The authors would like to thank Lucy Chammas, Martha Costa, Julie Kim, Nancy Heredia, and Jeremy Macedo for the extensive testing of many method modifications and refinement of the current BSelex platform.

The use of samples from human volunteers followed protocols approved by The Scripps Research Institute Institutional Review Board. Informed consent was obtained from the donors prior to the blood donation.
1. Reagent Preparation
<ol>
	<li>Peripheral blood mononuclear cells (PBMCs)
	<ol>
		<li>Recover peripheral blood mononuclear cells from normal human donors by Ficoll-Plaque Plus isolation. Cryopreserve at 50 million cells/mL in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). Freeze the cells at -80 &#176;C and transfer to liquid nitrogen for long term storage.</li>
	</ol>
	</li>
	<li>Labeling target antigen peptides for sorting
	<ol>
		<li>Generate or obtain peptides specific to the target protein (up to 70 residues long) with a terminal biotin.</li>
		<li>Label 4 nmol of each individual biotinylated peptide with streptavidin covalently attached to PE (R-phycoerythrin) or APC (allophycocyanin) in separate tubes. Prepare using a molar ratio of 9:1 peptide to streptavidin (SA), with a final concentration of roughly 3.2 &#181;M for SA-PE and 5.7 &#181;M SA-APC. Prepare Biotin tetramers as a negative control.</li>
		<li>Incubate each peptide mix overnight, in the dark, at 4 &#176;C with slow mixing.</li>
		<li>Remove unbound fluorophore by passing labeled peptides through microcolumns containing polyacrylamide beads.</li>
		<li>Store peptide tetramers up to 2 months at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Cell Sorting
<ol>
	<li>At least 1 h up to 16 h before the CD22+ isolation, thaw donor PBMCs and allow to rest at 37 &#176;C.
	<ol>
		<li>Remove vials of frozen PBMCs from the freezer and immediately transfer to a 37 &#176;C water bath. When the vials are almost thawed, transfer to the work area.</li>
		<li>Transfer the contents of each vial to a 50 mL tube containing prewarmed medium (keeping the donors separate). Add medium to a final volume of 50 mL. Gently mix the contents.</li>
		<li>Centrifuge at 370 x g for 6 min at room temperature. Discard the supernatant, resuspend the cells in 15 mL of RPMI complete medium and perform a cell count.</li>
		<li>Adjust cell concentration to 2.0 x 107 cells/mL with RPMI complete medium, and transfer cells to a T75 flask or larger and incubate at 37 &#176;C for at least 1 h and up to 16 h to allow recovery time for thawed cells.</li>
		<li>Collect the cells (pooling if desired) and centrifuge at 370 x g for 6 min at 4 &#176;C. Discard supernatant and resuspend the cells in 5 mL of ice cold MACS buffer (PBS, pH 7.6 containing 0.5% BSA and 2 mM EDTA), bring to 50 mL with MACS buffer and perform a cell count.</li>
		<li>Centrifuge the cells at 400 x g for 7 min at 4 &#176;C.</li>
		<li>Isolate the CD22+ B cells by positive selection via capture on CD22 microbeads. Use MACS buffer for all washes. Count and centrifuge 400 x g for 7 min at 4 &#176;C. The expected recovery is 5-10% of the total PBMC population.</li>
		<li>Resuspend cells at 40 million per mL FACS buffer (Tris buffer, pH 8.0 containing 0.5% BSA and 2 mM EDTA) and proceed with cell staining of the donors individually or as a pool.</li>
	</ol>
	</li>
	<li>Stain cells for memory B cell sorting
	<ol>
		<li>Remove an aliquot of cells for cytometer set up and compensation for each of the labeling fluorophores used. Include unstained cells as a control.</li>
		<li>Calculate the final staining volume for the number of CD22+ cells obtained (staining 2 million per 100 &#956;L).</li>
		<li>Add extracellular markers for B cells (CD19-PerCP-Cy5.5), IgG (IgG-FITC), and memory compartment (CD27-PECy7) as per manufacturers&#39;&#160;dilution recommendations, and mix gently. Aliquot 107 cells into a 1.5 mL tube for the negative control and transfer the remaining cells to a 15 mL tube.</li>
		<li>Add the dual labeled biotin-SA tetramers to the negative control tube at a final concentration of 36 nM each for PE and APC multiplied by the number of peptides in the sort tube and bring the volume to 0.5 mL final with FACS buffer (e.g., 10 peptides x 36 nM=360 nM).</li>
		<li>Add the peptide tetramers to the sort tube at 36 nM each for PE and APC and bring the cells to 2x106 per 100 &#956;L with TBS Buffer. Incubate at 4 &#176;C in the dark and with gentle rotation for 30-60 min.</li>
		<li>Wash 2x at 4 &#176;C, 400 x g, 7 min, removing an aliquot to count before the last wash. Filter cells using 5 mL filter cap tubes. Add DAPI (4&#39;,6-diamidino-2-phenylindole) stain to 0.3 &#181;M final concentration just prior to sorting as a marker of cell membrane integrity.</li>
	</ol>
	</li>
	<li>Single cell sorting via flow cytometry
	<ol>
		<li>Prepare 48 wells of 96-well PCR plates for sorting.
