Paul.xu: Created page with "Immunisations Approval for use of animals for immunisation was provided through the UCB Pharma, UK Animal Welfare and Ethical Review Body (AWERB) and the license was granted b..."

2018-03-21T14:49:13Z

Created page with "Immunisations Approval for use of animals for immunisation was provided through the UCB Pharma, UK Animal Welfare and Ethical Review Body (AWERB) and the license was granted b..."

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Immunisations Approval for use of animals for immunisation was provided through the UCB Pharma, UK Animal Welfare and Ethical Review Body (AWERB) and the license was granted by the UK Home Office. At the end of the study the mice were anesthetised with isoflurane, terminal bleeds taken and then sacrificed using a Schedule 1 method in accordance with the Animals Scientific Procedures Act (ASPA). Rabbits were terminally anaesthetised with a Euthatal solution in accordance with Animals Scientific Procedures Act (ASPA).

Five Balb/c mice were immunised subcutaneously with 100 μg of purified human IL-25 (IL7-E) (Peprotech) or human TNFR2 extracellular domain (residues 1–256) (generated in house) per injection with CFA for the first dose and IFA for subsequent boosts. The animals received 2 booster injections at 2 week intervals. Splenocytes and blood for sera analysis were prepared from animals harvested 7 days following the final boost. New Zealand White rabbits were immunised subcutaneously with 500 μg of purified mouse WISP-1-rabbit Fc fusion protein (generated in house) emulsified with CFA for the first dose and IFA for subsequent boosts. The animals received 2 booster injections at 3 week intervals. PBMC for FACS and sera analysis were prepared from whole blood of animals harvested 14 days following the final boost.

Preparation and staining of mouse B cells for sorting Mouse splenocytes were harvested using a gentleMACS Dissociator (Miltenyi Biotech). The cells were then pushed through a cell strainer (Fisher Scientific) to generate a single cell suspension before being used for FACS or being frozen in 10% DMSO in FCS. Spleens from all 5 mice were combined prior to FACS. Splenocytes were washed in a 10-fold excess of Hanks balanced salt solution without Mg2+ or Ca2+ (Life Technologies), 1% FCS (Life Technologies), 25mM HEPES (Sigma), 1mM EDTA (Sigma)(described as buffer solution below). Cells were then blocked using mouse Fc block reagent (Miltenyi Biotech) following the manufacturers’ instructions. Following this, B cells were enriched using anti-mouse CD45R microbeads (Miltenyi Biotech) and LS MACS columns (Miltenyi Biotech) according to manufacturers’ instructions. Following enrichment, cells were resuspended in buffer solution and stained with 1 μg (unless stated otherwise) per 108 cells of the following antibodies: Rat anti-mouse IgG brilliant violet 421 (BD biosciences), rat anti-mouse IgM PE-Cy7 (Bio legend), rat anti-mouse IgD APC-Cy7 (Bio legend), rat anti-mouse CD19 AF700 (BD biosciences) (2 μg per 108 Cells), and rat anti-mouse CD4, CD8, GR1 and F4/80 FITC (BD biosciences) (dump channel). Human IL-25 (Peprotech) and TNFR2 extracellular domain (residues 1–256) (generated in house) were labelled with PE and APC using Lightning Link PE and APC labelling kits (Innova Bioscience) following manufacturers’ guidelines. In each case, the two antigen preparations labelled with both fluorophores were pre-mixed and used at 2 μg total protein per 108 cells. Cells were incubated in the dark for 20 minutes at room temperature and then washed twice in buffer before being finally re-suspended in buffer for FACS sorting.

Preparation of rabbit B cells for sorting Rabbit whole blood was mixed 1:1 in PBS and the mixture layered onto Lympholyte mammal (Cedarlane) in a 50 ml sterile Falcon tube (Fisher Scientific). This was centrifuged at 1500 x g for 30 minutes at 5°C. The PBMC layer was removed using a pipette and used for FACS or frozen down in 10% DMSO in FCS. PBMCs were washed in a 10 fold excess of Hanks balanced salt solution without Mg2+ or Ca2+ (Life Technologies), 1% FCS (Life Technologies), 25mM HEPES (Sigma), 1mM EDTA (Sigma) (described as buffer solution below). Mouse anti rabbit IgM biotin (BD biosciences) was pre incubated with 5 fold excess PE-Cy7 streptavidin, following a 20 minute incubation a 10 fold excess of biotinylated human albumin was added to the mix to remove any free PE-Cy7 streptavidin. Mouse anti-rabbit CD4, CD8 and a Pan T-cell antibody (ABD serotec) were labelled with PerCP using a Lightning Link kit (Innova Biosciences) following the manufacturers’ instructions. Cells were resuspended in buffer and stained with 1 μg per 108 cells with the following antibodies: mouse anti-rabbit IgG PE (Southern biotech), mouse anti rabbit IgM biotin-streptavidin PE-Cy7 complex, mouse anti-rabbit CD4, CD8 and a Pan T-cell antibody PerCP. Mouse WISP-1 (R & D Systems) was labelled with Alexa 647 and Alexa 488 using Microscale labelling kits (Life technologies) following the manufacturer’s instructions. Both labelled antigen preparations were used at 1 μg per 108 cells. Cells were incubated in the dark for 20 minutes at room temperature and then washed twice in buffer before being finally resuspended in buffer for FACS sorting.

