**3.1. Cloning the variable VH and VL domains from hybridoma cells**

#### *3.1.1. Poly A+ RNA extraction*

The anti-PcrV IgG Mab166 hybridoma cell line [35] was cultured in a standard culture medium. After the cells had reached confluence in a 75 cm<sup>2</sup> flask, they were harvested by centrifugation at 600 rpm for 5 min. The cell pellet was homogenized in 2 mL of TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA), and total RNA extracted after chloroform fractionation, isopropanol precipitation, and washing with 70% ethanol. Poly A+ RNA was extracted with an oligotex mRNA spin-column (Qiagen, Valencia, CA).

a DNA sequencing service for DNA sequence acquisition and analysis. Sequencing of the immunoglobulin variable genes for Mab166 was analyzed by The International imMunoGe-

the correct reading frame. Assembly PCR was run with a set of primers to multiply VH-linker-VL. The assembled fragment was amplified using two oligonucleotide primers with either an *Nco*I or *Xba*I restriction enzyme site at the 5' end to facilitate cloning of the PCR product into a pBAD/ gene III plasmid (Thermo Fisher Scientific) (**Figure 1**). The ligation mixture was used to transform *E. coli* TOP10 cells (Thermo Fisher Scientific), and subsequently to transform *E. coli* LMG194.

scFv166 protein expression was induced in the *E. coli* plasmid-harboring transformants by adding L-arabinose to a final concentration of 0.004%. After 24 h culture at 26°C with agitation at 200 rpm, the cells were collected by centrifugation at 5000× g for 20 min and then incubated in phosphate-buffered saline (PBS) with 1 mM ethylenediaminetetraacetic acid for 10 min on ice to obtain the periplasmic fraction. The osmotically shocked lysate was centrifuged at 15,000× g for 20 min, passed through a 0.4-μm-pore-size filter and dialyzed overnight against

**Figure 1.** Expression vector pBAD/gIII::m166-HLL. The assembled scFv166 gene was subcloned into the pBAD/Gene III *E. coli* expression vector, downstream of, and in frame with, the gene III secretory leader sequence using *NcoI* and *XbaI*

Ser)3

**3.2. Expression and purification of recombinant single-chain antibody fragments**

cDNAs were each assembled into a single gene using a DNA linker frag-

Construction and Characteristics of a Recombinant Single-Chain Antibody Fragment...

linker peptide, thereby connecting the two cDNAs in

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123

neTics Database IMGT (http://www.imgt.org).

ment-encoding a glycine-serine (Gly4

*3.2.1. Expression and purification of scFv166*

The purified VH and VL

restriction sites.

#### *3.1.2. RNA oligo-capping*

To clone the variable VH and VL domains from the total RNA, the oligo-capping method reported by Maruyama and Sugano [40] using a GeneRacer™ kit (Thermo Fisher Scientific) was used. mRNA (250 ng) was incubated with calf intestinal phosphatase at 50 °C for 1 h to dephosphorylate non-mRNA or truncated mRNA species. After the reaction, phenol-chloroform extraction and ethanol precipitation were performed, and the dephosphorylated RNA was incubated with tobacco acid pyrophosphatase at 37°C for 1 h to remove the 5'-cap structure from the full-length mRNA. After phenol-chloroform extraction and ethanol precipitation, the synthetic RNA oligo (GeneRacer™ RNA Oligo, Thermo Fisher Scientific) was ligated to the decapped RNA with T4 RNA ligase at 37°C for 1 h. After phenol-chloroform extraction and ethanol precipitation, the RNA was suspended in diethylpyrocarbonatetreated water.

#### *3.1.3. Reverse transcription of mRNA*

The RNA-oligo ligated, full-length mRNA was reverse transcribed using a 54 base-pair primer containing an 18 nucleotide dT tail (GeneRacer™ Oligo-dT, Thermo Fisher Scientific) and avian myeloblastosis virus reverse transcriptase at 42°C for 1 h. After the reaction, the sample was diluted four times with sterile water.

#### *3.1.4. Construction of a single-chain antibody gene*

The cDNAs encoding V regions of the heavy and light (kappa) chains were PCR-amplified using a set of primers (VH forward: 5'-TGA GGA GAC GGT GAC TGA GGT TCC-3', VH reverse : 5'-CAG GTG CAG CTG AAG CAG TCA GG-3', Vk2 forward: 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3', Vk reverse : 5'-GAC ATC CAG ATG ACT CAG TCT CCA-3'). PCRs were run over 30 cycles (94°C for 30 sec, 60°C for 40 sec, and 72°C for 40 sec). VH and VL fragment-amplified PCR products were purified separately by agarose gel electrophoresis. The PCR products derived from the murine immunoglobulin VH and VL domain of Mab166 were subcloned into a pCR2.1 vector (TOPO cloningTM, Thermo Fisher Scientific) and submitted to a DNA sequencing service for DNA sequence acquisition and analysis. Sequencing of the immunoglobulin variable genes for Mab166 was analyzed by The International imMunoGeneTics Database IMGT (http://www.imgt.org).

The purified VH and VL cDNAs were each assembled into a single gene using a DNA linker fragment-encoding a glycine-serine (Gly4 Ser)3 linker peptide, thereby connecting the two cDNAs in the correct reading frame. Assembly PCR was run with a set of primers to multiply VH-linker-VL. The assembled fragment was amplified using two oligonucleotide primers with either an *Nco*I or *Xba*I restriction enzyme site at the 5' end to facilitate cloning of the PCR product into a pBAD/ gene III plasmid (Thermo Fisher Scientific) (**Figure 1**). The ligation mixture was used to transform *E. coli* TOP10 cells (Thermo Fisher Scientific), and subsequently to transform *E. coli* LMG194.

#### **3.2. Expression and purification of recombinant single-chain antibody fragments**

#### *3.2.1. Expression and purification of scFv166*

**3. Methods for construction of a single-chain antibody**

**3.1. Cloning the variable VH and VL domains from hybridoma cells**

medium. After the cells had reached confluence in a 75 cm<sup>2</sup>

The anti-PcrV IgG Mab166 hybridoma cell line [35] was cultured in a standard culture

trifugation at 600 rpm for 5 min. The cell pellet was homogenized in 2 mL of TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA), and total RNA extracted after chloroform

To clone the variable VH and VL domains from the total RNA, the oligo-capping method reported by Maruyama and Sugano [40] using a GeneRacer™ kit (Thermo Fisher Scientific) was used. mRNA (250 ng) was incubated with calf intestinal phosphatase at 50 °C for 1 h to dephosphorylate non-mRNA or truncated mRNA species. After the reaction, phenol-chloroform extraction and ethanol precipitation were performed, and the dephosphorylated RNA was incubated with tobacco acid pyrophosphatase at 37°C for 1 h to remove the 5'-cap structure from the full-length mRNA. After phenol-chloroform extraction and ethanol precipitation, the synthetic RNA oligo (GeneRacer™ RNA Oligo, Thermo Fisher Scientific) was ligated to the decapped RNA with T4 RNA ligase at 37°C for 1 h. After phenol-chloroform extraction and ethanol precipitation, the RNA was suspended in diethylpyrocarbonate-

The RNA-oligo ligated, full-length mRNA was reverse transcribed using a 54 base-pair primer containing an 18 nucleotide dT tail (GeneRacer™ Oligo-dT, Thermo Fisher Scientific) and avian myeloblastosis virus reverse transcriptase at 42°C for 1 h. After the reaction, the

The cDNAs encoding V regions of the heavy and light (kappa) chains were PCR-amplified using a set of primers (VH forward: 5'-TGA GGA GAC GGT GAC TGA GGT TCC-3', VH reverse : 5'-CAG GTG CAG CTG AAG CAG TCA GG-3', Vk2 forward: 5'-CCG TTT TAT TTC

were run over 30 cycles (94°C for 30 sec, 60°C for 40 sec, and 72°C for 40 sec). VH and VL fragment-amplified PCR products were purified separately by agarose gel electrophoresis. The PCR products derived from the murine immunoglobulin VH and VL domain of Mab166 were subcloned into a pCR2.1 vector (TOPO cloningTM, Thermo Fisher Scientific) and submitted to

reverse : 5'-GAC ATC CAG ATG ACT CAG TCT CCA-3'). PCRs

fractionation, isopropanol precipitation, and washing with 70% ethanol. Poly A+

extracted with an oligotex mRNA spin-column (Qiagen, Valencia, CA).

flask, they were harvested by cen-

RNA was

*3.1.1. Poly A+*

122 Antibody Engineering

*3.1.2. RNA oligo-capping*

treated water.

*3.1.3. Reverse transcription of mRNA*

CAG CTT GGT CCC-3', Vk

sample was diluted four times with sterile water.

*3.1.4. Construction of a single-chain antibody gene*

 *RNA extraction*

scFv166 protein expression was induced in the *E. coli* plasmid-harboring transformants by adding L-arabinose to a final concentration of 0.004%. After 24 h culture at 26°C with agitation at 200 rpm, the cells were collected by centrifugation at 5000× g for 20 min and then incubated in phosphate-buffered saline (PBS) with 1 mM ethylenediaminetetraacetic acid for 10 min on ice to obtain the periplasmic fraction. The osmotically shocked lysate was centrifuged at 15,000× g for 20 min, passed through a 0.4-μm-pore-size filter and dialyzed overnight against

**Figure 1.** Expression vector pBAD/gIII::m166-HLL. The assembled scFv166 gene was subcloned into the pBAD/Gene III *E. coli* expression vector, downstream of, and in frame with, the gene III secretory leader sequence using *NcoI* and *XbaI* restriction sites.

lysis buffer (50 mM NaH<sup>2</sup> PO4 , 300 mM NaCl, 10 mM imidazole, 0.05% Tween 20, pH 8.0). The lysate was mixed with nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) for 30 min at 4°C with gentle shaking. After the Ni-NTA agarose was collected by centrifugation (4000× g), it was resuspended in lysis buffer and packed onto the chromatography column.

The column was washed twice with washing buffer (50 mM NaH<sup>2</sup> PO4 , 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20, pH 8.0), and the bound scFv166 antibodies were eluted with elution buffer (50 mM NaH<sup>2</sup> PO4 , 300 mM NaCl, 250 mM imidazole, 0.05% Tween 20, pH 8.0). The eluate was dialyzed against PBS overnight and applied to an endotoxin removal column (Detoxi-Gel, Thermo Fisher Scientific) to get rid of the contaminating endotoxin. The purified antibodies were stored at −80°C until use.

In this study, we assembled the variable regions of the heavy and light chains of the anti-PcrV monoclonal IgG together with a glycine-serine linker in a single-chain antibody format. First, we assembled scFv166 in two different formats: one with VH-linker-Vk positioned between the two variable segments (**Figure 2**), the other with Vk -linker-VH positioned between two variable segments. The assembled scFv166 gene was subcloned into the *E. coli* pBAD/gene III expression vector, downstream of, and in frame with, the gene III secretory leader sequence. Expression of recombinant scFv166 was induced in *E. coli* by arabinose, after which it was purified via its C-terminal hexahistidine tag using Ni-NTA resin and conventional affinity column techniques. However, scFv-VL-linker-VH was highly insoluble, despite the expressed protein being detected in the whole lysates from *E. coli* cells after arabinose induction. Because scFv166 with its associated VH-linker-VL fragment was easier to purify as a soluble protein, we decided to focus on purifying it in that format. The purified scFv166 recognized the PcrV antigen in ELISAs and western immunoblots, as described in the next section.

#### **3.3. Protein gels and immunoblot analyses**

The purity of scFv166 was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining (**Figure 3**). Briefly, samples of *E. coli* lysate were loaded onto a 4–15% Tris-HCl gel (BioRad Laboratories Inc., Hercules, CA, USA) and, after electrophoresis, the gel was stained with Coomassie Blue. For the immunoblot analysis, after SDS-PAGE, the protein was transferred to a nitrocellulose membrane and immunostained with a horseradish peroxidase-conjugated anti-c-myc IgG antibody, after which the blot was developed with a chemiluminescent substrate (ECL, GE Healthcare Bioscience, Piscataway, NJ). Immunoblots of scFv166 and precipitated *P. aeruginosa* proteins were also performed (**Figure 4**). *P. aeruginosa* PA103 was cultured in tryptic soy broth deferrated with nitrilotriacetic acid for 24 h at 31°C and, after centrifugation at 5000× g for 20 min, the supernatant was harvested. Saturated ammonium sulfate solution was added (final concentration, 55%), and the solution was incubated on ice for 1 h, and then centrifuged (20,000 × g, 30 min). The precipitated proteins were resuspended in 100 μL of PBS. After adding 100 μL of SDS-PAGE sample buffer and boiling for 5 min, the sample was analyzed by SDS-PAGE. After electrophoresis, the proteins were blotted onto a nitrocellulose membrane, and then immunostained with scFv166 and a horseradish peroxidase-conjugated anti-c-myc IgG secondary antibody, and the blot was developed with ECL.

**3.4. Affinity determination of scFv166**

The affinity of scFv166 for its cognate antigen was determined by competition ELISA, and the result was compared with that of the hybridoma-derived parental Mab166, as described previously [41], **Figure 5**. Briefly, in the first step, the total antibody concentration range in which the absorbance correlates proportionately with the free antibody concentration was

**Figure 2.** scFv166 nucleotide sequence. After the gene III signal sequence (18 aa) and the short joint region (6 aa), the VH region (123 aa) is followed by the glycine-serine linker (15 aa), the VL region (108 aa), a cMyc-tag, and a hexahistidine-tag.

Construction and Characteristics of a Recombinant Single-Chain Antibody Fragment...

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Construction and Characteristics of a Recombinant Single-Chain Antibody Fragment... http://dx.doi.org/10.5772/intechopen.70316 125

**Figure 2.** scFv166 nucleotide sequence. After the gene III signal sequence (18 aa) and the short joint region (6 aa), the VH region (123 aa) is followed by the glycine-serine linker (15 aa), the VL region (108 aa), a cMyc-tag, and a hexahistidine-tag.

#### **3.4. Affinity determination of scFv166**

lysis buffer (50 mM NaH<sup>2</sup>

124 Antibody Engineering

tion buffer (50 mM NaH<sup>2</sup>

PO4

PO4

the two variable segments (**Figure 2**), the other with Vk

antibodies were stored at −80°C until use.

**3.3. Protein gels and immunoblot analyses**

antibody, and the blot was developed with ECL.

, 300 mM NaCl, 10 mM imidazole, 0.05% Tween 20, pH 8.0). The

, 300 mM NaCl, 250 mM imidazole, 0.05% Tween 20, pH 8.0).

PO4

, 300 mM NaCl, 20 mM

positioned between


lysate was mixed with nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) for 30 min at 4°C with gentle shaking. After the Ni-NTA agarose was collected by centrifugation (4000× g), it was

imidazole, 0.05% Tween 20, pH 8.0), and the bound scFv166 antibodies were eluted with elu-

The eluate was dialyzed against PBS overnight and applied to an endotoxin removal column (Detoxi-Gel, Thermo Fisher Scientific) to get rid of the contaminating endotoxin. The purified

In this study, we assembled the variable regions of the heavy and light chains of the anti-PcrV monoclonal IgG together with a glycine-serine linker in a single-chain antibody format. First,

variable segments. The assembled scFv166 gene was subcloned into the *E. coli* pBAD/gene III expression vector, downstream of, and in frame with, the gene III secretory leader sequence. Expression of recombinant scFv166 was induced in *E. coli* by arabinose, after which it was purified via its C-terminal hexahistidine tag using Ni-NTA resin and conventional affinity column techniques. However, scFv-VL-linker-VH was highly insoluble, despite the expressed protein being detected in the whole lysates from *E. coli* cells after arabinose induction. Because scFv166 with its associated VH-linker-VL fragment was easier to purify as a soluble protein, we decided to focus on purifying it in that format. The purified scFv166 recognized the PcrV

The purity of scFv166 was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining (**Figure 3**). Briefly, samples of *E. coli* lysate were loaded onto a 4–15% Tris-HCl gel (BioRad Laboratories Inc., Hercules, CA, USA) and, after electrophoresis, the gel was stained with Coomassie Blue. For the immunoblot analysis, after SDS-PAGE, the protein was transferred to a nitrocellulose membrane and immunostained with a horseradish peroxidase-conjugated anti-c-myc IgG antibody, after which the blot was developed with a chemiluminescent substrate (ECL, GE Healthcare Bioscience, Piscataway, NJ). Immunoblots of scFv166 and precipitated *P. aeruginosa* proteins were also performed (**Figure 4**). *P. aeruginosa* PA103 was cultured in tryptic soy broth deferrated with nitrilotriacetic acid for 24 h at 31°C and, after centrifugation at 5000× g for 20 min, the supernatant was harvested. Saturated ammonium sulfate solution was added (final concentration, 55%), and the solution was incubated on ice for 1 h, and then centrifuged (20,000 × g, 30 min). The precipitated proteins were resuspended in 100 μL of PBS. After adding 100 μL of SDS-PAGE sample buffer and boiling for 5 min, the sample was analyzed by SDS-PAGE. After electrophoresis, the proteins were blotted onto a nitrocellulose membrane, and then immunostained with scFv166 and a horseradish peroxidase-conjugated anti-c-myc IgG secondary

resuspended in lysis buffer and packed onto the chromatography column.

we assembled scFv166 in two different formats: one with VH-linker-Vk

antigen in ELISAs and western immunoblots, as described in the next section.

The column was washed twice with washing buffer (50 mM NaH<sup>2</sup>

The affinity of scFv166 for its cognate antigen was determined by competition ELISA, and the result was compared with that of the hybridoma-derived parental Mab166, as described previously [41], **Figure 5**. Briefly, in the first step, the total antibody concentration range in which the absorbance correlates proportionately with the free antibody concentration was

**Figure 3.** Expression and purification of scFv166. *E. coli* lysates were loaded onto a 4–15% gradient Tris-HCl gel and, after electrophoresis, the gel was stained with Coomassie Blue. For the immunoblot analysis, after polyacrylamide gel electrophoresis, the protein was blotted onto a nitrocellulose membrane and immunostained with a horseradish peroxidase-conjugated anti-c-Myc IgG antibody, and the blot was developed with a chemiluminescent substrate. The secreted scFv166 (298 amino acids) was detected as a 29.7 kD-band in the elute-1 and elute-2, designated by arrows. Soluble fraction: the osmotically shocked lysate; elute: the eluted solution from an Ni-NTA agarose column; soluble fraction after elution: the solution passed through an Ni-NTA agarose column (two sets of the lysate and the column elute were analyzed and labeled "−1" and "−2", respectively).

**4. Results**

**4.1. Aminoacid sequence of VH, VL of scFv166**

0

to immobilized PcrV were evaluated in a competition ELISA.

100

%Ab binding

The sequence of the Mab166 heavy chain region is shown in **Figure 2**. The DNA sequence of the 5'-untranslational region and a V-region segment in the heavy chain-containing complementarity determining regions (CDRs) 1 and 2 is identical (except two amino acids in the frame 3 region) to germline Musmus IGHV2S2 (IGHV subgroup 2, VH#101, Accession #J00502). The V-region sequence also shows the same level of homology as that reported for pseudogene, IGHV2S5 (Accession #M21165). Transcription starts 24 nucleotides downstream of the TATA box of germline IGHV2S2. Nucleotides differ from the germline sequence at 10 positions, and these cause the following amino acid changes: position #61 in CDR2 S->D, #87 in FR3 V->L, #95 Q->R, and #96 S->A, #97 N->T. The first 15 nucleotides in the D-region encode the first 5 unique amino acids in CDR3, and the region consists of 16 amino acids in total. The J-region DNA sequence is identical to the IGHJ4 germline sequence (Accession #V00770). The unique CDR3 sequence includes the Arg-Gly-Asp (RGD) sequence, which functions as a recognition sequence for adhesion receptors in many adhesive proteins including fibrinogen, fibronectin, von Willebrand factor, and vitronectin. The nucleotide sequence of the variable region of the kappa light chain, along with its predicted amino acid sequence, is shown in **Figure 2**. The CDRs are underlined, and the amino acids are numbered according to a convention. This kappa variable chain is a class II mouse kappa variable region. Although its sequence is not identical to any germline variable regions present in the data bank (The International ImMunoGeneTics Database IMGT), the DNA sequence of the 5'-untranslational region, and V-region of the kappa light chain shows the highest


competing PcrV antigen (mol/L)

**Figure 5.** Binding affinities of Mab166 and scFv166 in competition ELISA. Binding affinities (*K*d) of Mab166 and scFv166

scFV166 m166 IgG

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Construction and Characteristics of a Recombinant Single-Chain Antibody Fragment...

**Figure 4.** Immunoblot of *P. aeruginosa* proteins reacted with scFv166. Precipitated *P. aeruginosa* PA103 proteins were resuspended in 100 μL of PBS. After adding 100 μL of SDS-PAGE sample buffer and boiling for 5 min, the proteins in the sample were separated by SDS-PAGE with *E. coli*-derived recombinant PcrV (rPcrV) as a reference. After electrophoresis, the proteins were transferred to a nitrocellulose membrane, and immunostained with scFv166 and a horseradish peroxidase-conjugated secondary anti-c-Myc IgG antibody and the blot was developed with a chemiluminescent substrate. The bindings of scFv166 to both native PA103 PcrV (294 amino aicds, 32.4 kD) and recombinant PcrV (rPcrV, 306 amino acids, 33.8 kD) were detected as shown in arrows.

measured by indirect ELISA with the PcrV antigen coated at 1 μg/mL. In the second step, *K*d, the dissociation constant, was measured using binding equilibrium studies (competition ELISA) to determine the concentration that gives 50% inhibition of maximum binding.

**Figure 5.** Binding affinities of Mab166 and scFv166 in competition ELISA. Binding affinities (*K*d) of Mab166 and scFv166 to immobilized PcrV were evaluated in a competition ELISA.