		<ol>
			<li>Prepare a master mix: (2 &#956;L of 10x RT buffer, 0.5 &#956;L of RNase inhibitor, 7.5 &#956;L of sterile PCR-grade water) per sample. Aliquot 10 &#956;L per well, cover, and store at 4 &#176;C until ready for sort.</li>
		</ol>
		</li>
		<li>Run the negative control on the cell sorter, analyzing the entire sample.
		<ol>
			<li>Set flow cytometry gates to isolate the appropriate cell populations for single cell sorting.
			<ol>
				<li>Plot FSC area vs SSC area and set Gate R1 to isolate lymphocytes.</li>
				<li>Plot FSC height vs FSC width and set Gate R3 to exclude FSC doublets.</li>
				<li>Plot SSC height vs SSC width and set Gate R4 to exclude SSC doublets.</li>
				<li>Plot SSC height vs DAPI and set Gate R5 to isolate live cells (DAPI-).</li>
				<li>Plot IgG vs CD19 and set Gate R6 to isolate IgG+ B cells (CD19+ IgG+).</li>
				<li>Plot IgG vs CD27 and set Gate R7 to select memory B cells (CD27high).</li>
				<li>Plot Antigen-PE (Ag-PE) vs antigen-APC (Ag-APC) and set Gate R8 as an Ag-PE+/Ag-APC+ quadrant with a low number of events in the double positive gate.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Collect an equal number of memory cells from the antigen-positive (Ag+) sample for determination of signal to noise.</li>
		<li>Sort single cells having the phenotype CD19+ CD27+ Ag-PE+ Ag-APC+ (Gate R8) into prepared 96-well PCR plates.</li>
		<li>Cover plates with aluminum tape pads, centrifuge for 1 min at 400 x g, and store at -80 &#176;C for future cloning.</li>
	</ol>
	</li>
</ol>
3. Single Cell Cloning
<ol>
	<li>Reverse transcription reactions
	<ol>
		<li>Remove the single B cell sort plate from 80 &#176;C. Thaw on ice for 2 min. Spin the plate for 2 min at 3,300 x g to pool contents in the bottom of the wells before opening.</li>
		<li>Prepare a dNTP/buffer master mix with 10% non-ionic detergent and oligo(dT) as the reverse primer. Add enzyme mix to each well containing the single cell and DO NOT MIX.</li>
		<li>Incubate 5 min at 65 &#176;C. Add enzyme master mix containing 100 mM DTT, RNase inhibitor, and reverse transcriptase enzyme to bring total volume to 20 &#181;L. Incubate 10 min at 50 &#176;C, then 10 min at 80 &#176;C.</li>
		<li>Transfer to ice prior to starting PCR steps</li>
	</ol>
	</li>
	<li>Multi-step nested PCR reactions
	<ol>
		<li>Use separate nested PCR reactions are used for heavy chain (HC) and light chain (LC) amplification.</li>
		<li>For the first round of PCR amplification (Step I), use a pool of forward primers and a single reverse primer to amplify the HC and LC variable regions in separate PCR reactions (Figure 2). By design, the pool of forward primers will amplify a large percentage of germline leader (L) sequences and the reverse primer is specific to the downstream constant regions of each chain, including both the kappa (&#954;) and lambda (&#955;) for light chains16.