Mouse single B cell sorting Compensation for the mouse sorting experiments was carried out by labelling mouse splenocytes separately with rat anti-mouse CD45R labelled with the different flourophores that were used in the experiment. Comparison to an unstained control sample provided a clear positive and negative population for each fluorophore colour allowing compensation to be calculated using the BD FACS DIVA software.

FACS sorting was carried out on a BD FACS ARIA III, using a 100 μm sort nozzle at no greater than 2000 events per second. Five minutes prior to running and sorting the samples, 2% v/v 7AAD (BD biosciences) was added to the samples in order to identify and eliminate dead cells. Sorting was controlled using BD FACS DIVA software. Post sort analysis was performed using FlowJo software (Tree Star).

Lymphocytes were identified by size and granularity using FSC-A versus SSC-A. Single lymphocytes were identified and gated using FSC-W versus FSC-A. T-cells, macrophages and neutrophils were identified and eliminated from sorting gates (dump channel). CD19+, IgG+, IgM-, IgD- B cells were identified and gated. Finally a gate was drawn around the double-positive antigen-specific population. All antigen-specific single B cells were sorted at single-cell per well into reverse transcription mix chilled to 4°C in 96-well PCR plates.

Rabbit single B cell sorting Compensation for the rabbit sorting was done using anti-mouse compensation beads (BD bioscience) and a series of mouse antibodies covering all the fluorophores used in the experiment and a comparison made to an unstained cell sample. Compensation was again calculated using the BD FACS DIVA software.

FACS sorting was carried out on a BD FACS ARIA III, using a 100 μm sort nozzle at no greater than 2000 events per second. Five minutes prior to running and sorting the samples, 2% v/v 7AAD (BD biosciences) was added to the samples to identify and eliminate dead cells. Sorting was controlled using BD FACS DIVA software. Post sort analysis was performed using FlowJo software (Tree Star). Lymphocytes were identified by size and granularity using FSC-A versus SSC-A. Single lymphocytes were identified and gated using FSC-W versus FSC-A. T-cells were identified and eliminated (dump channel). IgG+, IgM- B cells were identified and gated on. Finally, a gate was drawn around the double-positive mWISP-1 antigen-specific population. All positive cells were sorted into reverse transcription mix chilled to 4°C at either 1 cell or 3 cells per well in a 96-well PCR plate.

cDNA generation and transcriptionally active PCR (TAP) Details of transcriptionally active PCR (TAP) including oligonucleotide and DNA fragment sequences are provided in published patent applications WO2010/097435 [34] and WO2010/097437 [35] and in Clargo et al. [9]. Briefly, cDNA from single B cells was prepared using Superscript III reverse transcriptase (Invitrogen) primed with oligo (dT). Antibody variable-region genes were then recovered via two rounds of PCR using either KOD DNA polymerase (EMD Millipore) or TaqPlus Precision DNA polymerase (Agilent) (mouse primary PCR). A primary PCR utilized gene-specific primers at both the 5’ and 3’ ends. The 5’ oligonucleotide set bound either at the 5’ end of the leader sequence (for rabbit variable regions) or at the 5’ end of the framework 1 region of the mature variable region sequence (for mouse variable regions). The 3’ reverse primer set annealed to CH1 or Cκ region respectively. In the secondary PCR, a single 5‘ forward oligonucleotide that annealed to a “tail” encoded at the 5’ end of the primary PCR product was used with a 3’ primer set that annealed in the J region. Not only did the secondary oligonucleotides introduce restriction sites to facilitate downstream cloning, but they also provided ~25 base-pair overlap regions; at the 5’ end with a human cytomegalovirus (HCMV) promoter fragment (plus a leader sequence for rat-derived fragments that were generated with the framework 1 primer set) and at the 3’ end with a heavy or light chain constant region fragment. Then, in a tertiary PCR, variable region DNA, HCMV promoter fragment and constant region fragment containing a poly-adenylation sequence were combined and amplified to produce two separate linear transcriptionally-active PCR (TAP) products, one encoding the heavy chain and the other the light chain. Rabbit variable regions were recombined with rabbit constant regions in the expression cassette whereas mouse variable regions were assembled with mouse constant regions to produce recombinant IgG molecules. PCR set up was performed using an Aviso Onyx liquid handling robot.

Recombinant antibody expression Heavy and light chain transcriptionally active PCR fragments were transiently co-transfected into Expi293 cells (Life technologies) using Expi293 fectamine (Life Technologies) as per manufacturers’ instructions. These were incubated for 7 days at 37°C in a 5% CO2 environment before the supernatants were harvested by centrifugation for further characterisation.

