Rapid production of antigen-specific monoclonal antibodies from a variety of animals
© Kurosawa et al; licensee BioMed Central Ltd. 2012
Received: 10 July 2012
Accepted: 28 September 2012
Published: 28 September 2012
Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging.
We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals.
Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.
The mouse hybridoma method has been used previously for the production of candidate monoclonal antibodies (mAbs) for therapeutic use . However, immune responses against highly conserved human proteins are often weak in mice, resulting in the production of low affinity and/or non-specific mAbs. To avoid the problem of human proteins being recognized as self-antigens in mice, the use of an evolutionarily distant animal from humans is essential to obtain better immunization against therapeutic target molecules. While a variety of animals have been used to produce polyclonal antibodies against human proteins, mAbs from animals other than rodents have not been routinely produced due to the difficulties in establishing immortalized antibody-producing cell lines by hybridoma, viral transformation or reprogramming [1–3]. Recently, the direct molecular cloning of cognate pairs of immunoglobulin gamma heavy chain (IgH) variable (VH), light chain kappa variable (VLκ) and light chain lambda variable (VLλ) genes from single antigen-specific plasma/plasmablast cells (ASPCs) using the polymerase chain reaction (PCR) has attracted attention as an alternative method for generating mAbs from immunized animals [4–7]. Although the use of ASPCs is best suited to the isolation of high affinity mAbs, since they go through the processes of somatic hypermutation and affinity maturation, the application of this method for species other than humans and mice is limited because the current plasma/plasmablast cell (PC) isolation protocols rely on a small number of identified PC-specific markers combined with the absence of one or more B cell differentiation antigens . Furthermore, expensive equipment and acquired technical skills are required to identify and isolate ASPCs from the bulk of the PC population . The manual VH and VL gene amplification from single cells followed by the construction of IgH and immunoglobulin light chain (IgL) gene expression plasmids are also limiting steps of this method [4, 9–12]. To achieve rapid and scalable automation for the generation of mAbs from a large numbers of single cells, we previously proposed a high-throughput single-cell-based immunoglobulin gene cloning method by developing a non-contact magnetic power transmission system (MAGrahd) for single-cell-based cDNA synthesis and a target-selective joint PCR (TS-jPCR) for IgH and IgL gene expression [13, 14].
PC identification without using antibodies against lineage markers
Isolation of ASPCs by FACS
Production of guinea pig mAbs against human insulin
The use of ASPCs is best suited to the generation of high affinity mAbs because somatic hypermutation of immunoglobulin genes allows a continual improvement in antibody recognition during the course of PC differentiation. However, the isolation of ASPCs is one of the most challenging types of cell separation due to the scarcity of ASPCs in lymph nodes and blood. Recently, several groups reported a microengraving method for the selection of ASPCs by using an array of on-chip micro wells [5, 6]. But the microwell-based method suffers from several problems, including the limited application of this method for species other than humans and mice and the limited numbers of cells that can be analyzed per experiment. FACS, on the other hand, enables the analysis of large numbers of cells at high speeds; however, the isolation of ASPCs by FACS had yet to be established. The main limitation in isolating ASPCs has been the non-specific binding of cells to fluorescently labeled antigens, which is caused by the use multiple antibodies against lineage-specific markers to enrich for PCs. To overcome these limitations, we developed ERIAA for detecting ASPCs from a variety of animals by FACS. Because ERIAA is based on weak cell surface IgG expression and abundant cytoplasmic rough ER, common features of PCs across animal species, a series of antibodies against lineage markers is not required for PC identification. By simply staining lymph node cells with antibodies against cell surface IgG and ER-tracker, we achieved PC identification from humans, mice, rabbits and rats with 70% to 90% purity, representing a 48-fold to 223-fold enrichment of PCs compared with the original lymphocyte populations. Furthermore, the reduction of background noise, due to not using several lineage-specific markers, allowed us to isolate ASPCs with a fluorescently labeled antigen. Unlike hybridoma and antibody display technology, ERIAA does not require a large number of lymphocytes. Because of the abundant PCs and small amount of red blood cells, lymph node is best suited for PCs isolation by ERIAA. Although the number of PCs in blood is significantly lower than that in lymph node, peripheral blood mononuclear cells can be used as a source in the case where human monoclonal antibodies from patients are required. In such a case, the use of ER-tracker in combination with known PC marker such as CD38 provides a high yield of PCs with very high purity. The isolation of ASPCs from spleen by ERIAA is less efficient due to the presence of non-plasma cells with strong ER-tracker signal (unpublished data).
