate (10 cm in diameter) containing 0.1 mg/mL carbenicillin, after which a 9-cm-diameter hydrophilic polyvinylidene difluoride (PVDF) filter (Durapore; Merck Millipore) was positioned atop the antigen-coated membrane. Transformed E. coli were grown in this 2YT medium, and 1-5 × 103 cells from the exponential growth phase were spread onto the filter and incubated at 37 °C for 16 h. After bacterial colonies had formed on the filter surface, 400 μL of 1 mM IPTG (Wako) solution was sprayed uniformly on the top membrane in the plate to induce scFv expression, and the plate was incubated at 30 °C for 6 h. Lastly, the filter harboring the colonies was removed, placed in a fresh 2YT plate containing 1% glucose and 0.1 mg/mL carbenicillin, and stored at 4 °C for later recovery of the bacteria. The nitrocellulose membrane was immersed in PBS containing 0.05% Tween-20 (PBS-T) and used for the detection of antigen-specific scFv expression. 2.7. Detection of antigen binding and identification of positive clones The membrane obtained after the preceding step was washed twice with PBST and blocked for 2 h in PBS containing 5% nonfat dry milk. To detect antigenbound scFvs, the membrane was incubated for 2 h with a horseradish peroxidase (HRP)-conjugated anti-His antibody (Wako; diluted 1:5000 in PBS-T), and then washed extensively in PBS-T. Subsequently, after development with a chemiluminescent HRP substrate kit (Immobilon Western; Merck Millipore), the chemiluminescence signal was detected using a Chemi-Stage CC16mini (KURABO, Japan), and each stained spot was analyzed using Labo-1D software
(KURABO). The filter harboring the colonies and the image presenting the chemiluminescence data were superimposed and the positive colonies corresponding to the chemiluminescence signals were identified. Positive clones were picked for colony-PCR as follows: initial denaturation at 94°C for 2 min, 30 cycles of 98°C for 10 s, 63°C for 15 s, and 68°C for 2 min, and a final elongation at 68°C for 2 min. The scFvs were amplified by PCR using Hot Start Taq DNA polymerase (Takara Bio) with the T7 forward and T7 reverse primers at a concentration of 1 µM each and were analyzed on a 0.75% agarose gel to check their size. These positive clones were picked using a toothpick, transferred to 2YT medium containing 0.1 mg/mL carbenicillin, and incubated at 37 °C for 16 h. The cells were pelleted by centrifugation at 5000 × g at 4 °C for 5 min. The plasmids were purified using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). scFv sequences were determined with an ABI Perkin Elmer 373A automated DNA sequencer (Applied Biosystems). 2.8. ELISA The selected positive clones were cultured at 37 °C in 2YT medium containing 0.1 mg/mL carbenicillin until they reached an OD600 of 0.6. Then, the cells were incubated at 30 °C for 6 h in the presence of 1 mM IPTG. Thereafter, they were centrifuged at 5000 × g at 4 °C for 5 min, and the supernatants were collected and analyzed by ELISA. Each well of 96-well ELISA plates was coated with 100 μL of 5 μg/mL rabbit IgG, and then a blocking solution (Blocking Reagent for ELISA; Roche Diagnostics, Swiss) was applied and the plates were incubated for 2 h. Subsequently, the plates were washed with PBS-T, after which 100 μL of the supernatant of bacterial cultures was added to each well. The wells were washed and then HRP-labeled anti-His antibody was added. The amount of the antigenspecific antibody present was measured using an HRP substrate kit (SigmaAldrich), and the plates were read using a microplate reader (Model 680; Bio-Rad Laboratories, Hercules, CA), at a wavelength of 450 nm. All experiments were conducted twice, and the average signal intensity was used in the analysis.
3. Results 3.1. Single-step colony assay performed using A10B To create the expression vector (pET-NXNN) used in this study (schematic in Fig. 1), the expression cassette (Fig. 1A) was inserted in the vector pET22b,
which is driven by the T7 promoter and contains a His-tag sequence for detection of scFv expression. To validate the methodology, a well-established scFv clone was used in the single-step method and the filter-sandwich assay. An scFv that recognizes the constant region of rabbit IgG was constructed from the sequence of a monoclonal IgG1 generated from the A10B hybridoma cell line (Shen et al., 2005b): scFv(A10B) is suitable for developing such methodologies because it is stable and is expressed at adequate levels. In the single-step colony assay (protocol shown schematically in Fig. 2), the expression-inducing reagent is applied to the colonies on the hydrophilic filter that is placed atop the antigencoated membrane in the agar plate. The E. coli colonies produce the antibody fragments as soluble periplasmic molecules, and the secreted antibody fragments that exhibit binding affinity for the antigen are captured on the antigen immobilized on the membrane. The upper filter carrying the viable colonies is stored for the recovery of positive clones, whereas the lower membrane harboring the captured antibody fragments is used for demonstrating antigen-binding activity. By superimposing and aligning the filter and the membrane image showing the chemiluminescent signal, positive clones are identified and picked for isolating the plasmids encoding the scFvs that exhibit affinity for the antigen. This method, which does not involve the transfer of the filter harboring the colonies for scFv expression, is named single-step colony assay. E. coli were transformed with scFv(A10B), and 1 × 103 bacteria were spread per 10-cm-diameter plate. The expression of scFv(A10B) by using our method and the filter-sandwich assay method was compared; the colonies on the filters were counted and the signal intensities of the spots representing the positive clones on the antigen-coated membrane were digitized and measured (Fig. 3). These quantitative comparisons are shown in Table 1. The number of colonies did not differ between the two methods, but the numbers of scFv-expressing colonies were 536 and 268 for our single-step method and the filter-sandwich method, respectively. Thus, in the single-step method, scFv expression was induced comparatively more effectively. Moreover, the mean signal intensity measured for the detected spots was 132 in the single-step assay but only 94 in the filter-sandwich assay; this result showed that scFv expression by each positive colony was higher in the single-step assay than in the filter-sandwich assay. Thus, our method ensures high expression of scFv by E. coli. In the singlestep assay, the ratio of scFv expression decreased when excess or insufficient IPTG was used, and the period for colony formation was also crucial, because if
the colonies were allowed to grow to >1 mm in size (>24 h incubation at 37 °C), the expression of scFv deceased drastically. 3.2. Single-step assay for library screening A single-step colony assay was used to select positive clones from the immune scFv library using rabbit IgG as the antigen (Fig. 4). E. coli cells were transformed with the scFv library and 3 × 104 bacteria were spread on 10 plates (3 × 103 bacteria per 10-cm-diameter plate). Following incubation for 16 h at 37 °C, 1703 colonies were observed in the plate (Fig. 4A). The colonies varied in diameter from 0.5 to 0.9 mm. Next, IPTG was sprayed on the upper filter to induce scFv expression. After incubation for 6 h at 30 °C, antigen binding of scFvs to the lower membrane was detected using chemiluminescence assay. Spots whose intensity was above 30 (arbitrary units) were considered as having a positive signal. In the plate shown in Fig. 4A, there were 12 such spots, amounting to 0.70% of the total number of colonies. Taking into account a total of 10 plates, the mean number of colonies was 1764 ± 64 and that of positive spots was 9.5 ± 4.2, or 0.54% (Table 2). Thus, positive clones expressing the anti-rabbit IgG scFvs were successfully identified using the single-step assay. 3.3. Selection of positive clones Of 12 positive spots, seven of those with the strongest signal intensities were selected for further study (indicated in Fig. 4A). The signal intensity of spots No.1 to No.7 was 854, 458, 503, 473, 410, 504, and 842, respectively. Each clone was identified by superimposing the filter and the membrane chemiluminescence image. Cells were picked from the upper filter, cultured, and scFv expression was induced. Cells were centrifuged at 5000 × g at 4 °C for 5 min. The supernatants were collected and examined for reactivity against the antigen (rabbit IgG) by ELISA (Fig. 4B). Every identified clone showed antigen-specific binding activity, whereas no binding to the uncoated ELISA plate blocked with BSA was detected. The two positive clones (No.3 and No.5) that showed the highest reactivity against the antigen were selected. Serially diluted supernatants from cultures of these two clones exhibited dose-dependent binding against the antigen, but did not bind to BSA. Thus, scFv clones exhibiting binding affinity and specificity toward the antigen were successfully established using the single-step colony assay.