		<ol>
			<li>Use 2.5 &#181;L of cDNA product as template for each PCR (Step I) reaction. Add primers to the final reaction concentration of 1 &#181;M for each. Double the 10x polymerase buffer to 2x concentration, bring the final volume to 25 &#181;L per reaction. Run HC PCR reactions using [94 &#176;C/4 min; 94 &#176;C /15 s, 55 &#176;C /20 s, 68 &#176;C/60 s; 68 &#176;C/3 min] for 50 cycles. Run LC PCR reactions using [98 &#176;C/4 min; 98 &#176;C /15 s, 72 &#176;C /20 s, 72 &#176;C/60 s; 72 &#176;C/3 min] for 50 cycles.</li>
		</ol>
		</li>
		<li>Use 2.5 &#181;L of the Step I product as template for each (Step II) PCR reaction. Add the pool of forward primers specific to the HC and LC (&#954; and &#955;) framework 1 region and a pool of reverse primers specific to the junction region of each antibody chain. Double the 10x polymerase buffer to 2x concentration, and run PCR reactions 50 cycles each using the same parameters as Step I.</li>
		<li>Run the completed PCR reactions on 1% agarose gel to visualize positive amplification hits. Recover paired amplicons (both heavy and light chain from the same cell) and isolate via gel extraction. Determine DNA fragment concentration via OD260 for accurate ligation mix calculations.</li>
		<li>Combine recovered heavy and light chain fragments with a linker fragment and the expression vector backbone using a 4-fragment ligation reaction using a commercial kit.</li>
		<li>Transform ligation reactions into chemically competent bacteria using a commercial kit. Once plated onto antibiotic plates, add 4 mL of growth media (with antibiotic) to the remaining transformation culture, and incubate 37 &#176;C overnight at 250 rpm. This is the &#34;ligation mix culture.&#34;</li>
		<li>Prepare miniprep DNA from the overnight ligation mix cultures using a commercial kit, and determine the resulting plasmid DNA concentration.</li>
	</ol>
	</li>
</ol>
4. ELISA Screen for Confirmation of Antigen-specificity
<ol>
	<li>Transfect 10 &#181;g of miniprep DNA with cationic lipid-based reagent into 10 mL of suspension 293 cells, and incubate for 3-4 days at 37 &#176;C (8% CO2) while rotating at 125 rpm. For harvest, centrifuge culture 10 min at 1,000 x g and recover clarified media.
	<ol>
		<li>Measure the IgG concentration in the supernatants via affinity to Protein A.</li>
		<li>Test each IgG (in supernatant) at 20 &#181;g/mL by enzyme-linked adsorbent assay (ELISA) against the individual peptides used for the sort, captured on a streptavidin plate or actin as a negative control. Use a goat anti-human peroxidase secondary antibody against human IgG Fab to detect recombinant clones. After subtraction of background, OD &#62; 0.5 is defined as screen positive.</li>
		<li>Confirm screen positive hits by performing an additional ELISA, using dilutions of IgG supernatant to generate a concentration curve starting at 20 &#181;g per mL, and plotting against OD for each antigen showing reactivity in the initial screen ELISA.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Su, K. Y., Watanabe, A., Yeh, C. H., Kelsoe, G., Kuraoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Culture+of+Human+Naive+and+Memory+B+Cells+for+Use+as+APCs.">Efficient Culture of Human Naive and Memory B Cells for Use as APCs.</a> Journal of Immunology. 197 (10), 4163-4176 (2016).</li><li>Corti, D., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+antiviral+antibodies.">Broadly neutralizing antiviral antibodies.</a> Annual Review of Immunology. 31, 705-742 (2013).</li><li>Steinitz, M., Klein, G., Koskimies, S., Makel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EB+virus-induced+B+lymphocyte+cell+lines+producing+specific+antibody.">EB virus-induced B lymphocyte cell lines producing specific antibody.</a> Nature. 269 (5627), 420-422 (1977).</li><li>Bloom, A. D., Nakamura, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+tetraploid,+immunoglobulin-producing+cell+line+from+the+hybridization+of+two+human+lymphocyte+lines.">Establishment of a tetraploid, immunoglobulin-producing cell line from the hybridization of two human lymphocyte lines.</a> Proceedings of the National Academy of Sciences of the United States of America. 71 (7), 2689-2692 (1974).</li><li>Olsson, L., Kaplan, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-human+hybridomas+producing+monoclonal+antibodies+of+predefined+antigenic+specificity.">Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity.</a> Proceedings of the National Academy of Sciences of the United States of America. 77 (9), 5429-5431 (1980).</li><li>Lechner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+Circulating+Immune+Cells+in+Neovascular+Age-Related+Macular+Degeneration.">Alterations in Circulating Immune Cells in Neovascular Age-Related Macular Degeneration.</a> Scientific Reports. 5, 16754 (2015).</li><li>Loken, M. R., Brosnan, J. M., Bach, B. A., Ault, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+optimal+lymphocyte+gates+for+immunophenotyping+by+flow+cytometry.">Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry.</a> Cytometry. 11 (4), 453-459 (1990).</li><li>Pascual, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+memory+to+hyperphosphorylated+tau+in+asymptomatic+individuals.">Immunological memory to hyperphosphorylated tau in asymptomatic individuals.</a> Acta Neuropathologica. 133 (5), 767-783 (2017).</li><li>van Ameijde, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+therapeutic+potential+of+a+naturally+occurring+human+antibody+targeting+a+phosphorylated+Ser(422)+containing+epitope+on+pathological+tau.">Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser(422) containing epitope on pathological tau.</a> Acta Neuropathologica Communications. 6 (1), 59 (2018).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Tiller, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+monoclonal+antibodies+from+single+human+B+cells+by+single+cell+RT-PCR+and+expression+vector+cloning.">Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.</a> Journal of Immunological Methods. 329 (1-2), 112-124 (2008).</li><li>Wrammert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+cloning+of+high-affinity+human+monoclonal+antibodies+against+influenza+virus.">Rapid cloning of high-affinity human monoclonal antibodies against influenza virus.</a> Nature. 453 (7195), 667-671 (2008).</li><li>Han, A., Glanville, J., Hansmann, L., Davis, M. 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The authors have nothing to disclose

The method presented here combines flow cytometry and single cell cloning, and the methods we describe here we based on methods previously developed by Tiller and colleagues11. Their work describes the recovery and cloning of monoclonal antibodies in order to study the B cell repertoire in humans at the level of individual cells. We have adapted the major components of their process to allow recovery of antigen-specific monoclonal antibodies from a population of memory B cells, including the multi-step amplification primer strategy. The major modification is the addition of labelled antigen &#34;bait&#34;. Additional adaptations have been made to the published protocol, including (but not limited to) modifying the cloning vector backbone into a single expression plasmid, additional primer coverage of the germline repertoire (both leader sequence and framework 1), transfection of suspension HEK293 cells for higher expression, and the use of high fidelity polymerases during PCR amplification.
For the methods described here, the most critical steps are near the transition between flow cytometry and single cell cloning. First, the proper placement of sorted single cells into the plate is essential. Setting the drop-delay correctly on the sorter is a key step. Environmental factors, such as low humidity, must also be taken into account, as we have found our recovery efficiencies drop significantly unless a static gun is used on the target sort plates. Following cell placement, plates are centrifuged to ensure cells have contacted the 10 &#181;L of buffer in bottom of the wells. All of these measures are critical for the molecular biology portion to be successful. If the conditions are sub-optimal for the reverse transcription reaction of a single cell, PCR amplification of even &#946;-actin can be difficult. The strength of the method presented is the ability to interrogate millions of cells and only sort the ones that match the antigen reactivity criteria against the antigen of choice. This requires the signal to noise ratio to be as high as possible, which is done by optimizing bait concentrations before sorting. Dual-labeled bait is used to reduce the recovery of false-positive cells, which can occur if one of the fluorophores has high background. Using more than one antigen bait can make the signal:noise optimization more difficult, but the ability to interrogate multiple peptides simultaneously is another benefit of this method.
