Name: the isolation, differentiation, and quantification of human antibody-secreting b cells from blood: elispot as a functional readout of humoral immunity
Uploaded: 2016-12-14T12:30:00
Description: Watch this Scientific Journal Video about The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity at JoVE

This study was supported by a research grant from the Ministry of Science and Technology of the Executive Yuan of Taiwan (NSC99-2320-B-002-011). I would like to acknowledge the excellent service provided by the Flow Cytometric Analyzing and Sorting Core of the First Core Laboratory in College of Medicine of National Taiwan University.

Human peripheral blood must be obtained from healthy donors under informed consent, and the use of blood samples must conform to the approved guidelines established by individual institutional review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cytometry (Figure 1) and ELISpot assays (Figure 3) was approved by the Internal Review Board of National Taiwan University Hospital (protocol number 201307019RINB).
1. Isolation and Purifica...

Isolation and Purification of Human Peripheral Blood B Cells
Normally, RBCs can be efficiently ruptured and cleared by lysis buffer (step 1.2). It is important not to incubate PBMCs with the RBC lysis buffer longer than 5 min, as cell viability might be affected by the ammonium chloride. Alternatively, RBCs and platelets can be simultaneously removed by the following protocol.
Mix fresh whole blood with acid-citrate-dextrose (ACD) buffer (39 mM citric acid, 75 mM sodiu...