3.4. Sequence analysis of selected clones The DNA sequence of each scFv from clones No.1 to No.7 obtained from the single-step colony assay was analyzed. The inferred amino acid sequences of selected clones are shown in Fig. 5. Every clone contained the complete scFv structure consisting of VH, linker, and VL. The length of VH from clones No.1 to No.7 was 125, 124, 120, 121, 127, 128, and 126 amino acids, respectively. The length of VL from clones No.1 to No.7 was 112, 113, 111, 112, 113, 112, and 116 amino acids, respectively. All sequences were unique, indicating that diversity of the scFv library was maintained through the single-step colony assay. These results indicate that genes encoding scFvs with binding affinity for the antigen could be successfully isolated using our method. 3.5. Application to other antigens To test whether the single-step colony assay technology could be widely used for developing monoclonal scFvs, we applied the method against different antigens (Table 2): 5 proteins and 2 peptides. The peptides were conjugated to keyhole limpet hemocyanin and injected into rats. E. coli were transformed with the scFv library, and bacteria were spread on 10 plates (3 × 103 bacteria per plate). Similar numbers of colonies appeared for each antigen. Colony diameter ranged from 0.5 to 0.9 mm; however, no correlation was observed between antigen type and colony size. To validate the positive clones, randomly selected 10 clones for each antigen were analyzed using colony PCR. The size of insert was checked by agarose gel electrophoresis. Percentage of clones which have full-length scFv inserts was shown in table2. All the clones except for one clone had full-length scFv inserts. Positive clones could be successfully identified with all tested antigens, accounting for approximately 0.87% of the total number of clones. The ratio did not differ substantially between protein and peptide antigens.
4. Discussion The colony-assay method for screening antibody libraries enables the unambiguous selection of positive clones exhibiting high binding specificity and affinity toward an antigen. Because the antigen-antibody reaction is directly revealed during the screening process in the colony assay, the clones that present positive signals express the scFvs showing affinity for the selected antigen, and false-negative clones are rarely identified (Kato and Hanyu, 2015).
Thus, for selecting clones in antibody-library screening, the colony assay presents notable advantages over the phage-display and panning method (Pini et al., 2002). However, the colony-assay method can be improved further for the selection of positive clones, particularly with regard to the handling of colonies during the assay and the reliability of antibody-fragment expression. In the colony assay, controlling the level of expression is critical (Rauth et al., 2010). Because scFv expression by itself is considerably toxic to E. coli, excess induction of expression leads the cell death and prevents the isolation of scFv genes by using the assay. Conversely, if scFv expression is insufficiently induced, the signal from positive clones becomes undetectably weak. In the case of the filter-sandwich assay, the concentration of the expression-inducing reagent (IPTG) added to cells cannot be precisely controlled because IPTG reaches the colonies by diffusing through the filter from the antigen-coated membrane and the agar plate. Moreover, in colony assays, the timing of the induction of expression is crucial, and the size of the colonies affects expression: If the colonies are extremely small, the signal from each colony is inadequate for detecting antigen binding, but if the colony diameter exceeds 0.5 mm, expression is not induced properly (Skerra et al., 1991). However, in the filter-sandwich assay, the strength of induction cannot be accurately determined, particularly during the step when the filter is transferred to the IPTG-containing plate to initiate the induction of expression, and the timing of IPTG exposure cannot be precisely controlled. These uncertainties related to induction in the filter-sandwich assay lead to unstable expression and failure of isolation of antibody-encoding genes. To overcome the aforementioned technical challenges, we have developed the single-step colony assay. In this method, the IPTG concentration is tightly controlled and exposure to IPTG amounts that induce toxic expression is avoided, and thus expression can be induced at optimal levels; this results in the reliable expression of scFvs and enables the successful isolation of scFv genes from positive clones. In the filter-sandwich assay, before the induction of antibody expression, the filter harboring colonies must be transferred without disturbing the colonies. This transfer requires delicate manipulation of the filter and frequently produces unwanted stress on the filter and occasionally disturbs the colonies themselves. These problems are not encountered in our single-step colony assay; the filter harboring the colonies does not have to be transferred before expression, and thus scFv expression can be induced safely without disturbing the colonies.