When recovery efficiencies are low, a set of nested &#946;-actin primers are used to confirm that template cDNA is present. The drawback to this approach is that it is difficult to determine whether there was no cell present in the well or the reverse transcription reaction failed, making troubleshooting difficult. Occasionally the opposite will occur and the efficiencies are higher than expected. The typical recovery for heavy chain is between 30-45% and light chains are higher, between 40-60%. Each PCR reaction (Step I &#38; II) uses 50 cycles to amplify from a single cell. The high number of total cycles also makes this method susceptible to contamination. Sequence analysis of amplicons can be used to determine whether a contaminant has been introduced, or an unusually high recovery rate has been legitimately achieved.
The utility of this method to recover native human antibodies against an antigen of choice has several significant limitations. First, only soluble proteins that can be labeled may be used as &#34;bait&#34;&#160;for the flow cytometry. For the sorts presented in the representative results, we used a series of synthesized overlapping peptides from tau protein, since using the whole protein proved difficult. The use of linear peptides is likely not optimal, since it may limit the identification of antibodies against non-linear or structural epitopes. However, we were able to identify several unique antibodies against tau and these are currently undergoing further evaluation8,9. Another limitation is the low throughput of the molecular biology recovery and cloning of IgGs. The initial sorting strategy allows for the screening of millions of cells, but the subsequent molecular biology processing consists of multiple stages. Ongoing optimization efforts to incorporate newer technologies, such as Gibson Assembly10 have streamlined several steps, but the bottleneck remains cloning individual heavy and light chain pairs.
The &#34;BSelex&#34;&#160;method utilizes the BCR expressed on the surface of memory B cells to identify cells that display antigen reactivity, and then recover these individual cells via flow cytometry11,12. Due to this reliance on the BCR, the method is restricted to the memory B cell compartment, and does not capture antibody secreting cells (ASCs) such as plasmablasts. However, this approach may be advantageous to recovering a broader repertoire of antigen-specific immunoglobins when compared to ASCs. Influenza vaccination studies demonstrate that while reactive ASCs can be dominated by a small number of expanded B cell clones, the antigen-specific memory B cell population is rarely clonal12. The T cell receptor (TCR) on the surface of T cells possesses similarities to the B cell receptor in gene arrangement and recombination to maximize diversity. Single cell approaches similar to what is described in the method presented here have been developed for assessing the T cell repertoire, including recovery and single cell cloning of alpha and beta chains13. However, TCR recognition requires peptides to be presented by MHC molecules, adding significant complexity to the labeled bait approach to identify peptide-specific T cells. Unlike B cells, T cells do not undergo affinity maturation of the entire variable region, so identification of the short CDR3 region and some flanking sequence is all that is required for identification and reconstitution. Finally, this method describes the identification of IgG molecules using flow cytometry, but it is also possible to use alternative phenotypic markers to identify B cells of different isotypes.
Currently, there are methods being developed to connect the enormous amount of data collected from next generation sequencing with functional analysis of antigen specificity, but these methods are still being refined. Despite its limitations, the method described here has been used to identify antibodies with potential therapeutic value for both infectious and non-infectious diseases8,14,15, and represents a reliable approach to recover relevant antigen-specific IgGs from humans, without extensive manipulation of B cells or extensive cell culture.

Antibodies are a growing class of therapeutic molecules, and the existing B cell repertoire in any human is a potential source of such antibodies. When recovered from a human donor, they require no adaptation or &#34;humanization&#34;, steps that are required for antibodies generated in other animal systems. Several methods exist for the identification and isolation of human antibodies, including B cell activation and proliferation1, immortalization via EBV transformation2,3, and generation of hybridoma cell lines4,5. However, all of these methods require extensive cell culture to screen and recover antigen-specific antibodies. Information about the human antibody repertoire has been greatly expanded with the development of next generation sequencing (NGS) technology, allowing for the identification of massive amounts of individual sequences present in donor samples. However, because NGS yields an agnostic view of all sequences present, it does not allow for the identification and isolation of antigen-specific antibodies, especially in the case of rare or low frequency antibodies.
