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. Author manuscript; available in PMC: 2012 May 31.
Published in final edited form as: J Immunol Methods. 2011 Feb 26;368(1-2):36–44. doi: 10.1016/j.jim.2011.02.010

Generation of Recombinant Guinea Pig Antibody Fragments to the Human GABAC Receptor

Adnan Memic 1, Veronica V Volgina 1, Hélène A Gussin 2, David R Pepperberg 2, Brian K Kay 1,3
PMCID: PMC3100176  NIHMSID: NIHMS287293  PMID: 21362428

Abstract

To generate monoclonal antibodies to the human ρ1 GABAC receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (VH) and light chain (VL) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the VH and VL regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned VH and VL regions were transferred together into a phagemid vector, yielding a library of 5×106 members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABAC, we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABAC receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABAC receptor.

Keywords: Fab, immunization, peptide antigen, phage-display, RT-PCR

INTRODUCTION

Phage libraries displaying various types of antibody fragments have been utilized extensively in research and for the development of therapeutics (Brissette and Goldstein, 2007; Siegel, 2008). Antibody fragments such as single-chain fragments of variable (V) regions (scFv), fragments of antigen binding (Fabs), or single-antigen binding variable domains of heavy-chain antibodies (i.e., VHH), can be displayed on the surface of M13 bacteriophage particles, from which binders are isolated from libraries by affinity selection (Yamashita et al., 2007; Siegel, 2008; Bostrom and Fuh, 2009; Nieri et al., 2009; Pansri et al., 2009).

Phage-displayed libraries can be generated in several ways. First, it is possible to construct very large libraries (i.e., billions of clones) with variable domains captured from non-immune donors. Such libraries, considered naïve, are often good sources of binding clones with desired specificity and affinity to almost any protein target (de Haard et al., 1999, Schofield et al., 2007; Pansri et al., 2009; Hust et al., 2010). Similarly, naïve libraries have recently been constructed by randomizing the complementary determining regions (CDRs) of an antibody scaffold, and have been a source of binders to a variety of targets (Barbas, 1995, Fellouse et al., 2005; Fellouse et al., 2007; Birtalan et al., 2008; Gao et al., 2009; Uysal et al., 2009). Alternatively, one can immunize animals and, after verifying an immunological response, recover the coding regions of immunoglobulins by reverse transcribing the mRNA prepared from isolated spleens or circulating B cells, and then amplifying immunoglobulin variable heavy chain (VH) and light chain (VL) regions using the polymerase chain reaction (PCR) (Shen et al., 2007). While immunized libraries are generally limited in the spectrum of their diversity, they have the advantage of yielding affinity-matured antibodies with excellent affinity and specificity (Rader et al., 2000, Heitner et al., 2001).

The GABAC receptor, a subfamily of GABAA receptors, is a ligand-gated chloride ion channel that is activated in vivo by the inhibitory neurotransmitter γ-aminobutyric acid (GABA). GABAC receptors are expressed in the retina and in other tissues of the central nervous system (Enz et al., 1996; Euler and Wassle, 1998; Lukasiewicz and Shields, 1998; Shen and Slaughter, 2001; Gibbs and Johnston, 2005). Native GABAC receptors are pentamers that consist of a combination of ρ subunits, three types of which (ρ1, ρ2, and ρ3) have been cloned from mammalian retina (Zhang et al., 1995; Milligan et al., 2004; Pan and Qian, 2005). The ρ1 subunits are capable of forming fully functional homopentamers when expressed in model cell systems such as Xenopus laevis oocytes (Qian et al., 1998) or human embryonic kidney 293 cells (HEK-293) (Wotring et al., 1999). Given the biological importance of the GABAC receptor, antibodies to the receptor could be useful in modulating channel activity and in characterizing ion channel function (e.g., Tipps et al., 2010). In the present study, we report the recovery of antibody fragments from guinea pigs immunized with a peptide within the extracellular N-terminal region of the ρ1 subunit of the GABAC receptor (Gussin et al., 2008) and expression of chimeric (guinea pig variable and human constant regions) Fab antibodies against ρ1. Such anti-peptide antibodies, stabilized in the context of the Herceptin Fab antibody constant regions, bind to the ρ1 GABAC receptor expressed in a neuroblastoma cell line.