Moreover, the omission of the intervening steps enables rapid screening and reduces contamination risk. In our use of the singe-step assay, the positive clones accounted for an average of 0.87% of the total number of clones. In traditional hybridoma methods, the number of 96-well microplates routinely screened for a single fusion is 20; this yields, on average, 10–20 positive hybridomas, although the final number strongly depends on the antigenicity of the antigens used and on the host animal. Thus, the two methods offer comparable ratios of positive clones. However, in the single-step assay, only one 10-cm-diameter plate is used, whereas in the hybridoma method, dozens of 96-well plates are required, and our method can be readily scaled up to identify high-expression clones. Therefore, the number of positive clones from the single-step assay could be higher than that from the hybridoma method. This would increase the chance of obtaining monoclonal antibody fragments with desired affinity, specificity, and function. The colony assay is used for selecting antibody fragments against various types of antigens, with the method being optimized to suit the specific purpose. Recombinat antibodies against EspA and Intimin of E. coli O157:H7 were established by colony filter screening (Kühne et al., 2004). Giovannoni et al. isolated anti-angiogenesis antibodies from a large combinatorial repertoire by means of iterative colony-filter screening: colonies located around positive signals were picked and the screening step was repeated, and the positive clones were selected after several rounds of the assay (Giovannoni et al., 2001). Neumann-Schaal et al. developed a colony-screening method in which E. coli colonies producing the required scFv were selected by cultivating the bacteria in the presence of ampicillin conjugated to the antigen of interest; the method relies on the neutralization of the conjugate by the produced scFv, which is secreted into the periplasm (Neumann-Schaal et al., 2013). They identified scFvs against biotin by growing the scFv library-expressing E. coli BL21(DE3) in presence of a biotin-ampicillin conjugate. Robert et al. developed subtractive colony-filter screening to select scFvs that recognize atherosclerotic but not normal aorta (Robert et al., 2006). The colony-lift assay was combined with phage display, with cell-coated filters being used to screen phage libraries for cell-binding clones (Radosević et al., 2003). Thus, the colony assay is a promising tool that could be further developed into a valuable method for screening antibody libraries. The single-step colony assay presented here enhances scFv expression during the assay, which increases the ratio of positive clones. Thus, the manner
in which expression is induced in our method is more appropriate than that in previous methods. However, special care is required during the induction procedure to ensure that the colonies on the filter do not diffuse, and the IPTG mist from the spray should be adequately fine and should spread equally to all the colonies on the filter. If a large amount of solution is sprayed, the colonies are disturbed and contamination might occur. The minimal possible amount of solution should be sprayed homogeneously. Moreover, for inducing expression, additional methods should be examined, such as the cold-shock system, which has provided favorable results in pilot experiments. For further improving the single-step assay, tight control of the expression level is critical, and various factors related to the expression vector must be optimized, such as the promoter, copy number, strength of ribosomal binding site, and temperature of incubation. Lastly, because rhamnose is less toxic than IPTG to E. coli (Giacalone et al., 2006), it should be tested as the expression inducer in the single-step assay.
5. Conclusions We have described here a simple and fast colony-assay method for screening scFv libraries. In this assay, E. coli colonies produce antibody fragments that are secreted as soluble molecules into the periplasm, and the bacterial colonies are formed on a hydrophilic filter that is positioned atop the antigen-coated membrane in an agar plate. The expression-inducing agent is applied in this configuration; thus, the method does not involve the transfer of filters harboring colonies for scFv expression. Furthermore, because the scFv expression level can be tightly controlled, this method ensures high scFv expression in the E. coli colonies. Our results suggest that the undisturbed colony growth, controlled protein expression, and the avoidance of cell death lead to an increased ratio of positive clones obtained from a library, and can thus be effective for successful isolation of genes from positive clones.
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Figure legends Fig. 1. Expression vector constructed in this study. A. A partial sequence of pET-NXNN, shown together with the cloning site and the restriction sites used in the cloning strategy. B. Schematic of scFv cloned into pET-NXNN. Black square, pel B leader; white rectangles, variable antibody domains; gray square, linker; dashed square, Histag; black circle, stop codon.
Fig. 2. Schematic depicting the single-step colony assay. The hydrophilic filter and the antigen-coated nitrocellulose membrane are placed in the 2YT plate, and bacteria transformed with the scFv(A10B) expression vector are spread on the filter. Colonies become visible after 8-h incubation at 37 °C, at which point scFv expression is induced by spraying 400 μL of 1 mM IPTG per plate. The expressed scFvs diffuse through the filter to the membrane, and the scFvs that exhibit binding affinity for the antigen are captured by the antigen immobilized on the membrane. After incubation for 6 h at 30 °C, the filter harboring the colonies is transferred to a fresh 2YT plate and the nitrocellulose membrane is developed to detect the antigen-binding scFvs. To identify positive
colonies, the filter and the membrane chemiluminescence image are superimposed and the colonies on the filter and the positive signals from the nitrocellulose membrane are aligned. The colonies corresponding to the positive signals are picked and cultured in the medium.
Fig. 3. Nitrocellulose membrane image showing positive clones of scFvs against rabbit IgG. A. Colonies of E. coli transformed with the scFv(A10B) expression vector were formed on the 9-cm-diameter hydrophilic filter placed atop the antigen-coated membrane in the 2YT plate. The expression-inducing reagent was applied in this configuration, and the bacterial colonies produced scFv(A10B) as soluble periplasmic molecules. The secreted scFv(A10B) was captured by the antigen immobilized on the membrane. The imaging result here shows the membrane harboring the captured scFv(A10B) and thus demonstrates the antigen-binding activity of the scFv. B. Result obtained using the filter-sandwich assay. Colonies of E. coli transformed with the scFv(A10B) expression vector were formed on a hydrophilic filter that was placed on 2YT plate. The filter harboring the colonies was then transferred onto an antigen-coated membrane that was soaked in the expression-inducing
reagent. The bacterial colonies produced scFv(A10B) as soluble periplasmic molecules, and the secreted scFv(A10B) was captured by the antigen immobilized on the membrane. The capture of scFv(A10B) on the membrane demonstrates the antigen-binding activity of the scFv.
Fig. 4. Screening of positive clones from the scFv library using the single-step assay. A. Antigen binding of scFvs to the membrane was detected by chemiluminescence. Seven positive spots that showed the strongest signal intensities were selected for further study. B. The seven positive clones identified in A were picked and cultured. The reactivity of culture supernatants against rabbit IgG and BSA (control) was investigated using ELISA. Data represent the mean of three replicates; error bars represent the standard deviation. C. Dose-dependent reactivities of supernatants from clones No.3 and No.5 were determined by ELISA. Supernatants from cultures of the two clones were serially diluted and their binding activity against rabbit IgG and BSA (control) was measured. Data represent the mean of three replicates; error bars represent the standard deviation.
Fig. 5. Amino acid sequences alignment of selected clones obtained from the single-step colony assay. VL, VH, and linker sequences are shown. Dots and dashes indicate identical amino acids and deletions, respectively.
