HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species
© Kivi et al. 2016
Received: 16 October 2015
Accepted: 4 January 2016
Published: 8 January 2016
The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories.
HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets.
HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.
Monoclonal antibodies (MAbs) are made by identical immune cells and target one particular epitope by monovalent or monospecific affinity. The high affinity and selective binding of MAbs to epitopes in target antigens makes them highly potent tools for use in biochemistry, molecular biology and medicine. The first working method described for the isolation of monoclonal antibodies was hybridoma technology, based on forming hybrid cell lines (hybridomas) by fusing an antibody-producing B-cell with a myeloma cell . The antibodies produced by a particular hybridoma clone share the same specificity. Thus, individual clones can be screened for the production of an antibody with the desired affinity. However, hybridoma technology has shortcomings: it takes a relatively long time (on the order of months) and has not been widely applied to organisms other than mice. Moreover, antibody sequence information is unavailable by this method. Thus, when a hybridoma-screened antibody is selected for further development (e.g., as a therapeutic product), the cDNA encoding the variable domains of the heavy (VH) and light chain (VL) must be isolated from the hybridoma cells. This step is required for the recombinant production of the final MAb product, as well as for improvements such as humanization, isotype conversion, and affinity maturation.
Alternatively, recombinant antibody isolation technologies usually do not include a hybrid cell line step, but instead clone the VH and VL domain sequences from the antibody-expressing source cells (e.g., B-cells from spleen, bone marrow or blood). Commonly, VH and VL cDNA is amplified by RT-PCR using mRNA isolated from the cells. By combinatorial strategies, a large repertoire of different VH and VL sequences are amplified from a population of cells (e.g., millions of B-cells isolated from an immunized animal). Thereafter, the amplified products are used for the construction of combinatorial libraries by the random pairing of the VH and VL domains. Thus, combinatorial strategies must involve a screening step for the identification of antibodies (VH and VL combinations) with the desired properties from large libraries. These screening methods involve in vitro antibody display techniques including phage display [2, 3], ribosome display , and in vivo display platforms such as bacterial, yeast, and mammalian cell-surface displays . Mammalian cell display has a number of advantageous features, especially for selecting antibodies for recombinant production . For example, using mammalian cells for MAb screening minimizes the loss of MAb during downstream production in mammalian cells, a complication sometimes associated with MAbs screened in phage, bacterial or even yeast display systems. Instead, performing antibody selection in mammalian cells ensures that the selected antibody is synthesized, modified, assembled and secreted through the same cellular pathways employed in the production phase.
Recombinant methods also include non-combinatorial strategies that are based on retrieving antibody sequences from a single B-cell or the clonally expanded progeny of a single cell. Using this technique, the original heavy and light chain pairs are maintained together during the isolation process (for a review, see ref. ). This is achieved by the amplification of rearranged VH and VL region cDNAs from one particular B-cell by technically challenging single-cell RT-PCR methods . Furthermore, isolated single B-cells can be amplified in cell culture. These methods include the immortalization of differentiated B-cells by the Epstein-Barr virus (EBV), retroviral oncogenes, or TLR ligands (reviewed in ref. ); by cultivation under specific conditions with irradiated thymoma cells [9, 10]; or by IL4 treatment and CD40 triggering . These techniques were developed to facilitate and increase the efficiency of non-combinatorial strategies (e.g., the enrichment of source B-cells that express antibodies with the desired affinity). However, all non-combinatorial methods involve either complicated single-cell manipulations and/or the establishment of sterile primary cell cultures from unsterile biological materials, as well as specialized equipment and/or expensive supplements. Any MAb development strategy can potentially include negative or “subtractive” screening steps to eliminate the recovery of antibodies recognizing the target antigen but also some non-desired off-target(s). This is especially useful for the development of very specific MAbs that can bind to specific protein modifications [12, 13] or recognize a specific member of a protein family with common domains, motifs and/or structures .