MATERIALS AND METHODS

Immunogen and guinea pig immunization

The immunogen was a 14-mer peptide (N-14), consisting of the amino acid sequence RQRREVHEDAHKQV, which is located within the N-terminal region of the human ρ1 subunit, outside the “core peptide” and within the potentially accessible “unstructured tail” of the protein (Gussin et al., 2008). Guinea pig was chosen as the species for antibody production, following earlier, unsuccessful attempts at obtaining specific anti-mammalian GABAC antibodies in rabbit and chicken (H. A. Gussin and H. Qian, unpublished observations). Peptide synthesis (purity >85%), conjugation to keyhole limpet hemocyanin (KLH) immunogenic carrier protein, animal immunization and serum collection were contracted to an outside source (Covance, Inc., Denver, PA). For coating of enzyme linked immunosorbent assay (ELISA) plates, a form of the N-14 peptide biotinylated at the N-terminus was used (Research Resources Center, University of Illinois at Chicago, Chicago, IL).

Library Construction

The strategy used for construction of the phage library is outlined in Figure 1. At the moment, only a limited number of expressed VH and VL genes from guinea pig (Cavia porcellus) have been sequenced (Kabat et al., 1991), which prevented primer design for amplification of immunoglobulins (Ig) from guinea pig lymphoid tissue. Although whole genome shotgun sequences of guinea pig are available (National Center for Biotechnology Information projects 12582 and 12583; http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=12583), the sequences were not usable for primer design because annotation of leader peptide, V-D, D-J junctions were lacking. In addition, it was unknown if the sequenced open reading frames (ORFs) in the genomic sequences were functional. Consequently, we decided to use degenerate primers, designed for amplification of murine VL and VH genes, as our staring point because guinea pig is closely related to mouse. However, these primers did not yield a PCR amplification product with guinea pig spleen cDNA. As an alternative approach, we designed degenerate primers based on human Ig sequences. Rabbit VH1 sequences (a1, a2, a3) are slightly closer to human (nucleotide identity ~78%) than to mouse VH gene (nucleotide identity ~73%) (Dr. K. L. Knight, personal communication), and we reasoned a similar situation might hold between guinea pig and human Ig sequences. The sequences of the degenerate primers (Fig. 2A and 2B) used to amplify and maximize the recovery of appropriate guinea pig VH and VL coding regions were designed using the database of human antibody sequences (http://vbase.mrc-cpe.cam.ac.uk/). Initially, several degenerate primers were used for PCR amplification of cDNA, prepared from RNA obtained from guinea pig B cells (described below), and DNA sequencing analysis of representative set clones showed that VH forward and reverse primers were incorporated, whereas VL clones contained forward primer sequences. We then modified our VL primers based on the nucleotides sequences we did obtain, and amplified the VL genes again. The resulting guinea pig VH and VL fragments were sequence-verified by comparison to mouse and human Ig (http://www.ncbi.nlm.nih.gov/igblast/), and to guinea pig genomic sequence (http://www.ncbi.nlm.nih.gov/nuccore/DS562855.1). Once we validated the output of our PCR reactions, they were scaled up for construction of the VH and VL libraries from the immunized guinea pig.

Fig. 1. Schematic representation of the library generation of antibody fragments.

Fig. 1

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First-strand cDNA from immunized guinea pigs was used to amplify individual heavy- and light-chain variable regions for the construction of antibody Fab fragments for combinatorial phage display. Blunt-ended PCR amplification products were then cloned into a TOPO vector, allowing quality control and the generation of two sub-libraries for further cloning. Finally, each variable region was sequentially inserted into the pCV3 vector to allow Fab phage display of chimeric guinea pig variable and human constant regions.

Fig. 2. Oligonucleotide primers and RT-PCR.

Fig. 2

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(A) Primer sequences used in the amplification of heavy-chain variable region genes for the generation of VH gene fragments. (B) Primer sequences used in the amplification of light-chain variable region genes for the generation of VL gene fragments. Primer sequences are shown 5′→3′, with the orientation of the primer indicated. Underlined sequences represent nucleotides required for pCV3 cloning. (C) Agarose gel electrophoresis of RT-PCR amplified guinea pig VH and VL coding regions. Size markers (right lane) are shown in kilobases (Kb) and base pairs (bp).