The purpose of the &#34;BSelex&#34;&#160;method is to identify antigen-specific antibodies from circulating peripheral blood mononuclear cells in human donors, and isolate and recover the sequences of these antibodies for further analysis. This method utilizes flow cytometry and cell sorting to take advantage of the B cell receptor (BCR) expressed on the surface of memory B cells. Millions of B cells can be screened for antigen-specificity via flow cytometry before the more low-throughput molecular biology methods are initiated. Paired heavy and light chain identification is not possible in most NGS methods, which analyze cell sequences in bulk. In the method we describe here, cells are isolated individually, and paired recovery of both heavy and light chain sequences is possible, which allows direct cloning and expression of the full IgG.

<table border="0" cellpadding="0" cellspacing="0" style="width:681px;" width="682">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MoFlo Astrio EQ, Cell Sorter</td>
			<td style="width:136px;">MoFlo</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">cell sorting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Biotinylated peptides</td>
			<td style="width:136px;">New England Peptide</td>
			<td style="width:119px;"></td>
			<td>Cell sorting &#34;bait&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Micro Bio-spin P30 gel column</td>
			<td style="width:136px;">BioRad</td>
			<td style="width:119px;">#7326223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Streptavidin (R-PE)</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:119px;">SA10041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Streptavidin (APC)</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:119px;">S32362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">CD22 MicroBeads, human&#160;</td>
			<td style="width:136px;">Miltenyi</td>
			<td style="width:119px;">#130-046-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LS columns</td>
			<td style="width:136px;">Miltenyi</td>
			<td style="width:119px;">#130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Pre separation filters&#160;</td>
			<td style="width:136px;">Miltenyi</td>
			<td style="width:119px;">#130-041-407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PerCp-Cy5.5 mouse anti-human CD19</td>
			<td style="width:136px;">BD Biosciences</td>
			<td style="width:119px;">BD#340951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PE-Cy7 mouse anti-human CD27&#160;</td>
			<td style="width:136px;">BD Biosciences</td>
			<td style="width:119px;">#560609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">FITC mouse anti-human IgG</td>
			<td style="width:136px;">BD Biosciences</td>
			<td style="width:119px;">#560952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">DAPI</td>
			<td style="width:136px;">Life Technologies</td>
			<td style="width:119px;">#D21490</td>
			<td></td>
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			<td height="21" style="height:21px;width:260px;">RNaseOUT</td>
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			<td style="width:119px;">#10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Bovine serum albumin, Fraction V</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">#A4503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RPMI media</td>
			<td style="width:136px;">Hyclone</td>
			<td style="width:119px;">#SH30096.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">HI FBS (Fetal bovine serum)</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:119px;">#10082147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Mastercycler Gradient</td>
			<td style="width:136px;">Eppendorf</td>
			<td style="width:119px;">Model #6325</td>
			<td>PCR machine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PCR 96-well plates</td>
			<td style="width:136px;">Phenix</td>
			<td style="width:119px;">MPS-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PCR Plate mats</td>
			<td style="width:136px;">Phenix</td>
			<td style="width:119px;">SMX-PCR96</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Advantage UltraPure PCR Deoxynucleotide Mix</td>
			<td style="width:136px;">Clontech</td>
			<td style="width:119px;">#639125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Superscript IV First-strand synthesis system</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:119px;">#18091050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Phusion High Fidelity Polymerase</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:119px;">F-531-L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Platinum polymerase</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:119px;">#10966108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Nuclease-free water</td>