Here, we describe the novel HybriFree technology, which combines elements of both combinatorial and non-combinatorial strategies and involves a screening step performed using mammalian cells. This method is universal and is in principle applicable to any species for which antibody sequence information is sufficient for designing primers for VH and VL cDNA amplification. The key property of HybriFree technology is its rapid and robust workflow: it takes approximately 10 days to go from source material collection to obtaining expression vectors for antigen-specific antibodies, together with sequence information regarding their VH-VL combinations. The method does not require the establishment of sterile B-cell cultures, single cell manipulations or specialized materials. Additionally, we have supplemented the method with an optional negative selection step to eliminate the recovery of antibodies directed to undesired off-targets.
Immunization of animals
Six- to eight-month-old Hy-Line chickens were immunized by four (weeks 0, 2, 4 and 18) intramuscular (im) injections of 0.5 mg protein antigen in complete (initial immunization) or incomplete (subsequent immunizations) Freund adjuvant. An antigen boost was given intravenously (iv) 2 weeks after the final injection as 0.1 mg protein in PBS.
Four- to six-week-old female BALB/c mice were initially immunized intraperitionally (ip) with ~50 μg of protein antigen in complete Freund adjuvant (week 0), followed with 5 ip administrations (weeks 3, 7, 16, and 2 times in week 17) with the same amount of antigen in PBS.
Approximately 5-month-old New Zealand rabbits were initially immunized by 2 subscapular injections (one in each side of the body). The protein antigen, or virus-like particles (VLPs), was administered 4–5 times in amounts of 0.1-0.4 mg in complete Freund (first immunization only) adjuvant or in incomplete Freund adjuvant. The response was boosted by an intravenous injection of 0.1 mg protein antigen.
All of the procedures performed using animals were in accordance with the European Union directive 86/609/EEC and were approved by the Estonian National Board of Animal Experiments (No. 115, 07.09.2012; No. 87, 28.08.2007).
Collection and preparation of spleen cells
After the confirmation of antigen-specific antibody response in egg yolk preparations (chickens; IgY) or in blood serum (mice and rabbits; IgG), spleens were collected 2–4 days after final immunization (i.e., the final boost). The animals were anesthetized, and cardiac puncture was used to collect blood. The spleen was removed and stored on ice until treatment (within one hour). For the preparation of cell homogenate, the spleen was homogenized in ice-cold PBS using a 40 μm cell dissociation sieve. The material was collected in a 50 ml cell culture tube and precipitated by centrifugation at 300 × g for 5 min at 4 °C. The cells were re-suspended in 50 ml of ice-cold PBS and centrifuged again. Finally, the precipitated cells were suspended in 5 ml ice-cold freezing medium (heat inactivated fetal bovine serum + 10 % DMSO), distributed into cryovials (1 ml per tube), and slowly frozen to −80 °C. For longer storage, the tubes were transferred into liquid nitrogen after 4–5 days.
Preparation of the cells for capturing: The frozen spleen cell suspension was thawed and transferred into 10 ml of RPMI1640 cell culture medium at ambient temperature. The cells were collected by centrifugation (200–300 × g, 5 min, room temperature), re-suspended in 10 ml of RPMI1640 supplemented with penicillin/streptomycin and 10 % of heat inactivated fetal bovine serum (rabbit or mouse splenocytes) or chicken serum (chicken splenocytes). The cells were seeded into a 100 mm cell culture dish and incubated ~1 h at 37 °C in a 5-8 % CO2 atmosphere. Then, free-floating cells were carefully collected, and the viability of the cells was evaluated using trypan blue exclusion. The cells were sedimented by centrifugation and re-suspended in the capture medium (RPMI1640 supplemented with 0.5 % BSA and 0.1 % NaN3). The chicken splenocyte preparations contained a high amount of erythrocytes; therefore, an additional Optiprep™ (60 % iodixanol, Axis-Shield PoC AS, Norway) gradient purification step was included by the sedimentation of lymphocytes along a density barrier  before re-suspension in the capture medium.
Capturing: MaxiSorp™ surface 96-wells (Thermo Fisher Scientific, US) were coated with the antigen (20 μg/ml in PBS, 4 °C, overnight) and blocked for 1–2 h with 2 % BSA in PBS. One hundred microliters of cell suspension containing 2 x 104 live cells in capture medium were loaded into a single well. The plate was centrifuged (200 × g, 5 min) and incubated for 45–60 min. The medium was discarded, and loosely attached cells were removed by washing 4–5 times with PBS, each time pipetting gently triturating (3–4 times) at the edge of the well. Finally, the plastic-bound cells were lysed in the wells using 200 μl of TriReagent® (Molecular Research Center, US) and 1–2 μg of yeast tRNA (Life Technologies, US) carrier added to each well. Total lysate of the human cell line U2OS was used as a carrier rather than tRNA in some experiments using chicken splenocytes.
RNA isolation and cDNA first-strand synthesis
The total RNA isolation was performed as recommended by the TriReagent® manufacturer. Material from 2 (mouse, rabbit) or 8 (chicken) wells was pooled together during the isolation. The final RNA samples were dissolved in 8 μl of nuclease-free water and subjected to cDNA first-strand synthesis using the SuperScript® III First-Strand Synthesis System according to the manufacturer’s instructions (Life Technologies, US). This preparation yielded 20 μl of cDNA reaction per sample.
PCR amplification of VH and VL regions
PCR reactions were performed in 50 μl for a total of 35–40 cycles using Phusion Green Hot Start II High-Fidelity DNA Polymerase (Life Technologies, US) with pre-optimized conditions for each reaction. The primers used for the amplification of antibody VH and VL regions were designed to maximally cover the variability of the VH and VL sequences. Primer pairs used for the amplification of chicken IgY regions were designed based on published data . The mouse primer cocktails used for the amplification of VH and VLκ sequences were designed based on published data  as well as V- and J-region cDNA sequences available in the international ImmunoGeneTics information system® (IMGT®) web resource . For the construction of scFv fragments, rabbit VH and VL primer cocktails were designed and prepared using previously published data . The sequences of the primers are listed in Additional file 1. The proprietary VH and VL primers were used for the construction of libraries expressing intact rabbit IgG molecules were designed using rabbit sequences stored at IMGT® . For cloning purposes or forming the coding sequence of the flexible linker (GGGGS)3 between the VH and VL domains, an additional 20–23 nucleotides were added to the 5’ ends of primers.
Circular Polymerase Extension Cloning and plasmid DNA isolation
The restriction- and ligation-independent Circular Polymerase Extension Cloning (CPEC) method  was used for library construction by the in-frame-directed cloning of amplified VH and VL regions into the pQMCF mammalian expression plasmid (Icosagen Cell Factory, Estonia). Briefly, the variable domain PCR products and linearized/dephosphorylated vector fragment(s) were separated and purified from a TAE-agarose gel. Approximately 100 ng of the vector together with inserts (~2 times molar excess of each) were used in the 20 μl CPEC reaction with Phusion High-Fidelity DNA Polymerase (Life Technologies, US). Totally 25 cycles of denaturation, annealing and extension were performed according to the the polymerase manufacturer’s instructions. Five microliters from the reaction were used for the transformation of competent TOP10 F’ or DH5α strain E. coli cells. Approximately 1/10 of the transformation mixture was used for the direct inoculation of 2 ml of selective carbenicillin-containing liquid growth medium, followed by the extraction of plasmid DNA (i.e., the library pool) from overnight culture. Another part of the transformation mixture was plated onto carbenicillin-containing solid medium to obtain individual clones. The bacterial clones were amplified in 0,75 ml of liquid medium, and plasmid DNA mini-preparations were purified using a Zyppy™-96 Plasmid Miniprep kit (Zymo Research, US) or a FavorPrep™ 96-Well Plasmid Kit (Favorgen Biotech Corp., Taiwan) according to the manufacturer’s instructions.
Cells, transfection and sample collection for mammalian screening
The Chinese hamster ovary (CHO-S from Thermo Fisher Scientific, US)-derived cell line CHOEBNALT85 (Icosagen Cell Factory, Estonia) was grown in serum-free chemically defined medium and was used for antibody screening. This cell line expresses EBV EBNA1 protein and mouse polyomavirus large T protein and is specifically designed for the prolonged and high level production of proteins in association with pQMCF vectors (USPTO Patent No: 7,790,446). The cells were transfected using chemical transfection Reagent 007 (Icosagen Cell Factory, Estonia) according to the published protocols . One microgram of plasmid DNA was transfected in 6-well plate format for analyzing library pools, and approximately 0.2 - 0.5 μg DNA per sample was used in a high-throughput 96-well plate transfection for screening individual clones. Seventy-two hours after transfection, the supernatants were collected for analysis. When necessary, scFv-Fc or human IgG1 concentrations in the samples were determined using FastELISA for Human IgG quantification (RD Biotech, France).
The ELISA plates (Nunc™ MaxiSorp™, Thermo Fisher Scientific, US) were coated at 4 °C overnight with antigen solution (2–5 μg/ml) or VLP suspension (20 μg/ml) in PBS, washed with washing solution (PBS containing 0.05 % Tween 20), and incubated 1–2 h with blocking solution (PBS containing 2 % BSA and 0.05 % Tween 20) at room temperature. After washing twice, the culture supernatants (diluted in blocking solution, if necessary) were incubated 1–2 h at room temperature. After washing 4 times, a second incubation was performed with goat anti-human IgG (for scFv-Fc) or anti-rabbit IgG antibody conjugated with HRP (LabAs, Estonia). The signals were developed with TMB VII substrate (Biopanda Diagnostics, UK). The reactions were stopped by the addition of 0.5 M H2SO4, and absorbance values were measured at 450 nm.
Protein sequences of identified antibody VH and VL were analyzed by exhaustive pairwise global alignments and the progressive assembly of alignments using Neighbor-Joining phylogeny for similarity determination. This was done using Clone Manager Professional (Scientific & Educational Software) and BioEdit Sequence Alignment  software. Complementarity determining regions (CDRs) in VH and VL amino acid sequences were determined using ref. [16, 18, 20].
Results and Discussion
Description of the HybriFree technology
The captured source cells are subjected to RNA isolation, cDNA preparation, and the amplification of VH and VL cDNAs (step 2, upper right in Fig. 1), followed by the construction of a combinatorial library in a mammalian expression vector (step 3, lower left in Fig. 1). Using high-throughput techniques, the plasmid DNA was prepared from single bacterial colonies grown in 96-well plates and transiently transfected into mammalian cells. Thereafter, antigen-specific antibody production is evaluated for each clone (step 4, lower right in Fig. 1). Finally, VH and VL cDNAs are sequenced for the selected positive clones. The whole process takes no more than 10 days.
Additionally, we introduced an optional pre-adsorption step to avoid the undesired cross-reactivity of the developed MAbs. In brief, the source cells are treated with an excess of off-target protein prior to capturing with desired target antigen. In this way, all binding sites on the cells that are cross-reactive with undesired off-targets are saturated and cannot bind to the target antigen anymore. In the following sections, this method is described in detail with proof-of-principle examples provided regarding the isolation of MAbs from rabbit, mouse, and chicken. Applying the pre-adsorption step for increased selectivity is also illustrated.
Capturing antigen-specific B cells
We have optimized the capturing of antigen-specific B cells in antigen-coated 96-wells (MaxiSorp™ surface) using a non-sterile crude suspension of intact spleen cells from different animals. However, the removal of plastic-adherent cells (e.g., macrophages) from lymphocytes during the first incubation step was found to increase the capture of antigen-specific lymphocytes as previously described .