Many protein targets are highly conserved between mice and humans and can therefore be recognized as self-antigens by a mouse host, making them less immunogenic. To avoid this problem of human proteins being recognized as self-antigens in mice, rabbit monoclonal antibody production by hybridomas was developed . Although a variety of animals can act as a potential host for producing mAbs by hybridoma, further application of hybridoma technologies to animals are expected to be limited due to difficulties in establishing a partner cell line for fusion. An alternative method for the generation of monoclonal antibodies by hybridoma is antibody display, which bypasses immune tolerance issues with highly conserved antigens. However, antibodies selected from synthetic repertoires often resulted in low affinity and/or non-specificity compared to antibodies derived from immunized animals. To overcome these limitations, phage antibody libraries were made from immunized animals and monoclonal antibodies with high binding affinity have been obtained [20–24]. Unlike antibody display technology, our method enables the preservation of original heavy-light chain pairings, which is useful in analyzing protective antibodies in infectious disease or identifying autoimmune antibodies in humans.
Our approach of utilizing ERIAA and high-throughput cloning enables the rapid generation of antigen-specific mAbs from a variety of animals. We verified the advantage of our technology by isolating hundreds of human insulin-specific mAbs from guinea pigs. Our methods will greatly contribute to the isolation of high performance mAbs needed for sensitive diagnostics and therapeutic purposes.
Antibodies against rat IgG (unconjugated, Dylight 594 conjugated, Dylight 650 conjugated and alkaline phosphatase (AP) conjugated), guinea pig IgG (unconjugated, Dylight 488 conjugated and AP conjugated), rabbit IgG (unconjugated, Dylight 594 conjugated, Dylight 650 conjugated and AP conjugated) and human IgG (Dylight 488 conjugated) were obtained from Abcam. Anti-mouse CD45R-PE, anti-mouse CD38-APC, anti-human CD20-PE and anti-human CD38-APC were obtained from Miltenyi Biotec (http://www.miltenyibiotec.com/en/default.aspx). All antibodies were diluted in 1 × phosphate-buffered saline (PBS) containing 0.5% (w/v) bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA) (PBS-BSA). Human insulin was obtained from Roche Applied Science (http://www.roche-applied-science.com/). Dynabeads® Oligo-(dT)25 magnetic beads and ER-Tracker™ Blue-White DPX and primers were purchased from Life Technologies. DNA sequencing was performed using an ABI 373XL DNA sequencer (Life Technologies). The synthetic peptides were purchased from Medical & Biological Laboratories (https://ruo.mbl.co.jp). Restriction enzymes and PrimeStar DNA polymerase with 1 × PrimeStar GC buffer were obtained from Takara Bio (http://www.takara-bio.com/). The framework (FW) regions and complementarity-determining regions (CDRs) were assigned using IMGT/V-QUEST (http://imgt.cines.fr/IMGT_vquest/share/textes/).
Animal experiments were approved by the Committee on Animal Experimentation at the University of Toyama. Female ICR mice (6 weeks old), Wistar rats, New Zealand white rabbits and Hartley guinea pigs were purchased from Japan SLC, Inc. (http://jslc.co.jp/). Groups of three animals were used to immunize each antigen. Mice were immunized three times (at intervals of 3 weeks) subcutaneously in the hind footpad with a 50 µl of 50:50 water-in-oil TiterMax Gold adjuvant emulsion (Sigma-Aldrich, http://www.sigmaaldrich.com/) containing 25 µg of egg albumin as an antigen. Rats and rabbits were immunized four times intramuscularly at the tail base with 200 µl of 50:50 water-in-oil TiterMax Gold adjuvant emulsion containing 100 µg of GFP. Guinea pigs were immunized four times intramuscularly at the tail base with a 200 µl of 50:50 water-in-oil TiterMax Gold adjuvant emulsion containing 100 µg of human insulin. At 1 week after the final immunization, the iliac or popliteal lymph nodes were surgically removed and used for the isolation of PCs. Human experiments were performed with the approval of the Clinical Research Ethics Committee at the University of Toyama and Itoigawa General Hospital. We obtained informed consent from all of the subjects. Human lymphocytes were prepared from surgically removed regional lymph nodes from patients with gallbladder and colon cancers.