Table 1 Quantitative analysis of the colony-assay results. Colonies growing on the filter and the spots representing the positive clones on the antigen-coated membrane (Fig. 3) were counted. Data represent the mean ± SD of 3 replicates. Number of
Number of
Average diameter
Signal intensities
colony
positive spots
Ratio (%)
of colony (mm)
/colony
601 ± 25
536 ± 7
89.3
0.8 ± 0.1
132 ± 15
0mM IPTG
632 ± 28
0
0
-
-
10mM IPTG
581 ± 31
142 ± 4
24.4
-
-
12h for colony formation
541 ± 12
502 ± 8
92.8
0.6 ± 0.1
-
24h for colony formation
612 ± 41
161 ± 4
26.3
1.2 ± 0.2
-
48h for colony formation
613 ± 19
48 ± 3
7.8
1.5 ± 0.2
-
622 ± 25
268 ± 5
30.7
0.4 ± 0.1
94 ± 11
Single-step colony assay (1mM IPTG, 16h for colony formation)
Filter-sandwich assay
Table 2 Number of colonies and percentages of positive clones obtained against various antigens. On each 10-cm-diameter plate, 3 × 103 E. coli cells were spread. Data represent the mean ± SD of 10 dishes. Antigen
Colonies
Positive clones
Ratio of positive clones (%)
scFv insert (%)
Rabbit IgG
1764 ± 64
9.5 ± 4.2
0.54
100
Mouse IgG
1528 ± 102
9.2 ± 2.8
0.60
100
Lysozyme
1530 ± 42
14 ± 2.6
0.92
100
Green Fluorescent Protein
1486 ± 58
11.5 ± 1.2
0.77
100
Glutathione S-transferase
1691 ± 32
30.3 ± 6.4
1.79
100
Peptide (HHHHHH)
819 ± 32
7.0 ± 3.2
0.85
90
Peptide (DYKDDDDK)
1985 ± 91
12.5 ± 5.4
0.63
100
S0168-1656(17)30291-2 http://dx.doi.org/doi:10.1016/j.jbiotec.2017.06.010 BIOTEC 7919
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Journal of Biotechnology
Received date: Revised date: Accepted date:
18-1-2017 19-4-2017 10-6-2017
Please cite this article as: Kato, Mieko, Hanyu, Yoshiro, Single-step colony assay for screening antibody libraries.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2017.06.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Single-step colony assay for screening antibody libraries
Mieko Kato,1,2 and Yoshiro Hanyu 2* 1
Bio-Peak Co., Ltd., 584-70 Shimonojo, Takasaki, 370-0854 Japan. 2 Structural Physiology Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan.
*Corresponding author: Yoshiro Hanyu Structural Physiology Research Group, Biomedical Research Institute, National Institutes of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305-8566 Japan Tel. +81-29-861-2716 Fax: +81-29-861-2706 E-mail: [email protected]
Highlights ・ An scFv selection method was developed based on colony-filter screening.
・The method does not involve transfer of filters with colonies before scFv expression.
・Controlled exposure to an expression-inducing agent causes enhanced scFv expression.
・Monoclonal scFvs were established using this single-step colony assay. ・Reactivity of scFv clones established using our method was evaluated.
Abstract We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3 × 103 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Abbreviations: scFv, single-chain Fv fragment; VL, light-chain variable region; VH, heavy-chain variable region; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; IPTG, isopropyl-β-Dthiogalactopyranoside; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. Keywords: scFv, Escherichia coli, Colony assay, Screening, Antibody library, Expression
1. Introduction A critical step in the establishment of clones of antigen-specific monoclonal antibody fragments is the screening of recombinant antibody libraries (Hoogenboom, 2005; Hust et al., 2014). For rapid screening of large antibody libraries, one of the most extensively used methods is the phage-display method (Hoogenboom et al., 1998; McCafferty et al., 1990). The display of antibody repertories on the surface of bacteriophages and their selection through panning (Parmley and Smith, 1988) allow the isolation monoclonal antibodies (Haque and Tonks, 2012; Schofield et al., 2007). Moreover, in vitro screening performed using this method enables the establishment of monoclonal antibody fragments against various types of antigens, including toxic antigens (Geyer et al., 2012). Phage-display technology allows the handling of extremely large libraries, such as those including 1011 molecules (Bradbury and Sblattero, 2000; Schirrmann et al., 2011); by comparison, the number of hybridomas screened in hybridoma technology is in the order of 103. However, the specific antigen-binding activity is typically not the only driving force exploited during the panning process (Hammers and Stanley, 2014; Nelson, 2010). Multiple rounds of panning have been documented to frequently give rise to a strong bias for antibodies directed against immunodominant epitopes and abundant proteins (de Wildt et al., 2000), which results in the loss of diverse binding specificities and of valuable antibody clones. A common problem encountered is that several factors influence the selection of antigen-specific clones and produce undesired effects; these factors include high expression efficiency and high folding efficiency despite poor antigen-binding activity, hydrophobic binding properties of the phage particle itself, and enhanced tolerance with host cells. Thus, although phage-display screening is a powerful tool for enriching clones from diverse libraries, the method is inadequate for identifying single clones through several rounds of panning. Considerable effort is required to identify single clones exhibiting high specificity and affinity against an antigen after the bio-panning step (Rauth et al., 2010). As an antibody-screening tool, colony-lift immunoassay is superior to—and can
be used instead of—the phage-display method (Kato and Hanyu, 2015; Pini et al., 2002). The detection of antibody fragments released by bacteria was performed by a phage plaque assay in earlier experiments (Caton and Koprowski, 1990; Huse et al., 1989). Libraries of antibody fragments were expressed in Escherichia coli (E. coli) by using phage vectors (Mullinax et al., 1990; Persson et al., 1991). Then the active fragments secreted from viable E. coli colonies were detected by colony-lift immunoassay (Rodenburg et al., 1998). Colonies of E. coli engineered to express antibodies are grown on a plate, and these colonies are lifted onto nitrocellulose membranes to prepare replicas of the colonies (Radosević et al., 2003). The membranes are next transferred onto antigencoated membranes and then placed on plate containing an expression-inducing reagent, such as isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed antibodies diffuse out and react with the antigen and are detected, and thus the colonies that express an antibody that can react with the antigen are identified. The advantage of this method is that the antibody-antigen binding can be directly observed during the screening process, which reduces the selection of falsenegative clones. Dreher et al. improved the colony assay by developing the filter-sandwich colony-screening assay (hereafter the filter-sandwich assay) (Dreher et al., 1991; Skerra et al., 1991). Antibody-expressing E. coli colonies are grown directly on hydrophilic filters, which are then transferred onto antigen-coated membranes soaked with IPTG solution and placed on IPTG plates, to induce antibody production. The antibodies produced by the colonies diffuse out and bind to antigen on the membrane and are detected using immunological methods. The major advantage of this methodology is that it does not involve the lifting of colonies, which requires delicate handling, and Giovannoni et al. used this method to successfully establish several monoclonal antibodies (Giovannoni et al., 2001). However, the induction of expression in the filter-sandwich assay requires further improvement: expression induction is not tightly controlled because the induction reagent (IPTG) that is applied reaches the cells by diffusing through the filter from the antigen-coated membrane. Consequently, the IPTG concentration to which the cells are exposed is not precisely controlled. However, its concentration is critical for colony assay because exposure to excess IPTG induces cell death and prevents the isolation of genes from positive clones, whereas exposure to insufficient IPTG induces inadequate antibody expression for the detection of signals from positive clones.