We have most carefully optimized the number of source cells used per single capture reaction. The number of bound cells must be sufficient for the robust one-step amplification of the VH and VL regions. Even if the amplified VH and VL regions correspond to only those antibodies that bind to the target antigen, it is necessary to maintain the diversity of library within limits suitable for a mammalian screen. To this end, we have found that 2–6 x 104 mammalian splenocytes and 8–16 x 104 chicken splenocytes per cDNA sample is optimal. We have determined that to increase the variability of the recovered MAbs, setting up more capturing reactions/well is preferable to increasing the cell number per well. We also observed that ~45 min capture time in ambient temperature is sufficient for cells to bind to the antigen, without being so long that major degradation of cellular mRNA without de novo synthesis becomes an issue.
Supplementing the capture medium with 0.1 % NaN3 improved the yields of amplified PCR products, especially for chicken splenocytes and/or when cell capture was initially performed at a higher temperature. This is illustrated in Fig. 2d by VH and VL amplification from splenocytes collected from chicken immunized with mouse CD48 protein. The cells were captured on an antigen-coated surface at 37 °C using RPMI1640 + 0.5 % BSA or the same medium supplemented with 0.1 % of NaN3 as the capture medium. The yield of the VH and VL products was much higher for reactions supplemented with NaN3. The success of RNA isolation and cDNA synthesis was confirmed by amplification from human β-actin-spliced mRNA (an equal amount of human cell line lysate was used in both samples as a carrier in RNA isolation). We speculate that the favorable impact of NaN3 may be caused by the blocking of antibody internalization and/or mRNA degradation during capture.
ScFv-Fc library construction
Library pool samples (a mixture of all library members) and single clone DNA minipreparations (using a convenient high-throughput 96-well method) were prepared from E. coli cells directly transformed with CPEC reaction product. PCR analysis of bacterial colonies showed the high efficiency of the cloning, and routinely >90 % of CPEC clones contained directed VH-VL insertion in the vector (data not shown).
Mammalian cell screening of antigen-specific scFv-Fc molecules
In the examples provided below, we used the CHO-derived cell line CHOEBNALT85 for screening. These cells grow in serum-free chemically defined medium and ensure high transfection efficiencies in a variety of scales, including a 96-well high-throughput format. In association with the pQMCF vector, CHOEBNALT85 cells ensure relatively high transient production levels of IgG-like molecules (typically tens of micrograms per milliliter at 72 h time-point using a 2 ml culture scale). Moreover, we routinely use the same cells for recombinant antibody production at larger scales. Using the same cells for screening and production decreases the odds of subsequent problems with productivity. However, any mammalian cell line that can be efficiently transfected with plasmid DNA expression vector is usable for screening by this method. In the examples described here, the expression of scFv-Fc was controlled using the Rous sarcoma virus long terminal repeat promoter.
Hit rates (proportion of antigen recognizing clones) achieved from positive library pools in different HybriFree experiments
18/118 (15 %)
After B-cell capturing as described here, the screening was done using E. coli based system
71/140 (50 %)
3/43 (7 %)
3/23 (13 %)
7/93 (8 %)
Full-length protein was used for immunization but the B-cell capturing and screening were performed using only C-terminal domain (one-third)
19/190 (10 %)
After B-cell capturing as described here, the screening was done using E. coli based system
12/78 (15 %)
7/24 (29 %)
7/46 (15 %)
13/93 (14 %)
16/188 (9 %)
Summarized results obtained from 2 rabbits
80/186 (43 %)
25/94 (27 %)
104/234 (44 %)
Summarized results obtained from 3 experiments
11/46 (24 %)
55/94 (59 %)
see section “Screening natural intact IgG molecules” for more details
43/125 (34 %)
High-throughput methods used for the preparation and transfection of plasmid DNA usually result in some variability in DNA concentration, and thus also in transfection efficiency. This can lead to differences in scFv-Fc expression/secretion level, also influenced by the actual DNA sequence (codon usage) of VH and VL cDNA. Thus, the differences in signals observed in 96-well screening do not necessarily reflect differences in the quality or affinity of the individual MAbs. Therefore, normalization is necessary for a head-to-head comparison of the selected MAbs. This is illustrated in Fig. 4 using 10 positive MAbs against human artemin screened from the mouse library. Normalized ELISA curves are shown in Fig. 4e, and the readouts of the same clones in an initial high-throughput screen are illustrated in Fig. 4f.
Screening intact IgG molecules
The modification in this method is illustrated by the development of rabbit MAbs against mouse CD48 protein. VH and VL fragments were created from spleen cells captured on mouse CD48-coated wells. The cloning reaction was performed using pQMCF IgG vector containing rabbit IgG heavy and light (kappa) chain constant region codon-optimized cDNAs. The newly formed IgG heavy and light chains were expressed under the control of the RSV LTR and CMV promoters, respectively. Initial restriction analysis of library pool DNA and colony PCR from 38 individual bacterial colonies indicated 100 % efficiency of pQMCF IgG vector assembly from the 4 fragments (data not shown). The Western blot analysis of the culture medium sample from library pool DNA-transfected CHO85EBNALT cells confirmed the predominance of intact rabbit IgG molecules assembled from the separately expressed heavy and light chains (Fig. 5b). The analysis of the same library pool transfection medium sample in mouse CD48 ELISA demonstrated the presence of antigen-specific antibodies (Fig. 5c). Finally, the screening of 95 clones in CHO85EBNALT cells resulted 39 % of clones with ELISA readouts 15–45 times over negative control and considered as “positive” (Fig. 5d).
Some positive (H2, A2, B2) and negative (F8, F12) clones (readouts indicated in Fig. 5d) were selected for sequencing of heavy and light chain cDNAs. All sequenced clones had essentially identical VH sequence, suggesting that the binding with the antigen was mainly determined by the VL domain. In this, the differences between the sequences of positive and negative clones were observed, most recognizable in CDR3 region (Fig. 5e). We have been observed that very high percentage of positive clones in the screening often associate with the limited number of different VH or/and VL sequences that are combined in the library. It may indicate that only few best binders have remained associated with the antigen coated surface during the repeated washing steps in B-cell capturing. Alternatively, particular cDNA product among others (e.g. this one provides most optimal template for the primer mix during the initial rounds of PCR) may be preferrably amplified. Irrespective of the excact mechanism, we have determined that increasing the variability of the recovered MAbs can be achieved by setting up more capturing reactions. This is clearly preferable to increasing the cell number per well.
Selective capturing using the pre-adsorption of source cells
We propose that this pre-adsorption step is useful for screening antibodies from organisms immunized with complex antigens. For example, using the viral surface proteins pseudotyped VLPs rather than purified surface proteins alone for immunization generally gives better odds of recovering a virus-neutralizing antibody . Using the same VLPs for capturing antigen-expressing cells as well as for screening yields opportunities to isolate and clone such antibodies. However, using VLPs for immunization usually results in a mixed response, including many MAbs specific to off-targets (e.g., directed to structural components of the VLPs). Thus, it may be useful to inhibit the recovery of the off-target antibodies by pre-adsorption of the antibody-expressing source cells with non-pseudotyped VLPs.
Production, sequence analysis and applications of the developed MAbs
We describe the novel HybriFree method for the development of monoclonal antibodies from an animal species for which coding sequence information of the antibody variable domain is available. This robust and rapid method includes the enrichment of source material by capturing specific antibody-producing cells for the construction of a combinatorial VH-VL library screened after expression in mammalian cells. The resultant expression vectors can be directly used to establish medium-scale transient production in mammalian cell factories without the need for additional cloning steps.
We thank Tiiu Männik, Urve Toots, Silver Olesk, Anneli Plink, Mihkel Allik, Kai Virumäe, Karl Mumm, Fernando Rodriguez Castaneda, Sulev Kuuse, Viia Kõiv and the Veterinary Clinic of the Estonian University of Life Sciences for their kind help with experimental work. We also thank Andrei Nikonov for very useful advice that helped us on improving the VH and VL PCR.
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