Isolation of ASPCs by ERIAA
Cells (1 to 5 × 106/ml) prepared from mouse or human lymph nodes were suspended in 1 ml PBS-BSA and stained with fluorescently labeled antibodies against IgG for 30 minutes at 4°C with rotation. After washing with PBS-BSA, the cells were stained with anti-mouse CD45R-PE and CD38-APC or anti-human CD20-PE and CD38-APC, respectively. The cells were washed twice with PBS and incubated with 1 ml PBS containing ER-tracker (1 µM) for 5 minutes at room temperature. The cell suspension was then diluted with 4 ml PBS and analyzed by FACS. To achieve single-cell sorting of ASPCs, the cells (1 to 5 × 106/ml) prepared from rabbit, rat or guinea pig tissue were suspended in 1 ml PBS-BSA and stained with fluorescently labeled antigen (0.1 µg/ml) and fluorescently labeled antibody against IgG at 4°C for 30 minutes with gentle agitation. After washing with PBS, the cells were stained with ER-tracker as described above. The forward-versus-side-scatter (FSC vs SSC) lymphocyte gate (R1) was applied to exclude dead cells. The PCs (IgGlow ERhigh, R2 gate) were further subdivided into fractions according to their binding levels of fluorescently labeled antigens to define the ASPCs (IgGlow ERhigh antigen+) and non-specific PCs (IgGlow ERhigh antigen-). Single-cell sorting was performed using a JSAN flow cytometer equipped with an automatic cell deposition unit (http://baybio.co.jp/english/top.html) with fluorescently labeled antibodies against IgG monitored in the FL-l (mouse, human and guinea pig) or FL-5 (rabbit and rat) channel, fluorescently labeled antigen in the FL-1 (GFP Dylight 488) or FL-2 (insulin-Cy3) channel and ER-tracker in the FL-7 channel.
FACS-sorted cells were deposited onto propyltriethoxysilane-coated glass slides (Matsunami Glass, http://www.m-osaka.com/en/exhibitors/231/), fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.1% Triton X-100. The cells were stained with a fluorescently labeled antibody against IgG and fluorescently labeled antigen for 60 minutes at room temperature. The images were captured using an Olympus IX71 fluorescence microscope equipped with a SPOT RT3 digital microscopy camera (SPOT Imaging Solutions, http://www.spotimaging.com/) and compiled using Adobe Photoshop CS5 software. A minimum of 100 cells were counted for analysis in each experiment, and each experiment was performed in triplicate.
DNA fragments encoding the human, rabbit, rat and guinea pig immunoglobulin gamma (IgG), kappa (IgK) and lambda (Igλ) constant regions were amplified using lymphocyte cDNAs as the templates with primers for IgG (IgGC S and IgGC AS), IgK (IgKC S and IgKC AS) and Igλ (IgλC S and IgλC AS), respectively. The amplified DNA fragments digested with XhoI and NotI were inserted into the corresponding sites of pJON-IgG . The DNA fragments encoding the human insulin A and B chains were subcloned into the SpeI/XhoI site of the pET42b vector. The human insulin B chain mutants B1-13 and B1-20 were generated using the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies; http://www.genomics.agilent.com/) with wild-type human insulin B chain DNA as the template. The primers used were as follows: B(1-13) forward 5'-TGCGGCTCACACCTGGTGGAAAAACTTGCGGCCGCACTCGAG-3', B1-13 reverse 5'-CTCGAGTGCGGCCGCAAGTTTTTCCACCAGGTGTGAGCCGCA-3', B1-20 forward 5'-GCTCTCTACCTAGTGTGCGGGAAACTTGCGGCCGCACTCGAG-3' and B1-20 reverse 5'-CTCGAGTGCGGCCGCAAGTTTCCCGCACACTAGGTAGAGAGC-3'. Insulin epitope-glutathione S-transferase (GST) fusion proteins were produced in Escherichia coli BL21 (DE3) and purified using Superflow Ni-NTA columns (Qiagen, http://www.qiagen.com/default.aspx). The antigens were labeled with DyLight Fluor Labeling Reagents (Thermo Scientific, http://www.thermoscientific.com/) and purified using Bio-Gel columns (BIO-RAD, http://www.bio-rad.com/).
Molecular cloning of VH and VL genes from single cells
Primers used in this study
human IgGV AS1
5' RACE PCR human VH first
human IgGV AS2
5' RACE PCR human VH second
human IgKV AS1
5' RACE PCR human VLκ first
human IgKV AS2
5' RACE PCR human VLκ second
human IgλV AS1
5' RACE PCR human VLλ first
human IgλV AS2
5' RACE PCR human VLλ second
rat IgGC S
Rat IgG constant gene cloning
rat IgGC AS
Rat IgG constant gene cloning
rat IgKC S
Rat IgK constant gene cloning
rat IgKC AS
Rat IgK constant gene cloning
rat IgGV AS1
5' RACE PCR rat VH first PCR
rat IgGV AS2
5' RACE PCR rat VH second PCR
rat IgKV AS1
5' RACE PCR rat VLκ first PCR
rat IgKV AS2
5' RACE PCR rat VLκ second PCR
rat IgH cassette S
Rat IgH cassette amplification
rat IgLk cassette S
Rat IgLk cassette amplification
rab IgGC S
Rabbit IgG constant gene cloning
rab IgGC AS
Rabbit IgG constant gene cloning
rab IgKC S
Rabbit IgK constant gene cloning
rab IgKC AS