IgG expression assessment IgG levels in expi293 supernatants were assessed for both mouse and rabbit via ELISA. Briefly, goat anti-species Fc fragment-specific polyclonal antibodies (Jackson Immunoresearch) were coated onto 96 well ELISA plates at 2 μg/ml. Plates were blocked with 1% PEG5000 (Sigma) in PBS before four half-log serial dilutions of each transient expression sample were prepared and applied to the plates along with a dilution series of a purified rabbit or mouse IgG standard. Following washing, goat anti-species Fab fragment-specific antibodies conjugated to horseradish peroxidase (Jackson Immunoresearch) were added at a 1:5000 dilution. Plates were developed with 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) (Sigma) before being read at 630nm on a synergy 2 microplate reader (BioTek). Sample concentrations were then calculated by interpolating sample values from the standard curves.

Recombinant IgG antigen-binding assays Binding of mouse-derived antibodies to target (IL-25 or TNFR2) was determined by ELISA. Briefly, 96-well ELISA plates were coated with 2 μg/ml of the target protein. Plates were blocked with 3% BSA (Life Technologies) in PBS. Recombinant TAP samples containing mouse IgG were added to the plates and finally the plates were revealed with goat anti-mouse Fc fragment-specific antibodies conjugated to horseradish peroxidase (Jackson Immunoresearch) at a 1:5000 dilution. Plates were developed with TMB before being read at 630nm on a Synergy 2 microplate reader (Bio Tek). Positive binders were determined based on the production of an absorbance value which was at least 4-fold higher than that produced by samples containing no antibody (background).

Binding of rabbit-derived antibodies was determined via homogenous fluorescence assay. Briefly, mouse WISP-1 (R & D systems) was biotinylated using Lightning link biotin (innova Bioscience) according to manufacturers’ instructions and coated onto SuperavidinTM beads (Bangs laboratories). Coated beads were then incubated with TAP supernatants containing recombinant rabbit IgG and anti-rabbit Fc-Alexa Fluor 647 (Jackson Immunoresearch) at a 1:5000 dilution for 1 hour. Plates were then read on an FMAT ABI 8200 (Applied Biosystems). Binders were determined as antibodies that showed a mean fluorescence signal greater than zero on WISP-1-coated beads and no signal on uncoated beads.

Secondary PCR product sequencing and diversity assessment To determine the sequence diversity from the sorted mouse B-cells, we performed DNA sequencing of the secondary PCR products. This was achieved using the secondary forward oligonucleotide for both the heavy and kappa chains as a primer. CDR3 sequences were extracted and used to assess diversity. Sequence diversity was assessed using a Principal Components Analysis (PCA) [[36|36]] similar to that described in Clargo et al. [9]. This provides a means to reduce the dimensionality of the data and generate an easy to interpret 2-dimensional data plot that illustrated the extent of diversity in our recombinant antibody panel. The relative position of the data points on the 2-D plot can be considered to be directly proportional to sequence identity across the data set based on the VH CDR3. Identical sequences resulted in the co-localization of data points on the 2-D plot (shown with multiple identifier flags) and related sequence families (those containing 80% sequence identity or higher in VH CDR3) have been identified either by using a single colour for the data points (IL-25Fig) or by collectively circling data points (TNFR2Fig). All other sequences were considered unique.

For the TNFR2 antibody panel, information regarding sequence uniqueness and functionality could be visualized on the same plot. TNF-blocking activity was indicated by shape type and colour of the data point (blue diamonds represent blocking antibodies, red squares represent non-blockers and black circles were antibodies where blocking data could not be reliably generated) and affinity was indicated by size of the data point (larger being of higher affinity).

BIAcore analysis Surface plasmon resonance was performed using a BIAcore T200 (GE Healthcare). All experiments were performed at 25°C. Goat F(ab’)2 anti-mouse Fcγ (Jackson Immunoresearch) was immobilised on a CM5 Sensor Chip (GE Healthcare) via amine coupling chemistry to a capture level of ~5000 response units. HBS-EP+ buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20, (GE Healthcare) was used as the running buffer. A 10‒50 μl injection of each antibody sample (0.1‒5 μg/ml) was used to achieve ~100 RU of capture. Two 3 min injections of human TNFR2 (generated in-house) at 10 nM and 100nM were passed over the captured antibody. Affinity was estimated following a 15 min dissociation phase. To determine blocking activity, binding of TNFR2 was followed by a 1 min injection of human TNF (generated in house) at 150 nM, at a flow-rate of 30 μl/min. The surface was regenerated at a flow-rate of 10 μl/min by two 60 s injections of 40 mM HCl and a 30s injection of 5 mM NaOH. Double referenced background subtracted binding curves were analyzed using the T200 Evaluation software (version 1.0) following standard procedures. Kinetic parameters and estimated affinity values for most antibodies were determined from fitting a BIAcore algorithm using a single cycle kinetics 1:1 binding model except 4G3, 3C11, 3A9 and 3F7 which were fitted using a bivalent model and affinity estimates derived from ka1 and kd1.
[[Category:Antibodies]]
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152282

Cloning antibody from B cells - Revision history

Paul.xu: Created page with "Immunisations Approval for use of animals for immunisation was provided through the UCB Pharma, UK Animal Welfare and Ethical Review Body (AWERB) and the license was granted b..."