Here, we describe a simple and fast colony-assay method for screening singlechain Fv fragment (scFv) libraries. In this assay, E. coli colonies produce antibody fragments as soluble molecules that are secreted into the periplasm. Bacterial colonies are formed on a hydrophilic filter that is positioned atop the antigencoated membrane in an agar plate, and the expression-inducing reagent is applied to the filter in this configuration. Thus, this method does not involve the transfer of filters harboring colonies before scFv expression. The method ensures high scFv expression by E. coli colonies and enables the isolation of positive clones from libraries.
2. Materials and Methods 2.1. Immunization Approximately 6-week-old female Wister rats, obtained from SLC (Tokyo, Japan), were intraperitoneally (i.p.) injected with 100 μg of the antigen, 3 times once every 2 weeks; the first injection was in complete Freund’s adjuvant (SigmaAldrich, St. Louis, MO) and the second and third were in incomplete Freund’s adjuvant (Sigma-Aldrich) in a volume of 0.2 mL. The rats were intravenously (i.v.) boosted with 100 μg of the antigen in normal phosphate-buffered saline (PBS) one week after the third i.p. injection and then sacrificed 3 days after the i.v. boost, following which their spleens were aseptically removed. All animals were cared for and maintained in accordance with the guidelines of the National Institute of Advanced Industrial Science and Technology. 2.2. Amplification of VH and VL fragments RNA from the collected rat spleens was purified using NucleoSpin RNA II (Macherey-Nagel, Germany), and the corresponding cDNA was synthesized using PrimeScript Reverse Transcriptase (Takara Bio, Japan) and used as the template for PCR amplification of VL and VH sequences; the amplification protocol was the following: initial denaturation at 94 °C for 2 min, followed by 25 cycles of 98 °C for 3 s, 56 °C for 15 s, and 68 °C for 2 min, and a final elongation at 68 °C for 2 min. PCR was performed by using KOD FX polymerase (Toyobo, Japan) with 10 pM of the primers reported by Sepulveda and Shoemaker (Sepulveda and Shoemaker, 2008). NcoI and XhoI restriction sites were added to the 5ʹ end of the forward and reverse VH primers, and NheI and NotI sites were added to the
5ʹ end of the forward and reverse VL primers, respectively. 2.3. Preparation of expression vector A schematic of the DNA construct that we prepared is shown in Fig. 1 together with the restriction sites. The vector used for the single-step assay, pET-NXNN, was designed and constructed based on pET22b(+) (Merck Millipore, Darmstadt, Germany) for periplasmic protein expression. DNA fragments (Fig. 1A) used for inserting the linker and the restriction sites for cloning VH and VL fragments were synthesized by GenScript (Piscataway, NJ). The generated fragment was digested with NcoI and Bpu1102I (New England Biolabs, MA) and inserted into pET22b(+) (Fig. 1B). 2.4. Construction of scFv library VL and VH sequences PCR-amplified from spleen cDNA of immunized rats were purified and cloned into pET-NXNN. First, pET-NXNN and VL fragments were digested with NheI and NotI (New England Biolabs) and purified using NucleoSpin Gel and PCR clean-up (Macherey-Nagel). The VL library was constructed by ligating VL fragments and the vector, and E. coli DH5 chemically competent cells (Nippon Gene, Japan) were transformed with the resulting vectors. The transformed bacteria were incubated in 6 mL of SOC medium for 1 h at 37 °C, after which the cells were plated on 2YT plates supplemented with 1% glucose and 0.1 mg/mL carbenicillin (Wako, Japan) and cultured overnight. The cells were then collected and suspended in 10 mL of 2YT medium, and the vectors containing the VL fragments (termed pET-NX-VL) were purified using NucleoSpin Plasmid EasyPure (Macherey-Nagel). Lastly, pET-NX-VL and VH fragments were digested with NcoI and XhoI (New England Biolabs), and the scFv library was constructed by ligating the VH fragments to the vector. E. coli DH5 cells were transformed with these vectors and cultured as described above, and then the cells were collected and suspended in 10 mL of 2YT medium, supplemented with 0.1 mg/mL carbenicillin, and used for the colony assay. 2.5. Construction of expression vector for anti-rabbit IgG scFv (scFv(A10B)) For the colony assay, we used scFv(A10B), which recognizes the constant region of rabbit IgG; scFv(A10B) consists of VH and VL sequences joined by the linker sequence GGGGSGGGGSGGGGS (Shen et al., 2005a). The DNA for scFv(A10B), which was synthesized by GenScript, was digested with NcoI and
NotI (New England Biolabs) and inserted into the pET-NXNN vector for the colony assay. E. coli DH5 cells were transformed with this vector. The transformed bacteria were incubated in 6 mL of SOC medium for 1 h at 37 °C and then plated on 2YT plates supplemented with 1% glucose and 0.1 mg/mL carbenicillin and cultured overnight. Cells were collected and suspended in 10 mL of 2YT medium, supplemented with 0.1 mg/mL carbenicillin, and used for the colony assay. 2.6. Colony growth and scFv expression The procedure used in the single-step colony assay is depicted schematically in Fig. 2. The antigen-coated membrane was prepared as follows. A 9-cmdiameter nitrocellulose membrane was coated with the antigen by incubating it for 6 h in PBS containing rabbit IgG (Sigma-Aldrich; 10 μg/mL). Subsequently, the membrane was blocked for 2 h in PBS containing 5% nonfat dry milk, washed twice in PBS, and placed on a 2YT plate (10 cm in diameter) containing 0.1 mg/mL carbenicillin, after which a 9-cm-diameter hydrophilic polyvinylidene difluoride (PVDF) filter (Durapore; Merck Millipore) was positioned atop the antigen-coated membrane. Transformed E. coli were grown in this 2YT medium, and 1-5 × 103 cells from the exponential growth phase were spread onto the filter and incubated at 37 °C for 16 h. After bacterial colonies had formed on the filter surface, 400 μL of 1 mM IPTG (Wako) solution was sprayed uniformly on the top membrane in the plate to induce scFv expression, and the plate was incubated at 30 °C for 6 h. Lastly, the filter harboring the colonies was removed, placed in a fresh 2YT plate containing 1% glucose and 0.1 mg/mL carbenicillin, and stored at 4 °C for later recovery of the bacteria. The nitrocellulose membrane was immersed in PBS containing 0.05% Tween-20 (PBS-T) and used for the detection of antigen-specific scFv expression. 2.7. Detection of antigen binding and identification of positive clones The membrane obtained after the preceding step was washed twice with PBST and blocked for 2 h in PBS containing 5% nonfat dry milk. To detect antigenbound scFvs, the membrane was incubated for 2 h with a horseradish peroxidase (HRP)-conjugated anti-His antibody (Wako; diluted 1:5000 in PBS-T), and then washed extensively in PBS-T. Subsequently, after development with a chemiluminescent HRP substrate kit (Immobilon Western; Merck Millipore), the chemiluminescence signal was detected using a Chemi-Stage CC16mini (KURABO, Japan), and each stained spot was analyzed using Labo-1D software