RNA was extracted from B cells of guinea pigs immunized with the N-14 peptide. Frozen B cells were provided by the vendor at a concentration 106 cells per mL, of which RNA was extracted from 100 μL, using the RNAaqueous®-4PCR kit (Ambion, Austin, TX), following the manufacturer’s instructions. The quality of the RNA was assessed by agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg of total RNA with random decamers provided in the RNAqueous®-4PCR and RETROscript® kits (Ambion), and was directly used as a template for PCR. PCR conditions were as follows: 5 μL of 10x AccuPrime Pfx Reaction mix, containing either the VH or VL sets of primers at 0.3 μM, 5 μL of template cDNA (50–100 ng), 0.5 μL AccuPrime Pfx DNA polymerase, and water to 50 μL; amplification: 95°C for 15 s, 58°C for 30 s, and 68°C for 1 min (35 cycles), final extension: 68°C for 6 min. Fig. 2C shows the agarose gel electrophoresis of these PCR products (i.e., amplified guinea pig VH and VL coding regions). For cloning, 4 μL of each PCR reaction were incubated with the pCR® II-Blunt-TOPO® vector (Invitrogen, Carlsbad, CA), and after desalting, the DNA sample was transformed into the TG1 strain of Escherichia coli by electroporation (Dower et al., 1988). The transformants were selected through growth on Petri plates containing super optimal broth (SOC) and 50 μg/mL kanamycin, and to assess the quality of the sub-libraries, we sequenced seven randomly picked clones from the VH and the VL sub-libraries. The colonies (105 colonies for VH and 5×103 colonies for VL) for each sub-library were recovered separately and plasmid DNA extracted with the Wizard Plus SV kit (Promega, Madison, WI).

These two sub-libraries were used for final Fab library construction. The bulk VL and VH coding regions were excised from the extracted plasmid DNA and ligated sequentially into the pCV3 vector with Sfi I, and Not I and Asc I restriction enzyme sites, respectively. Both ligations were performed at 16°C for 16 h with 20 units of T4 ligase, according to the manufacturer’s instructions (New England BioLabs, Beverly, MA), with an insert/vector ratio of 6 to1. To construct the pCV3 vector, we inserted the coding regions for the Herceptin Fab (Gerstner et al., 2002), which were synthesized commercially (Blue Heron, Seattle, WA), between the Nru I and Eco RI sites of the phagemid vector, pAPIII6 (Haidaris et al., 2001). In this construct, the PhoA promoter drives transcription, and the stII signal peptide sequences, preceding both light and heavy chains, traffic the two chains into the bacterial periplasm (Simmons et al., 2002) for phage-display. Two Sfi I restriction sites flanking the VL coding region, and Not I and Asc I restriction sites flanking the VH coding region, were introduced by Kunkel mutagenesis (Kunkel, 1985). Thus, the pCV3 vector allows bicistronic expression of human Fab, where V regions for both light and heavy chains can be exchanged using specific restriction enzyme cleavage sites.

Phage Selection Experiments

TG1 bacterial cells carrying unique Fab phagemid DNA were grown to mid-log density, and then infected by the M13K07 helper virus (New England BioLabs) and incubated for 2 h with mild shaking. Following infection, selection antibiotics (100 μg/ml ampicillin and 70 μg/ml kanamycin) were added to the medium to select for both the helper virus and the phagemid genomes, and the culture was grown overnight at 30°C, with vigorous shaking in a baffled flask. The bacterial cells were then pelleted, and secreted phage particles were recovered from the supernatant through precipitation in 8% polyethylene glycol (PEG 8000) and 1 M NaCl for ~1 h at 4°C. The phage particles were centrifuged at 17,500xg for 20 min, and the pellet of phage resuspended in 1 mL of 0.5% casein in phosphate buffered saline (PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 1.5 mM KH2PO4, pH 7.2) and used for affinity selection experiments.