			<td style="width:136px;">QIAGEN</td>
			<td style="width:119px;">#129114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Oligonucleotide primers</td>
			<td style="width:136px;">IDT</td>
			<td style="width:119px;">assorted</td>
			<td>Primers for all PCR steps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Gel Extraction Kit</td>
			<td style="width:136px;">QIAGEN</td>
			<td style="width:119px;">#28706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">PCR Purification kit</td>
			<td style="width:136px;">QIAGEN</td>
			<td style="width:119px;">#28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Miniprep Kit</td>
			<td style="width:136px;">QIAGEN</td>
			<td style="width:119px;">#27106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">NEBuilder HiFi DNA Assembly Cloning Kit</td>
			<td style="width:136px;">New England Biolabs</td>
			<td style="width:119px;">E5520S</td>
			<td>Gibson assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Expi293 Expression Media</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:119px;">#A14351-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">ExpiFectamine 293 Transfection Kit</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:119px;">#A14525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Opti-MEM Media</td>
			<td style="width:136px;">Invitrogen</td>
			<td style="width:119px;">#31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">CO2 incubator (Multitron)</td>
			<td style="width:136px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Octet&#160;RED384 System</td>
			<td style="width:136px;">Pall/ Forte Bio</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Pierce Streptavidin Coated High Binding Capacity Clear 96-well Plates</td>
			<td style="width:136px;">Thermo Scientific</td>
			<td style="width:119px;">#15500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Corning Costar 96-well Polystyrene Plate High Binding Half Area</td>
			<td style="width:136px;">Corning</td>
			<td style="width:119px;">#3690</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Goat Anti-human IgG Fab Secondary Antibody</td>
			<td style="width:136px;">Jackson Labs</td>
			<td style="width:119px;">#109-036-097</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Goat Anti-mouse HRP Secondary Antibody</td>
			<td style="width:136px;">Jackson Labs</td>
			<td style="width:119px;">#115-035-072</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">SureBlueTM TMB Microwell Peroxidase Substrate (1-Component)&#160;</td>
			<td style="width:136px;">KPL</td>
			<td style="width:119px;">#52-00-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">TMB Stop Solution</td>
			<td style="width:136px;">KPL</td>
			<td style="width:119px;">#50-85-06</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell screening method for the selection and recovery of antibodies with desired specificities from enriched human memory b cell populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

This method covers a multi-step process to isolate antigen-specific antibodies from human donors. In the representative data shown here, cells were incubated with a pool of fluorescently-labeled peptides representing several different domains of the tau protein, including phosphorylated peptides to mimic putative phosphorylation sites. These peptides were used as &#34;bait&#34;&#160;to identify cells that are reactive with tau epitopes(s) of interest. In preparation for sorting, a panel of fluorescently-labeled phenotypic markers were used to identify different cell populations within the enriched B cell population. A series of cytometry gates were devised to isolate the target memory B cells (Figure 1). Lymphocytes were isolated based on their cell size and granularity using forward scatter (FSC) and side scatter (SSC) plots in flow cytometry6,7. Following exclusion of multiple cells (&#34;doublets&#34;) and dead cells, phenotypic markers allowed the segregation of IgG+ memory B cells via IgG, CD19 (B cell) and CD27 (memory). In this approach, the CD27 marker does not distribute into two discrete populations, so the top 45% of CD27+ expressing cells are included for the final gate. Finally, cells double-positive for both APC and PE fluorophores distribute into the upper right quadrant of the gating graph, indicating reactivity to both labeled versions of peptides. The cells that fell within the drawn gate were isolated and sorted into individual wells of a 96-well plate. The use of antigen with two different labels increases the signal-to-noise ratio and reduces the number of false positives in the subsequent molecular biology process. The sorted cells represented about 0.1% of the memory B cells in the final gate, and 0.001% of the starting cell sample.