B cells play a central role in the development of humoral immunity. They initially develop in the bone marrow and enter the blood stream as na&#239;ve B cells, which can migrate into the lymphoid tissues, such as the spleen, lymph nodes, and tonsils, for further development. Upon Ag encounter, some na&#239;ve B cells migrate into lymphoid follicles, where germinal center B cells can differentiate into memory B cells and plasmablasts (PBs)/plasma cells (PCs). While most PBs/PCs egress into the blood stream, a few eventually reside in the bone marrow to undergo terminal differentiation into long-lived PCs1. B cells in circulation are heterogeneous, and at steady state, PBs/PCs are rare in peripheral blood2. As a result of the availability of lineage-specific surface markers, flow cytometry has become a popular method for the identification and characterization of the B-cell subsets in peripheral blood. An extended application of flow cytometry is the addition of a cell sorter function, which permits the separation and isolation of individual subsets of B cells with high purity. Based on the expression of specific surface receptors at different developmental stages, human circulating B cells are generally classified into three main subpopulations: na&#239;ve B cells (CD19+CD27-CD38-), memory B cells (CD19+CD27+CD38-), and PBs/PCs (CD19+CD27+CD38+)3-4 (Figure 1). Na&#239;ve B cells by nature have not encountered Ags. However, they can be differentiated into IgM+CD27+ memory B cells. Although na&#239;ve B cells are homogeneous in expressing B-cell antigen receptor (BCR)-associated molecules (e.g., CD19, CD20 and CD22) they are heterogeneous in their immunoglobulin repertoire5. The majority of CD27+ memory B cells can be differentiated into CD27+/hiCD38+ PBs/PCs6. In addition, memory B cells and PBs/PCs are polyclonal and exhibit developmental and functional heterogeneity4-7. PBs/PCs in circulation are normally short-lived and do not express CD138, but those made to settle down in the bone marrow will terminally differentiate and become long-lived. Terminally differentiated PCs express CD138 and down-regulate CD27 molecules on their surfaces8. Since both PBs and PCs are capable of secreting Abs, in many occasions they are collectively denoted as ASCs. In contrast, neither na&#239;ve B cells nor memory B cells can produce appreciable amounts of Abs9-10. Nevertheless, when isolated, both na&#239;ve and memory B cells can be differentiated into ASCs in 3 - 10 days when placed in the proper culture conditions6, 11-15. In fact, ASCs derived from in vitro differentiation share similar surface expressions of CD27 and CD38 with those directly isolated from peripheral blood6. In addition, the ASCs differentiated in vitro express a low level of surface CD20, similar that of circulating PBs/PCs6. Although the culture-derived ASCs are all short-lived, they can secrete Abs, indicating that they are functionally competent and able to contribute to the humoral immunity.
Both ELISA and ELISpot are by far the most commonly applied methods with which to obtain functional information on the humoral immune response. ELISA is a 96-well plate-based assay, and it is frequently used to measure the titers of serum Ag-specific Abs and other analytes (e.g., cytokines). It is convenient and scalable. ELISA is designed to use a solid-phase enzyme assay to detect the presence of Abs or other substances, such as serum, in a liquid sample16. The readouts from serum ELISAs have been widely used to represent the immune response of the body. A tool necessary for the acquisition of readouts from ELISA assays is a spectrophotometric microplate reader. The reader can determine the optical density (O.D.) of the end products typically resulting from the reaction of horseradish peroxidase (HRP)-conjugated detection Abs and their specific substrates17. With regard to reporting the humoral immune response, serum Ab levels determined by ELISA denote the collective, but not individual, performance of ASCs in the body. In addition, ELISA fails to take into account the participation by memory B cells, which do not secrete Abs.
Like ELISA, ELISpot is a widely used method for detecting and monitoring the immune response in peripheral blood samples17-18. ELISpot is a technique related to a sandwich ELISA. In it, cells are placed into the polyvinylidene difluoride (PVDF) membrane-backed wells of 96-well microplates for a short-term culture. The ELISpot assay is analogous to performing western blotting on a microplate and developing the spots on the PVDF membrane in each well. An automated ELISpot reader system or a stereomicroscope for manual counting is required. The main advantage of ELISpot in detecting an immune response is its superb sensitivity in the quantification of ASCs and cytokine-secreting cells. It reports their functional activities in humoral and cellular immunity, respectively. In the measurement of humoral immune function, serum Ab levels determined by ELISA and the number of ASCs enumerated by ELISpot are often correlated, but the data readouts from these two assays have some differences in functional implications19-20. The main advantage of ELISpot is its sensitivity of method. The level of serum Ab titers as reported by ELISA is presented semi-quantitatively as O.D. readouts, denoting the relative Ab level, or more quantitatively, as concentration readouts when a known amount of the proper isotypes of Abs is included for reference. In contrast, the results of ELISpot are presented as the absolute number of ASCs in a cell pool of interest (e.g., unfractionated peripheral blood mononuclear cells (PBMCs) and purified B cells from PBMCs). ELISpot can detect a single ASC, but ELISA requires Ab amounts from ASCs to reach optimized assay-dependent concentrations prior to measurement. Hence, ELISpot is obviously superior to ELISA in sensitivity of quantification. Moreover, ELISpot is also suitable for quantifying the in vitro differentiated ASCs from activated memory B cells. Memory B cells do not secrete Abs but can differentiate into ASCs upon activation; they therefore have no contribution to serum Abs detected by ELISA. Thus, ELISpot is the method of choice in the measurement of the immune response of circulating memory B cells after activation in culture. It allows for the monitoring of the maintenance of long-term humoral immunity.

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			<td>BD Vacutainer K2E</td>
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			<td>Anti-human CD45-FITC</td>
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			<td>Anti-mouse/rat/human CD27-PerCP Cy5.5</td>
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			<td>Anti-human CD27-PerCP Cy5.5</td>
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			<td>Anti-human CD19-FITC</td>
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			<td>Anti-human CD20-FITC</td>
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			<td>biotinylated anti-human CD27</td>
			<td>Biolegend</td>
			<td>302804</td>
			<td>clone O323</td>
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			<td>biotinylated anti-human CD27</td>
			<td>eBioscience</td>
			<td>13-0279-80</td>
			<td>clone O323</td>
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			<td>7-aminoactinomycin D (7-AAD)</td>
			<td>BD Biosciences</td>
			<td>559925</td>
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			<td>CpG (ODN 2006)&#160;</td>
			<td>InvivoGen</td>
			<td>tlrl-2006</td>
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			<td>Recombinant human IL-2</td>
			<td>PeproTech</td>
			<td>200-02</td>
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			<td>Recombinant human IL-10</td>
			<td>PeproTech</td>
			<td>200-10</td>
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			<td>Recombinant human IL-21</td>
			<td>PeproTech</td>
			<td>200-21</td>
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			<td>Recombinant human sCD40L</td>
			<td>PeproTech</td>
			<td>310-02</td>
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			<td>Protein A of S. aureus Cowan (SAC)</td>
			<td>Sigma-Aldrich</td>
			<td>82526</td>
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			<td>Pokeweed mitogen (PWM)</td>
			<td>Sigma-Aldrich</td>
			<td>L9379</td>
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			<td>MultiScreen filter plates, 0.45 &#181;m pore size</td>
			<td>Merck Millipore</td>
			<td>MSIPS4510</td>
			<td>sterile, clear 96-well filter plate with hydrophobic PVDF membrane</td>
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			<td>BCIP/NBT solution</td>
			<td>Sigma-Aldrich</td>
			<td>B6404</td>
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			<td>BCIP/NBT single reagent, alkaline phosphatase substrate</td>
			<td>Merck Millipore</td>
			<td>ES006</td>
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			<td>Human IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>009-000-003</td>
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			<td>Human IgG, Fc fragment</td>
			<td>Jackson ImmunoResearch</td>
			<td>009-000-008</td>
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			<td>F(ab&#39;)2 fragment of goat anti-human Ig (IgG+IgM+IgA)</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-006-127</td>
			<td></td>
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			<td>Goat anti-human IgG-alkaline phosphatase, Fc&#947; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-055-008</td>
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			<td>Goat anti-human IgM-alkaline phosphatase, Fc&#181; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-055-095</td>
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			<td>Goat anti-human IgG-peroxidase, Fc&#947; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-035-008</td>
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			<td>Goat anti-human IgM-peroxidase, Fc&#181; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-035-095</td>
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			<td>BD ELISPOT AEC substrate kit</td>
			<td>BD Biosciences</td>
			<td>551951</td>
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			<td>C.T.L. ImmunoSpot analyzer</td>
			<td>C.T.L.</td>
			<td></td>
			<td></td>
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the isolation, differentiation, and quantification of human antibody-secreting b cells from blood: elispot as a functional readout of humoral immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

PBMCs were depleted of RBCs and adherent cells (steps 1.2 to 1.7). An aliquot (2 x 106) of cells were subjected to a flow cytometric analysis to illustrate the populations of na&#239;ve B cells, memory B cells, and PBs/PCs in peripheral blood (Figure 1). In this donor&#39;s PBMCs, about 10% of the lymphocytes were CD19+ B cells. In the B-cell compartment, the percentage of CD19+CD27- na&#239;ve B cells was around 50%. On the oth...

Watch this Scientific Journal Video about The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity at JoVE

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

The overall goal of this ELISpot methodology is to allow the quantification of Human Antibody-secreting B cells from the blood. This method can help answer key questions in the bio-medical field about the immunology of B cells and vaccine research. The main advantage of this technique is that it allows the detection and measurement of anti-body secreting B cells at the single cell level.Demonstrating the procedure will be Tsung-Chih Tseng, a graduate student from my laboratory. Immediately after obtaining a 10 ml blood sample from median cubital vein into a 15 ml tube containing potassium supplemented EDTA, invert the tube several times to prevent clot formation. Then add 35 ml of autoclaved red blood cell lysis buffer to the cells for a five minute incubation at room temperature.When light can be observed through the tube, centrifuge the blood and confirm the presence of a white pellet. Remove the residual lysis buffer with 10 ml's of autoclaved PBS and re-suspend the pellet in 10 ml of RPMI 1640 medium. Plate the cells in a 10 cm culture dish for a 30 minute incubation at 37 degrees celsius and five percent carbon dioxide.Then gently swirl the culture dish a few times and transfer the floating cells into a plastic 15 ml conical tube. After two washes by centrifugation, re-suspend the pellet at five to 10 times tenth of the six cells per ml concentration in 200 micro liters of cold PBS buffer and add 5 micro liters of biotinylated anti-human anti-body cocktail specific for blood cells per one times tenth of the sixth cells. After a 30 minute incubation on ice, wash the cells in a 10 fold excess volume of sterile PBS and re-suspend the pellet in five micro liters of streptavidin conjugated micro beads per one times tenth of the six cells for a 30 minute incubation on ice.At the end of the incubation add two ml of PBS buffer to the cells and place the tube onto a magnetic stand for eight minutes at room temperature. When the micro beads have attached to the wall of the tube carefully transfer the supernatant into a new conical tube and add 2 more ml of PBS buffer to the cells. After the second supernatant collection, collect the pooled cells by centrifugation and transfer the cells to a five ml polystyrene tube and fresh PBS buffer at a one times tenth of the seven cells per ml concentration.After preforming non specific blocking, add one microgram of each selection anti-body per one times tenth of the six cells to the tube with gentle mixing for a 30 minute incubation on ice. During the last five minutes of the incubation add five micro liters of 7-AAD to the cells. Then add two milli liters of fresh PBS to the cells and vortex before centrifuging.Re-suspend the pellet in fresh sorting buffer at a one to five times tenth of the seven cells per milli liter concentration and filter the cells through a 40 micron cell strainer into a new five milli liter polystyrene tube. Then sort the cells by flow cytometry into three 15 ml tubes containing 5mls of fresh RPI medium to collect the naive B cells, memory B cells and plasmablast plasma cells. When all of the cells have been harvested, seed the subsets into individual wells of a 12 well cell culture plate at a one to ten times tenth of the fifth cells per milli liter concentration in fresh RPMI medium.Next activate the B cells with five micrograms of CPG ODN 2006 per one times tenth of the sixth cells per milli liter per well in the cell culture incubator for five days. At the end of the activation, add 30 micro liters of 35%ethanol and distilled water to each well of the ELISpot plates for 30 seconds. Then replace the ethanol in each well with 150 micro liters of autoclaved double distilled water and incubate the plates at room temperature for five minutes to remove the residual ethanol.Perform a PBS wash followed by the addition of 50 micro liters of the polyclonal FAB2 fragment of anti-human IG to each well. Wash the plates two times with PBS as just demonstrated. Block the non specific binding in each well with 200 micro liters of fresh PBS with BSA for two hours at room temperature.After another PBS wash series, incubate the wells in 100 micro liters of fresh RPMI 1640 medium at 37 degrees celsius. Then replace the medium in each ELISpot well with the appropriate number of activated B cells. Bring the final volume of each well to 150 micro liters with fresh RPMI medium then return the ELISpot plate to the cell culture incubator for eight to 14 hours.At the end of the incubation period, discard the medium and wash the wells with 200 micro liters of PBS plus tween20 for five, three minute washes. After the last wash, add the appropriate detection anti-bodies to their corresponding wells and incubate the plates for two hours in the dark. After another PBS wash series add 50 micro liters of BCIPMBT substrate to each well for five to 15 minutes at room temperature.When purple spots appear, stop the reaction with 100 micro liters of double distilled water per well and rinse the plate with running tap water. Then air dry the ELISpot membrane's protected from light. In this donor PBMC sample, after red blood cell lysis and adherent cell depletion about 10 percent of the lymphocytes were CD19 positive B cells.In the B cell compartment about 50 percent of the cells were CD27 negative naive B cells and 50 percent were CD27 expressing memory B cells. CPG treatment of purified human B cells and CD19+CD27+for five days as just demonstrated typically results in the generation of 50 to 200 IgM and 10 to 50 IgG anti-body secreting cells from one times ten to the fourth activated B cells. Once mastered, this ELISpot technique can be completed in three hours if it is performed properly.Before beginning this procedure, it is important to remember to code the place with appropriate capture re-agents for detecting the anti-body secreting cells. After its development, this technique paved the way for researchers in the field of vaccinology to explore vaccine efficacy in large scale trials and longitudinal follow ups. After watching this video you should have a good understanding of how to purify human B cells from the blood.Separate naive and memory B cells for their differentiation and perform an ELISpot essay to qualify anti-body secreting cells. Don't forget that working with human blood can be hazardous and that precautions such as wearing gloves should always be taken while performing this procedure.

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