Rabbit IgK constant gene cloning
rab IgGV AS1
5' RACE PCR rabbit VH first PCR
rab IgGV AS2
5' RACE PCR rabbit VH second PCR
rab IgKV AS1
5' RACE PCR rabbit VLκ first PCR
rab IgKV AS2
5' RACE PCR rabbit VLκ second PCR
rab IgH cassette S
Rabbit IgH cassette amplification
rab IgLk cassette S
Rabbit IgLk cassette amplification
g-pig IgGC S
Guinea pig IgG constant gene cloning
g-pig IgGC AS2
Guinea pig IgG constant gene cloning
g-pig IgKC S
Guinea pig IgK constant gene cloning
g-pig IgKC AS
Guinea pig IgK constant gene cloning
g-pig IgλC S
Guinea pig Igλ constant gene cloning
g-pig IgλC AS
Guinea pig Igλ constant gene cloning
g-pig IgGV AS1
5' RACE PCR guinea pig VH first
g-pig IgGV AS2
5' RACE PCR guinea pig VH second
g-pig IgKV AS1
5' RACE PCR guinea pig VLκ first
g-pig IgKV AS2
5' RACE PCR guinea pig VLκ second
g-pig IgλV AS1
5' RACE PCR guinea pig VLλ first
g-pig IgλV AS2
5' RACE PCR guinea pig VLλ second
g-pig IgH cassette S
Guinea pig IgH cassette amplification
g-pig IgLk cassette S
Guinea pig IgLk cassette amplification
g-pig IgLλ cassette S
Guinea pig IgLλ cassette amplification
Nhe polyC S
5' RACE PCR first
5' RACE PCR second
TS-jPCR was performed as previously described . The IgH and IgL DNA cassettes were amplified from the respective pJON plasmids using PCR with primers for IgG (IgH cassette S and Cassette AS), IgK (IgLk cassette S and Cassette AS) and Igλ (IgLλ cassette S and Cassette AS). The amplified DNA cassettes were purified using S-400 spin columns. The PCR-amplified V gene fragments were joined to their respective DNA cassettes to build linear IgH and IgL genes by TS-jPCR. The primers used were Joint S and Joint AS. The PCR primers are detailed in Table 1.
The cognate pairs of IgH and IgL genes produced by TS-jPCR were cotransfected using the FuGENE HD Transfection Reagent (Roche, http://www.roche.com/research_and_development.htm) into 293FT cells grown in 96-well culture dishes. At 3 days after transfection, the cell culture supernatants were analyzed for the secretion of recombinant antibodies. The antibody concentrations and reactivities were determined by enzyme-linked immunosorbent assay (ELISA), as described previously . Large-scale recombinant mAbs were prepared using the FreeStyle™ 293 Expression System (Life Technologies) according to the manufacturer's protocol. The antibodies produced were purified using HiTrap™ Protein A HP columns (GE Healthcare, http://www3.gehealthcare.com/en/Global_Gateway). The protein concentrations were determined spectrophotometrically at 280 nm. The integrity of the produced antibodies was verified using SDS-PAGE with an IgG as the reference.
where Ao and Ai are the signal of total antibody incubated in the absence and presence of a given concentration of antigen, respectively, and ao is the total concentration of antigen in the antigen-antibody mixture.
Mapping of the antibody epitopes was performed using competitive ELISA. Briefly, epitope-GST fusion proteins (100 nM) and a series of peptides (250 nM) was incubated in PBS with the guinea pig mAbs (1 nM) overnight at 4°C. The peptide/antibody mixture was then transferred to an insulin-coated plate and tested for reactivity against insulin, as described above. The antibody epitopes were also determined by western blot analysis using bacterially expressed GST-fused insulin A chain, B chain and B chain truncation mutants (1-20 and 1-13). The peptides used corresponded to amino acid residues 1 to 10, 6 to 15 and 12 to 21 of the A chain and 1 to 10, 6 to 15, 11 to 20, 16 to 25 and 20 to 30 of the B chain of human insulin.
Formalin-fixed, paraffin-embedded mouse pancreatic tissue samples were subjected to double immunohistochemical staining for insulin and glucagon. The primary antibodies used were a rabbit monoclonal anti-human glucagon antibody (Cell Signaling Technology, http://www.cellsignal.com/) to identify α cells and guinea pig monoclonal anti-human insulin antibodies to identify β cells. The insulin signal was visualized with goat anti-guinea pig antibody-AP and the VECTOR Red Alkaline Phosphatase Substrate Kit (Vector Labs, http://www.vectorlabs.com/). The glucagon signal was visualized with goat anti-rabbit IgG Dylight 488. The tissue samples were embedded in ProLong Gold Antifade Reagent with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies), subjected to fluorescence microscopy and analyzed using 2D Deconvolution MetaMorph software (Molecular Devices, http://www.moleculardevices.com/).
We thank the members of our laboratory for fruitful discussions and help. This research was supported in part by grants from the Hokuriku Innovation Cluster for Health Science and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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