(KURABO). The filter harboring the colonies and the image presenting the chemiluminescence data were superimposed and the positive colonies corresponding to the chemiluminescence signals were identified. Positive clones were picked for colony-PCR as follows: initial denaturation at 94°C for 2 min, 30 cycles of 98°C for 10 s, 63°C for 15 s, and 68°C for 2 min, and a final elongation at 68°C for 2 min. The scFvs were amplified by PCR using Hot Start Taq DNA polymerase (Takara Bio) with the T7 forward and T7 reverse primers at a concentration of 1 µM each and were analyzed on a 0.75% agarose gel to check their size. These positive clones were picked using a toothpick, transferred to 2YT medium containing 0.1 mg/mL carbenicillin, and incubated at 37 °C for 16 h. The cells were pelleted by centrifugation at 5000 × g at 4 °C for 5 min. The plasmids were purified using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). scFv sequences were determined with an ABI Perkin Elmer 373A automated DNA sequencer (Applied Biosystems). 2.8. ELISA The selected positive clones were cultured at 37 °C in 2YT medium containing 0.1 mg/mL carbenicillin until they reached an OD600 of 0.6. Then, the cells were incubated at 30 °C for 6 h in the presence of 1 mM IPTG. Thereafter, they were centrifuged at 5000 × g at 4 °C for 5 min, and the supernatants were collected and analyzed by ELISA. Each well of 96-well ELISA plates was coated with 100 μL of 5 μg/mL rabbit IgG, and then a blocking solution (Blocking Reagent for ELISA; Roche Diagnostics, Swiss) was applied and the plates were incubated for 2 h. Subsequently, the plates were washed with PBS-T, after which 100 μL of the supernatant of bacterial cultures was added to each well. The wells were washed and then HRP-labeled anti-His antibody was added. The amount of the antigenspecific antibody present was measured using an HRP substrate kit (SigmaAldrich), and the plates were read using a microplate reader (Model 680; Bio-Rad Laboratories, Hercules, CA), at a wavelength of 450 nm. All experiments were conducted twice, and the average signal intensity was used in the analysis.
3. Results 3.1. Single-step colony assay performed using A10B To create the expression vector (pET-NXNN) used in this study (schematic in Fig. 1), the expression cassette (Fig. 1A) was inserted in the vector pET22b,
which is driven by the T7 promoter and contains a His-tag sequence for detection of scFv expression. To validate the methodology, a well-established scFv clone was used in the single-step method and the filter-sandwich assay. An scFv that recognizes the constant region of rabbit IgG was constructed from the sequence of a monoclonal IgG1 generated from the A10B hybridoma cell line (Shen et al., 2005b): scFv(A10B) is suitable for developing such methodologies because it is stable and is expressed at adequate levels. In the single-step colony assay (protocol shown schematically in Fig. 2), the expression-inducing reagent is applied to the colonies on the hydrophilic filter that is placed atop the antigencoated membrane in the agar plate. The E. coli colonies produce the antibody fragments as soluble periplasmic molecules, and the secreted antibody fragments that exhibit binding affinity for the antigen are captured on the antigen immobilized on the membrane. The upper filter carrying the viable colonies is stored for the recovery of positive clones, whereas the lower membrane harboring the captured antibody fragments is used for demonstrating antigen-binding activity. By superimposing and aligning the filter and the membrane image showing the chemiluminescent signal, positive clones are identified and picked for isolating the plasmids encoding the scFvs that exhibit affinity for the antigen. This method, which does not involve the transfer of the filter harboring the colonies for scFv expression, is named single-step colony assay. E. coli were transformed with scFv(A10B), and 1 × 103 bacteria were spread per 10-cm-diameter plate. The expression of scFv(A10B) by using our method and the filter-sandwich assay method was compared; the colonies on the filters were counted and the signal intensities of the spots representing the positive clones on the antigen-coated membrane were digitized and measured (Fig. 3). These quantitative comparisons are shown in Table 1. The number of colonies did not differ between the two methods, but the numbers of scFv-expressing colonies were 536 and 268 for our single-step method and the filter-sandwich method, respectively. Thus, in the single-step method, scFv expression was induced comparatively more effectively. Moreover, the mean signal intensity measured for the detected spots was 132 in the single-step assay but only 94 in the filter-sandwich assay; this result showed that scFv expression by each positive colony was higher in the single-step assay than in the filter-sandwich assay. Thus, our method ensures high expression of scFv by E. coli. In the singlestep assay, the ratio of scFv expression decreased when excess or insufficient IPTG was used, and the period for colony formation was also crucial, because if
the colonies were allowed to grow to >1 mm in size (>24 h incubation at 37 °C), the expression of scFv deceased drastically. 3.2. Single-step assay for library screening A single-step colony assay was used to select positive clones from the immune scFv library using rabbit IgG as the antigen (Fig. 4). E. coli cells were transformed with the scFv library and 3 × 104 bacteria were spread on 10 plates (3 × 103 bacteria per 10-cm-diameter plate). Following incubation for 16 h at 37 °C, 1703 colonies were observed in the plate (Fig. 4A). The colonies varied in diameter from 0.5 to 0.9 mm. Next, IPTG was sprayed on the upper filter to induce scFv expression. After incubation for 6 h at 30 °C, antigen binding of scFvs to the lower membrane was detected using chemiluminescence assay. Spots whose intensity was above 30 (arbitrary units) were considered as having a positive signal. In the plate shown in Fig. 4A, there were 12 such spots, amounting to 0.70% of the total number of colonies. Taking into account a total of 10 plates, the mean number of colonies was 1764 ± 64 and that of positive spots was 9.5 ± 4.2, or 0.54% (Table 2). Thus, positive clones expressing the anti-rabbit IgG scFvs were successfully identified using the single-step assay. 3.3. Selection of positive clones Of 12 positive spots, seven of those with the strongest signal intensities were selected for further study (indicated in Fig. 4A). The signal intensity of spots No.1 to No.7 was 854, 458, 503, 473, 410, 504, and 842, respectively. Each clone was identified by superimposing the filter and the membrane chemiluminescence image. Cells were picked from the upper filter, cultured, and scFv expression was induced. Cells were centrifuged at 5000 × g at 4 °C for 5 min. The supernatants were collected and examined for reactivity against the antigen (rabbit IgG) by ELISA (Fig. 4B). Every identified clone showed antigen-specific binding activity, whereas no binding to the uncoated ELISA plate blocked with BSA was detected. The two positive clones (No.3 and No.5) that showed the highest reactivity against the antigen were selected. Serially diluted supernatants from cultures of these two clones exhibited dose-dependent binding against the antigen, but did not bind to BSA. Thus, scFv clones exhibiting binding affinity and specificity toward the antigen were successfully established using the single-step colony assay.
3.4. Sequence analysis of selected clones The DNA sequence of each scFv from clones No.1 to No.7 obtained from the single-step colony assay was analyzed. The inferred amino acid sequences of selected clones are shown in Fig. 5. Every clone contained the complete scFv structure consisting of VH, linker, and VL. The length of VH from clones No.1 to No.7 was 125, 124, 120, 121, 127, 128, and 126 amino acids, respectively. The length of VL from clones No.1 to No.7 was 112, 113, 111, 112, 113, 112, and 116 amino acids, respectively. All sequences were unique, indicating that diversity of the scFv library was maintained through the single-step colony assay. These results indicate that genes encoding scFvs with binding affinity for the antigen could be successfully isolated using our method. 3.5. Application to other antigens To test whether the single-step colony assay technology could be widely used for developing monoclonal scFvs, we applied the method against different antigens (Table 2): 5 proteins and 2 peptides. The peptides were conjugated to keyhole limpet hemocyanin and injected into rats. E. coli were transformed with the scFv library, and bacteria were spread on 10 plates (3 × 103 bacteria per plate). Similar numbers of colonies appeared for each antigen. Colony diameter ranged from 0.5 to 0.9 mm; however, no correlation was observed between antigen type and colony size. To validate the positive clones, randomly selected 10 clones for each antigen were analyzed using colony PCR. The size of insert was checked by agarose gel electrophoresis. Percentage of clones which have full-length scFv inserts was shown in table2. All the clones except for one clone had full-length scFv inserts. Positive clones could be successfully identified with all tested antigens, accounting for approximately 0.87% of the total number of clones. The ratio did not differ substantially between protein and peptide antigens.
4. Discussion The colony-assay method for screening antibody libraries enables the unambiguous selection of positive clones exhibiting high binding specificity and affinity toward an antigen. Because the antigen-antibody reaction is directly revealed during the screening process in the colony assay, the clones that present positive signals express the scFvs showing affinity for the selected antigen, and false-negative clones are rarely identified (Kato and Hanyu, 2015).
Thus, for selecting clones in antibody-library screening, the colony assay presents notable advantages over the phage-display and panning method (Pini et al., 2002). However, the colony-assay method can be improved further for the selection of positive clones, particularly with regard to the handling of colonies during the assay and the reliability of antibody-fragment expression. In the colony assay, controlling the level of expression is critical (Rauth et al., 2010). Because scFv expression by itself is considerably toxic to E. coli, excess induction of expression leads the cell death and prevents the isolation of scFv genes by using the assay. Conversely, if scFv expression is insufficiently induced, the signal from positive clones becomes undetectably weak. In the case of the filter-sandwich assay, the concentration of the expression-inducing reagent (IPTG) added to cells cannot be precisely controlled because IPTG reaches the colonies by diffusing through the filter from the antigen-coated membrane and the agar plate. Moreover, in colony assays, the timing of the induction of expression is crucial, and the size of the colonies affects expression: If the colonies are extremely small, the signal from each colony is inadequate for detecting antigen binding, but if the colony diameter exceeds 0.5 mm, expression is not induced properly (Skerra et al., 1991). However, in the filter-sandwich assay, the strength of induction cannot be accurately determined, particularly during the step when the filter is transferred to the IPTG-containing plate to initiate the induction of expression, and the timing of IPTG exposure cannot be precisely controlled. These uncertainties related to induction in the filter-sandwich assay lead to unstable expression and failure of isolation of antibody-encoding genes. To overcome the aforementioned technical challenges, we have developed the single-step colony assay. In this method, the IPTG concentration is tightly controlled and exposure to IPTG amounts that induce toxic expression is avoided, and thus expression can be induced at optimal levels; this results in the reliable expression of scFvs and enables the successful isolation of scFv genes from positive clones. In the filter-sandwich assay, before the induction of antibody expression, the filter harboring colonies must be transferred without disturbing the colonies. This transfer requires delicate manipulation of the filter and frequently produces unwanted stress on the filter and occasionally disturbs the colonies themselves. These problems are not encountered in our single-step colony assay; the filter harboring the colonies does not have to be transferred before expression, and thus scFv expression can be induced safely without disturbing the colonies.
Moreover, the omission of the intervening steps enables rapid screening and reduces contamination risk. In our use of the singe-step assay, the positive clones accounted for an average of 0.87% of the total number of clones. In traditional hybridoma methods, the number of 96-well microplates routinely screened for a single fusion is 20; this yields, on average, 10–20 positive hybridomas, although the final number strongly depends on the antigenicity of the antigens used and on the host animal. Thus, the two methods offer comparable ratios of positive clones. However, in the single-step assay, only one 10-cm-diameter plate is used, whereas in the hybridoma method, dozens of 96-well plates are required, and our method can be readily scaled up to identify high-expression clones. Therefore, the number of positive clones from the single-step assay could be higher than that from the hybridoma method. This would increase the chance of obtaining monoclonal antibody fragments with desired affinity, specificity, and function. The colony assay is used for selecting antibody fragments against various types of antigens, with the method being optimized to suit the specific purpose. Recombinat antibodies against EspA and Intimin of E. coli O157:H7 were established by colony filter screening (Kühne et al., 2004). Giovannoni et al. isolated anti-angiogenesis antibodies from a large combinatorial repertoire by means of iterative colony-filter screening: colonies located around positive signals were picked and the screening step was repeated, and the positive clones were selected after several rounds of the assay (Giovannoni et al., 2001). Neumann-Schaal et al. developed a colony-screening method in which E. coli colonies producing the required scFv were selected by cultivating the bacteria in the presence of ampicillin conjugated to the antigen of interest; the method relies on the neutralization of the conjugate by the produced scFv, which is secreted into the periplasm (Neumann-Schaal et al., 2013). They identified scFvs against biotin by growing the scFv library-expressing E. coli BL21(DE3) in presence of a biotin-ampicillin conjugate. Robert et al. developed subtractive colony-filter screening to select scFvs that recognize atherosclerotic but not normal aorta (Robert et al., 2006). The colony-lift assay was combined with phage display, with cell-coated filters being used to screen phage libraries for cell-binding clones (Radosević et al., 2003). Thus, the colony assay is a promising tool that could be further developed into a valuable method for screening antibody libraries. The single-step colony assay presented here enhances scFv expression during the assay, which increases the ratio of positive clones. Thus, the manner
in which expression is induced in our method is more appropriate than that in previous methods. However, special care is required during the induction procedure to ensure that the colonies on the filter do not diffuse, and the IPTG mist from the spray should be adequately fine and should spread equally to all the colonies on the filter. If a large amount of solution is sprayed, the colonies are disturbed and contamination might occur. The minimal possible amount of solution should be sprayed homogeneously. Moreover, for inducing expression, additional methods should be examined, such as the cold-shock system, which has provided favorable results in pilot experiments. For further improving the single-step assay, tight control of the expression level is critical, and various factors related to the expression vector must be optimized, such as the promoter, copy number, strength of ribosomal binding site, and temperature of incubation. Lastly, because rhamnose is less toxic than IPTG to E. coli (Giacalone et al., 2006), it should be tested as the expression inducer in the single-step assay.
5. Conclusions We have described here a simple and fast colony-assay method for screening scFv libraries. In this assay, E. coli colonies produce antibody fragments that are secreted as soluble molecules into the periplasm, and the bacterial colonies are formed on a hydrophilic filter that is positioned atop the antigen-coated membrane in an agar plate. The expression-inducing agent is applied in this configuration; thus, the method does not involve the transfer of filters harboring colonies for scFv expression. Furthermore, because the scFv expression level can be tightly controlled, this method ensures high scFv expression in the E. coli colonies. Our results suggest that the undisturbed colony growth, controlled protein expression, and the avoidance of cell death lead to an increased ratio of positive clones obtained from a library, and can thus be effective for successful isolation of genes from positive clones.
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Figure legends Fig. 1. Expression vector constructed in this study. A. A partial sequence of pET-NXNN, shown together with the cloning site and the restriction sites used in the cloning strategy. B. Schematic of scFv cloned into pET-NXNN. Black square, pel B leader; white rectangles, variable antibody domains; gray square, linker; dashed square, Histag; black circle, stop codon.
Fig. 2. Schematic depicting the single-step colony assay. The hydrophilic filter and the antigen-coated nitrocellulose membrane are placed in the 2YT plate, and bacteria transformed with the scFv(A10B) expression vector are spread on the filter. Colonies become visible after 8-h incubation at 37 °C, at which point scFv expression is induced by spraying 400 μL of 1 mM IPTG per plate. The expressed scFvs diffuse through the filter to the membrane, and the scFvs that exhibit binding affinity for the antigen are captured by the antigen immobilized on the membrane. After incubation for 6 h at 30 °C, the filter harboring the colonies is transferred to a fresh 2YT plate and the nitrocellulose membrane is developed to detect the antigen-binding scFvs. To identify positive
colonies, the filter and the membrane chemiluminescence image are superimposed and the colonies on the filter and the positive signals from the nitrocellulose membrane are aligned. The colonies corresponding to the positive signals are picked and cultured in the medium.
Fig. 3. Nitrocellulose membrane image showing positive clones of scFvs against rabbit IgG. A. Colonies of E. coli transformed with the scFv(A10B) expression vector were formed on the 9-cm-diameter hydrophilic filter placed atop the antigen-coated membrane in the 2YT plate. The expression-inducing reagent was applied in this configuration, and the bacterial colonies produced scFv(A10B) as soluble periplasmic molecules. The secreted scFv(A10B) was captured by the antigen immobilized on the membrane. The imaging result here shows the membrane harboring the captured scFv(A10B) and thus demonstrates the antigen-binding activity of the scFv. B. Result obtained using the filter-sandwich assay. Colonies of E. coli transformed with the scFv(A10B) expression vector were formed on a hydrophilic filter that was placed on 2YT plate. The filter harboring the colonies was then transferred onto an antigen-coated membrane that was soaked in the expression-inducing
reagent. The bacterial colonies produced scFv(A10B) as soluble periplasmic molecules, and the secreted scFv(A10B) was captured by the antigen immobilized on the membrane. The capture of scFv(A10B) on the membrane demonstrates the antigen-binding activity of the scFv.
Fig. 4. Screening of positive clones from the scFv library using the single-step assay. A. Antigen binding of scFvs to the membrane was detected by chemiluminescence. Seven positive spots that showed the strongest signal intensities were selected for further study. B. The seven positive clones identified in A were picked and cultured. The reactivity of culture supernatants against rabbit IgG and BSA (control) was investigated using ELISA. Data represent the mean of three replicates; error bars represent the standard deviation. C. Dose-dependent reactivities of supernatants from clones No.3 and No.5 were determined by ELISA. Supernatants from cultures of the two clones were serially diluted and their binding activity against rabbit IgG and BSA (control) was measured. Data represent the mean of three replicates; error bars represent the standard deviation.
Fig. 5. Amino acid sequences alignment of selected clones obtained from the single-step colony assay. VL, VH, and linker sequences are shown. Dots and dashes indicate identical amino acids and deletions, respectively.
Table 1 Quantitative analysis of the colony-assay results. Colonies growing on the filter and the spots representing the positive clones on the antigen-coated membrane (Fig. 3) were counted. Data represent the mean ± SD of 3 replicates. Number of
Number of
Average diameter
Signal intensities
colony
positive spots
Ratio (%)
of colony (mm)
/colony
601 ± 25
536 ± 7
89.3
0.8 ± 0.1
132 ± 15
0mM IPTG
632 ± 28
0
0
-
-
10mM IPTG
581 ± 31
142 ± 4
24.4
-
-
12h for colony formation
541 ± 12
502 ± 8
92.8
0.6 ± 0.1
-
24h for colony formation
612 ± 41
161 ± 4
26.3
1.2 ± 0.2
-
48h for colony formation
613 ± 19
48 ± 3
7.8
1.5 ± 0.2
-
622 ± 25
268 ± 5
30.7
0.4 ± 0.1
94 ± 11
Single-step colony assay (1mM IPTG, 16h for colony formation)
Filter-sandwich assay
Table 2 Number of colonies and percentages of positive clones obtained against various antigens. On each 10-cm-diameter plate, 3 × 103 E. coli cells were spread. Data represent the mean ± SD of 10 dishes. Antigen
Colonies
Positive clones
Ratio of positive clones (%)
scFv insert (%)
Rabbit IgG
1764 ± 64
9.5 ± 4.2
0.54
100
Mouse IgG
1528 ± 102
9.2 ± 2.8
0.60
100
Lysozyme
1530 ± 42
14 ± 2.6
0.92
100
Green Fluorescent Protein
1486 ± 58
11.5 ± 1.2
0.77
100
Glutathione S-transferase
1691 ± 32
30.3 ± 6.4
1.79
100
Peptide (HHHHHH)
819 ± 32
7.0 ± 3.2
0.85
90
Peptide (DYKDDDDK)
1985 ± 91
12.5 ± 5.4
0.63
100