To screen the library for antibody fragments binding the N-14 peptide, two microtiter plate wells (Nunc, Thermo Fisher Scientific, Rochester, NY) were coated overnight with 0.4 μg of streptavidin at 4°C, washed three times with PBS, and then incubated with biotinylated peptide (100 μL of 10 μg/mL solution) for 1 h at room temperature. Non-specific binding sites in the wells were blocked with 150 μl of blocking buffer (SuperBlock, Thermo Fisher Scientific) for 30 min at room temperature. After washing out the wells five times with PBS-0.05% Tween 20 (PBST), 50 μL of phage library (1011 colony forming units) and 50 μL of PBST was added. After 2 h incubation, the wells were washed five times with PBST, incubated with 100 μL of 100 mM glycine (pH 2) to elute the bound phage particles, followed by pH neutralization of the eluate with 6 μL of 2 M Tris (pH 10). The recovered phage particles were used to infect log-phase TG1 cells (10 min at 37°C), and the cells were spread over four 90 mm Petri plates, containing 2xYT (16 g tryptone, 10 g yeast extract, and 5 g NaCl in one liter of water, with the pH adjusted to 7.0 with NaOH), 1.5% agar, 2% glucose, and 100 μg/mL ampicillin. The next day, the colonies were scraped together and 2 drops were used to inoculate a 40 mL culture of 2XYT, containing 100 μg/mL ampicillin. When the culture reached log-phase, 1 mL of culture was transferred to a 15 mL tube and ~ 1010 plaque forming units of M13K07 helper phage was added, and incubated at 37°C for 1 h with a slow agitation. The cells were then pelleted by centrifugation, and resuspended in 30 mL of 2xYT containing 100 μg/mL ampicillin and 100 μg/mL kanamycin, and incubated overnight at 30°C, with vigorous aeration. The culture was transferred to an Eppendorf tube, spun 20 min at 10,000xg, and the supernatant transferred to a clean Eppendorf tube, and adjusted to 8% polyethylene glycol (PEG 8000) and 1 M NaCl for ~1 h at 4°C. The phage particles were centrifuged at 17,500xg for 20 min, and the pellet of phage resuspended in 1 mL of 0.5% casein in PBS. The resuspended phage was transferred to a 1.5 mL microcentrifuge tube, spun at 17,500xg for 10 min, and used for the next round of affinity selection. The second and third rounds of selection were conducted in the same as the first round, except that neutravidin-coated wells were used. The output of the third round of affinity selection was diluted, used to infect TG1 cells, and plated on Petri plates containing 2xYT, 1.5% agar, and 100 μg/mL ampicillin to generate individual colonies.

Enzyme-linked immunosorbent assay (ELISA)

We analyzed by ELISA the binding of individual phage clones to the N-14 peptide, obtained from the phage particle preparations as described above. Neutravidin coated 96-well plates (Themro Fisher Scientific) were incubated with biotinylated peptide (100 μL of 10 μg/mL solution), for 1 h at room temperature. Treated wells were then blocked with 150 μl of SuperBlock for 30 min at room temperature, with mild shaking. Following washing of all wells three times with PBST, 100 μL of the phage preparation (1013 phage particles/mL) was added to the wells and incubated for 1.5 h at room temperature, with mild shaking. The wells were then washed four times with PBST. One hundred μL of horseradish peroxidase (HRP)-conjugated anti-M13 phage antibody (GE Healthcare, Piscataway, NJ), diluted 1/4,000 in blocking buffer, was added to each well and incubated at room temperature for 1 h with mild shaking. The wells were washed five times with PBST, and 85 μL of solution containing 2′,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (Sigma Aldrich, St. Louis, MO) and hydrogen peroxide, were added to each well and incubated at room temperature, typically for 20 min. The optical absorbance of duplicate wells was measured at 405 nm. In the competition ELISA experiments, increasing concentrations of the free N-14 peptide were pre-incubated with phage particles for 1 h at room temperature, before addition to microtiter plate wells; all other steps were as described above. The coding regions of positive clones were sequenced.

Neuroblastoma Cell Culture and Immunofluorescence

Neuroblastoma cells, either stably expressing the human GABAC ρ1 receptor (SHp5-ρ1 cells; gift from Dr. David S. Weiss (University of Texas Health Science Center at San Antonio, San Antonio, TX), or non-expressing control cells (SHSY5Y; ATCC, Manassas, VA), were cultured in a 1:1 blend of Dulbecco’s Modified Eagle Medium: Ham’s F-12 nutrient mixture (DMEM/F12) (Hyclone-Fisher Scientific), which was supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (0.1 mg/mL). The cells were grown to 60–80% confluence in 8-well Lab-Tek II chamber slides (Nunc-Thermo Fisher Scientific, Rochester, NY) that were pre-coated with gelatin. Cells were washed twice with PBS, incubated with 450 μL of phage preparations (1013 phage particles/mL) in a 1:1 mix of PBS and SuperBlock, for 2 h at 4°C, and washed twice in PBS before fixation in 1% paraformaldehyde in PBS (10 min). After two further washes with PBS, the fixed cells were incubated with a 1/500 dilution (in SuperBlock) of biotinylated mouse anti-M13 antibody (Affinity BioReagents, Thermo Fisher Scientific), followed by staining with streptavidin-conjugated Dy-Lite488 (Thermo Scientific), diluted 1/500 in SuperBlock. Finally, slides were washed five times with PBS, mounted with Vectashield H-1000 (Vector Laboratories, Burlingame, CA), and analyzed by epifluorescence microscopy at 40x or 63x magnification.

RESULTS

The starting point of the present investigation was the observed reactivity with ρ1-GABAC-expressing cells and cell lysates, of a polyclonal antibody raised in guinea pig against a 14-mer peptide of the ρ1 GABAC extracellular domain (Gussin et al., 2008). Using the spleen obtained from the immunized guinea, we undertook preparation of a Fab library containing the guinea pig VH and VL regions fused to the constant regions of human Ig light and heavy chains (Fig. 1). To achieve this, RNA was extracted from the guinea pig spleen B cells and cDNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) with random decameric oligonucleotides. The heavy and light chain V regions were amplified from cDNA with degenerate primers (Fig. 2A, B); these primers were designed in part based on human antibody sequences, and also in part based on a representative set of guinea pig Ig sequences cloned and sequenced within this project. DNA sequences of the clones demonstrated that the VH and VL gene segments from the guinea pig were functional and rearranged, and contained mutations (with respect to each other) indicative of affinity maturation. Both the VH and VL coding segments exhibited a high degree of sequence diversity, as indicated by comparison of their CDRs (Fig. 3).

Fig. 3. Comparison of the amino acid sequences of the VL and VH coding regions.

Fig. 3

Fig. 3

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The amino acid sequences are compared with respect to each other and to germ-line guinea pig sequences. (A) Comparisons of VL sequences and (B) VH sequences were performed by ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw/). Sequence gaps were introduced to maximize homology and are indicated by dashed lines. Sequence 2N cont2 was derived from guinea pig genomic contigs 36478 and 47208 (http://www.ncbi.nlm.nih.gov/nuccore/DS562855.1). Framework regions and CDRs were assigned by using the Kabat database option in IgBLAST, (http://www.ncbi.nlm.nih.gov/igblast/) and are marked by the arrows beneath the alignments.

The first step in the construction of the chimeric Fab library involved subcloning the amplified and cloned guinea pig VL coding regions, into a phage-display vector. This phagemid vector, pAPIII6 (Haidaris et al., 2001), displays proteins at the N-terminus of the minor capsid protein (pIII) of the bacteriophage M13 particles (Kehoe and Kay, 2005). Rather than display the VH and VL protein segments as an scFv, we decided to create Fabs, using constant regions of the Herceptin Fab (Kelley et al., 1992; Gerstner et al., 2002) and the variable regions of the immunized guinea pig. This route was chosen because the Herceptin Fab has been optimized for display on phage particles. After cloning the guinea pig VL segments into the phagemid vector, the guinea pig VH regions were serially transferred into the construct, yielding 5 × 106 independent transformants.

Phage particles displaying chimeric Fabs were affinity-selected with the 14-mer peptide segment of GABAC. A biotinylated form of the peptide was captured on streptavidin-coated microtiter plate wells for three rounds of affinity selection. While peptide-specific clones were not detectable in the initial chimeric Fab library, they became apparent after the third round of panning. Four distinct antibodies (H6, A10, A11 and D12) were isolated from our selections. Selection of biotinylated peptide-specific antibodies was confirmed by ELISA (Fig. 4), in which individual clones were screened against the biotinylated peptide using an anti-M13 antibody conjugated to HRP for detection. A competitive ELISA, in which the peptide was used to displace soluble forms of the Fabs, yielded IC50 values for the antibody fragments in the high nanomolar range (700–800 nM; data not shown).

Fig. 4. ELISA screening of individually selected clones.

Fig. 4

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Culture supernatants containing recombinant phage particles were screened for binding to the N-terminal peptide of the GABAC receptor using an anti-phage antibody conjugated to horseradish peroxidase. “Neg” refers to binding by a negative control phage that displays the herceptin Fab. Absorbance was read at 405 nm. The data are from representative clones tested in duplicate, with error bars indicating the standard deviation. All clones tested showed greater than a two-fold signal-to-noise ratio, whereas phage particles displaying Fabs of unrelated specificity did not exhibit any binding.

The results obtained with the antibodies selected from the chimeric Fab library demonstrate the feasibility and utility of generating monoclonal antibodies from an immunized guinea pig. As indicated by the sequence alignments (Fig. 5), the same or related clones were selected from the chimeric Fab library. When comparing binders, we observed that one or more amino acid substitutions were present outside the CDR regions (in addition to those substitutions present in the CDRs), suggesting that they represented affinity-matured clones arising through B cell selection. However, we cannot rule out the possibility that these sequences represented different germline V genes, as a database of guinea pig VH and VL germline genes is not available. Two clones (A10 and D12) expressed the same parent Herceptin light chain, but different heavy chains, consistent with the possibility that the light chain is less important than the heavy chain with respect to binding to the N-14 peptide. In these two clones, a large loop (8 amino acids) was observed in CDR1 of the VH sequences. By contrast, for the heavy chain sequences, sequence diversity was greatest in CDR3 of the heavy chain. The alignment of the binder sequences also revealed residues that were invariant among their framework and CDR regions (Fig. 5).

Fig. 5. Comparison of the amino acid sequences of the full-length VL and VH coding regions for those Fab fragments binding to the N-terminal peptide of GABAC.

Fig. 5

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The sequences are grouped according to the amino acid alignment preformed by ClustalW2. Sequence gaps were introduced to maximize homology and are indicated by dashed lines. Framework regions and CDRs were mapped by using Kabat database and are marked by the arrows beneath the alignments. (A) Comparison of VL sequences and (B) VH sequences; note that the VL sequences for A10 and D12 are identical and that CDR3 was assigned based on CDR3 assignments for human Ig.

To test for immunological recognition of the native GABAC receptors in living cells, we prepared phage particles for staining cells by immunofluorescence. We used phage particles for probing the cells, rather than soluble Fabs, because we found the Fab preparations to lose solubility over time. To avoid possible endocytosis of the phage particles, we carried out the cell staining experiments at 4°C. We observed strong staining with antibodies H6 and D12 against the surfaces of GABAC-expressing neuroblastoma cells, whereas non-expressing cells did not exhibit staining (Fig. 6). Clones A10 and A11 did not show a significant fluorescence signal when incubated with either GABAC-expressing or non-expressing cells (data not shown). As a negative control for staining, GABAC-expressing and non-expressing cells were incubated with phage displaying the herceptin Fab (Fig. 6C). This control did not exhibit substantial cell staining. Thus, two of our Fabs are able to bind the N-14 motif on the surface of neuroblastoma cells.

Fig. 6. Immunofluorescence staining of GABAC-expressing and non-expressing neuroblastoma cells when incubated with phage particles displaying guinea pig Fab fragments.

Fig. 6

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(A) Left panel shows immunofluorescent staining and phase contrast images of the cells upon treatment with phage H6. Binding of phages to the expressing neuroblastoma cells was detected by green fluorescence as detected by streptavidin-Dy-Lite488 against a biotinylated anti-phage antibody. Right panel shows immunofluorescent staining and phase contrast images of non-expressing neuroblastoma cells treated with the same phage preparation. (B) Immunofluorescence staining and phage contrast images of phage D12. (C) Immunofluorescence staining and phase contrast images of cells stained with phage particles displaying the herceptin Fab. Size bar corresponds to 15 μm.

DISCUSSION

We report the successful selection of GABAC antibodies by recovery of immunoglobulin VH and VL coding segments from a guinea pig immunized with a 14-mer peptide of the N-terminal extracellular domain of the GABAC receptor. In this effort, chimeric Fabs, derived from guinea pig variable regions and constant human regions, were displayed on the surface of bacteriophage M13 particles. Four clones were isolated from a phage library, using the biotinylated 14-mer peptide for affinity-selection, and recognized the N-14 peptide derived from the GABAC receptor in an ELISA. For two of the Fabs (A10, A11) staining of the GABAC-expressing neuroblastoma cells was weak or absent, suggesting that some of Fabs exhibit low affinity to epitopes present in the N-14 peptide. However, the other two of these recovered clones (H6 and D12) exhibited binding to neuroblastoma cells expressing functional homopentameric ρ1 GABAC receptors.

The scarcity of expressed guinea pig Ig genes available in GenBank necessitated design of oligonucleotide primers for PCR amplification of their VH and VL regions. We found degenerate primers based on human VH and VL to be suitable for PCR, most likely as a consequence of a high degree of nucleotide similarity between guinea pig and human immunoglobulins. One benefit of recovering VH and VL coding regions from immunized guinea pigs is that we have obtained the nucleotide sequence and primary structure of functionally expressed guinea pig VH and VL segments. This information may facilitate further study of the guinea pig’s Ig genes and its B cell repertoire.

Several possibilities may account for the observation that the four chimeric Fabs have dissociation constants in the high nanomolar range. First, the oligonucleotide pimers may not have amplified the complete set of guinea pig immunoglobulin regions, which may have included the high affinity antibodies. Second, the best binding VH region may not have paired up with the best binding VL region in a phage-display clone. Third, inclusion of the Herceptin constant regions may have altered the folding or placement of the two variable regions, relative to each other. Higher affinity antibody fragments should be achievable through random mutagenesis (Chowdhury and Pastan, 1999; Fermer et al., 2004; Groves et al., 2006) or chain shuffling (Kranz and Voss, 1981; Andris-Widhopf et al., 2000).

From the sequence alignments of the four isolated Fabs, it appears that affinity maturation was present in variable regions of both heavy- and light-chains of the guinea pig antibodies, resulting in numerous amino acid substitutions. Although the heavy- chain of the antibodies showed a higher diversity in the CDR regions, the framework regions were more conserved among one another. The light chains from two peptide- specific chimeric Fab fragments (D12 and A10) were identical, and represented the parental cloning vector sequence of the Herceptin antibody variable region. Chimeric D12 Fab recognized peptide and native protein, and was highly specific as shown by cell staining, unlike the A10 antibody, which demonstrated weak or no cell staining. This functional difference between D12 and A10 Fab may be due to the utilization of different VH genes or, alternatively, to the efficiency of pairing of light and heavy chains. This observation also implies that the light chain is less important than the heavy chain with respect to binding to the 14-mer peptide antigen. Thus, an interesting aspect of the binding of the Fab to the 14-mer peptide and to ρ1 GABAC is the relative contribution of each variable region to antigen binding and specificity. The contribution of an individual variable region (VL versus VH) can vary from one immune recognition event to another; nevertheless, antibody specificity may be primarily set by a single heavy or light chain (Gulliver et al., 1994, Chang and Siegel, 1998).

In summary, our study establishes the utility of recombinant guinea pig antibodies for generating monoclonal antibodies to cell surface receptors. The binding activity of clones H6 and D12 to functional homopentameric ρ1 GABAC receptors provides the ability to in vivo target neural tissues that express this receptor. In addition, such antibodies have the potential to modulate receptor activity (Tipps et al., 2010) for clinically relevant applications.

Acknowledgments

We thank Ms. Nanthanit Jaruseraneee and Ms. Feng Feng for help with the cell culture experiments, Dr. Mark Sullivan (University of Rochester) for providing the pAPIII6 vector, Dr. David S. Weiss (University of Texas Health Science Center at San Antonio) for providing the ρ1 GABAC-expressing neuroblastoma cells, Mr. Michael Scholle for initial design of the Herceptin Fab gene, Dr. Haohua Qian (National Eye Institute, NIH), and Dr. K. L. Knight (Loyola University at Chicago) for helpful technical discussions, and Dr. Thomas Cunningham and Mr. Michael Kierny for editorial comments. This work was supported by grants from the National Institutes of Health (R01 EY016094; P01 GM075913, U54 CA119343, and P30 EY001792), the Daniel F. and Ada L. Rice Foundation (Skokie, IL), Hope for Vision (Washington, DC), the American Health Assistance Foundation (Clarksburg, MD), and Research to Prevent Blindness (New York, NY).

Abbreviations

bp

base pairs

CDR

complementary determining regions

cDNA

complementary DNA

ELISA

enzyme linked immunosorbent assay

EDTA

Ethylenediaminetetraacetic acid

Fabs

Fragments of antigen binding

GABA

γ-aminobutyric acid

HRP

horseradish peroxidase

Ig

immunoglobulin

Kb

kilobase

PBS

phosphate buffered saline

PBST

PBS with 0.05% Tween 20

PEG

Polyethylene glycol

PCR

polymerase chain reaction

RT-PCR

reverse transcriptase-polymerase chain reaction

scFvs

single-chain Fragments of variable regions

VH

variable domains of immunoglobulin heavy chain

VL

variable domains of immunoglobulin light chain

Footnotes

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