The first readout of single cell cloning is confirmation of amplification of the respective heavy and light variable chains (Figure 2). Since paired recovery of both amplicons is desired, the PCR reactions are evaluated side-by-side on agarose gel, and matched pairs are excised from the gel and DNA fragments extracted. Typical efficiency of amplification is 30-50% heavy chain and 50-70% light (kappa) chain. Recovery of paired amplicons is usually between 25-40% efficiency. These efficiencies vary between donor pools, and the representative data (Figure 3) is an example of very efficient amplification from 24 single cells (42% paired recovery). Following IgG cloning, the IgG expression vector is transfected into human embryonic kidney (HEK-293) suspension cells, which are used to maximize expression. The use of serum-free medium reduces contaminating proteins from the recombinant antibody prep, and helps minimize noise in subsequent binding assays. The recovered antibodies are screened against the original panel of tau peptides and scored for reactivity by ELISA (Figure 4). An initial threshold of OD = 0.5 above background is used to indicate positive antigen reactivity, and &#946;-actin protein is used as a control for non-specific binding. If the flow cytometry and sorting steps use a mixed pool of peptides, the screening ELISA is the first step of deconvoluting specific reactivities of the recovered IgGs. Three of the 10 IgGs assayed demonstrated reactivity against phosphorylated CBTAU22.1 peptide, and one was reactive to non-phosphorylated CBTAU27.1 (Figure 4A). Additional confirmation was completed using a concentration curve of the same recombinant antibody samples against the peptides identified in the initial screen, and an additional peptide as a negative control (Figure 4B). For each positive hit, an individual plasmid clone was isolated from the transformed pool and reconfirmed by the same ELISA method. Only Clone 34 is shown the data presented, and while reactivity to non-phosphorylated CBTAU27.1 was confirmed, lower affinity binding was also observed with the phosphorylated 27.1 peptide (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59809/59809fig1.jpg" /> 
Figure 1: Isolation of antigen-reactive single cells via flow cytometry. (A) Shown is a representative plot where the lymphocyte population was contained within the drawn gate. (B) Gates based upon forward and side scatter (FSC-height v FSC-width (R3); SSC- height v SSC- width(R4)) were used to exclude doublets. Only cells within drawn gates were evaluated in subsequent plots. DAPI+ cells were considered dead and excluded (R5). In this experiment, 3.7 x 107&#160;&#34;live&#34;&#160;cells were interrogated. The majority of live cells were B cells (CD19+), and IgG+ cells (4.66%) were isolated from this population (R6). The top 43.2% of CD27+-expressing memory cells were included in the selection gate (R7). (C) Quadrant 2 (Q2) contained cells reactive to both labeled antigens (peptide-APC v peptide-PE), and these cells were sorted and recovered individually into a 96-well plate. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59809/59809fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59809/59809fig2.jpg" /> 
Figure 2: PCR amplification recovers IgG heavy and light variable sequence for cloning. (A) Both heavy (VH) and light (Vk or Vl) chain variable regions are recovered using nested PCR reactions. Step I primers amplify forward from the native leader sequence and reverse from within the constant region. Multiple arrows represent a pool of between 7-12 primers, which are used to ensure broad coverage of possible germlines. Step II primers are nested, specific to the extreme ends of the variable open reading frame, and add sequence to the ends of the amplicons that are homologous to adjacent sequence in the expression vector. (B) The linker consists of the constant light chain (Ck or Cl), followed by the heavy chain promoter and a non-native signal peptide sequence. The amplicons, linker, and plasmid backbone are simultaneously ligated via overlapping homologous sequence generated during the Step II PCR amplification, and isolated as an intact plasmid. The recombinant heavy and light chains in the final expression vector are driven by independent (and identical) CMV promoters, and are translated as separate proteins. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59809/59809fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59809/59809fig3.jpg" /> 
Figure 3: IgG heavy and light variable chain amplicons are recovered from single cells. The nested PCR reactions (Step II) were directly loaded onto 1% agarose gel and visualized. Heavy chain (H) and kappa light chain (L) PCR reactions from the same cell were loaded in adjacent wells so paired amplicons were easily observed. Successful heavy and light chain products are roughly 400 bp and 350 bp respectively. Successful recovery of paired amplicons are denoted by an (*). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59809/59809fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59809/59809fig4.jpg" /> 
Figure 4: Antigen specificity is confirmed from recombinant IgGs. Plasmids containing the recovered heavy and light chain sequences are transfected into HEK cells for expression. After four days, recombinant antibodies are assessed for reactivity by ELISA. (A) IgG concentration in the clarified media was measured and diluted to 20 &#181;g/mL. The pool of peptides used for sorting were individually assessed using 40 pmol per well in streptavidin plates. All IgGs were tested in duplicate wells. (B) Clones that displayed OD450 measurement &#62;0.5 were re-assessed (Clones #32, #34, #35) using 1:5 dilution steps against the peptides identified in the screen. (C) Once confirmed as a hit, a single plasmid clone was isolated from the clone #34 transformed ligations, transfected, and reconfirmed by ELISA. The same was done for clones #32 and #35 (data not shown). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59809/59809fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations at JoVE.com

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

single-cell-screening-method-for-selection-recovery-antibodies-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.5K Views.  Janssen Pharmaceutical Companies of Johnson and Johnson. The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

Video: Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

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Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood