Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

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668 | VOL.6 NO.5 | 2011 | nature protocols

IntroDuctIonWe have previously developed two methods for detecting antigen-specific antibody (Ab)-secreting cells (ASCs) using microwell array chips1–4: (i) detecting intracellular Ca2 + mobilization induced by antigen binding to cell-surface B-cell receptors (BCRs) with a Ca2 + -dependent fluorophore and (ii) detecting binding of fluorophore-labeled antigens to BCRs. However, the application of these methods was limited because they could not distinguish target-cell signals from background noise. This background noise hampered the detection of very rare (i.e., 1 in 10,000 cells) target cells. The method presented here can be used to detect molecules secreted from single cells resident in microwells on a chip. In the case of human ASCs, secreted Abs diffuse from the wells, are trapped by human IgG-specific Abs precoated on the chip and are then visual-ized using fluorophore-labeled antigens. The diffused Abs form fluorescent doughnut-like signals that can easily be distinguished from nonspecific signals if the binding of the antigen to the Ab is specific. Here we describe the development of this protocol, the principles behind the method and the application of this method to live single-cell analysis.

Development of the protocolTo achieve high-throughput single-cell separation and analysis, a microwell array chip was developed by us and our collaborators5. This microwell array chip has a regular array of up to 234,000 wells, with a size and shape optimized for the accommodation of a single lymphocyte in each well, enabling us to analyze cells on a single-cell basis. By using this microwell array chip, we have been able to detect antigen-specific B cells using three approaches: (i) monitor-ing intracellular Ca2 + changes induced by an antigen binding to cell-surface BCRs1,3,4, (ii) monitoring the binding of fluorescently labeled antigens to BCRs2 and (iii) monitoring antigen-specific Ab secretion from ASCs6. It is difficult, however, for the first two meth-ods to distinguish specific signals from nonspecific noise because intracellular Ca2 + levels change in response to various stimuli besides antigen binding, and because fluorescently labeled anti-gens tend to bind nonspecifically to the cell surface. Consequently,

when the proportion of antigen-specific B cells is very low, antigen-specific signals tend to be hidden by nonspecific signals. However, we have developed the third method to identify antigen-specific ASCs by detecting antigen-specific Ab secretion. In developing this method, we used the principles of the enzyme-linked immunospot (ELISPOT) assay to detect Ab secretion. Because the ELISPOT assay is an excellent method for monitoring the secretion of various molecules, it has been widely used to analyze the Ab or cytokine secretion from single cells7–10. By combining ELISPOT principles with a cell-based chip containing a regular array of thousands of microchambers containing single cells, we successfully developed a chip-based procedure to detect Abs secreted from ASCs. The Abs secreted from a well diffuse onto the chip surface and bind to IgG-specific Abs coated on the chip surface. Bound antibodies are then visualized using fluorescently labeled antigens, resulting in the formation of a circular, doughnut-like signal on the chip surface around the well (Fig. 1). Specific signals can easily be distinguished from nonspecific noise; therefore, the reliability and specificity of chip analysis are greatly improved. Furthermore, we improved the sensitivity of this method by enhancing the protein-binding proper-ties of the silicon chip surface. In summary, this protocol combines the strengths of ELISPOT and microwell array chips, allowing the identification and recovery of target antigen-specific ASCs from among a large number of lymphocytes.

Comparison with current methods for detecting antigen-specific B cellsOur protocol (the ‘immunospot array assay on a chip’, or ISAAC, method) substantially improves the sensitivity and specificity of detecting antigen-specific B cells compared with our previously devel-oped methods using the microwell array chip. Alternatively, single antigen-specific B cells can be analyzed and separated using a fluores-cence-activated cell sorter, but it is difficult to distinguish antigen- specific signals from nonspecific noise using fluorescence-activated cell sorting, thus leading to difficulties in identifying very rare antigen- specific B cells. Recently, Wrammert et al.11 detected plasmablasts

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot arrayAishun Jin1,2, Tatsuhiko Ozawa1, Kazuto Tajiri3, Tsutomu Obata4, Hiroyuki Kishi1 & Atsushi Muraguchi1

1Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. 2Department of Immunology, College of Basic Medical Science, Harbin Medical University, Harbin, China. 3The Third Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. 4Central Research Institute, Toyama Industrial Technology Center, Toyama, Japan. Correspondence should be addressed to H.K. ([email protected]) or A.J. ([email protected]).

Published online 28 April 2011; doi:10.1038/nprot.2011.322

Here we report a new method for isolating antigen-specific antibody-secreting cells (ascs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ascs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. this protocol can be completed in less than 7 h, including 3 h of cell culture. the method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (elIspot) but it also overcomes the limitations of elIspot in recovering ascs that can be used to produce antigen-specific human monoclonal antibodies. this method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.

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nature protocols | VOL.6 NO.5 | 2011 | 669

screening for antigen-specific ASCs12. Fundamentally, this method is based on ELISA because secreted antibodies that diffuse into the cell culture medium are detected. In this procedure, appropriate cell dilutions for analyzing ASCs at the single-cell level must be prepared because more than 100 cells can be accommo-dated in each well. Furthermore, constant secretion of large amounts of Abs may be required to reach a sufficient, detectable concentration because of the large well volume in this system. Comparatively, our method seems to offer a more rapid, effi-cient and suitable protocol for detecting primary ASCs, although the microengrav-ing method also enables the detection of these cells. The ISAAC method described here is more suitable for detecting antigen-specific cells not only for the analysis of immune responses but also in the develop-ment of therapeutic antibodies.

Applications of the methodThe ISAAC method can be used for multi-ple purposes. First, it can be used to produce candidate human antibodies for therapeutics from human peripheral blood lymphocytes (PBLs). As reported previously6, the ISAAC method enables the isolation of antigen- specific mAbs directly from human B cells within a week, without producing hybri-doma cells or immortalizing B cells with the Epstein-Barr virus. Given that this method enables ASC screening with multiple antigens and selection of ASCs secreting high-affinity antibodies, useful immunotherapeutic agents can easily be screened and developed. In addi-

tion, because ISAAC can detect antigen-specific human ASCs and retrieve them for the production of antigen-specific antibodies in a single day, this method is suitable for the development of a broad range of therapeutic mAbs against viruses—such as HIV, influenza and hepatitis C—that continuously change their surface proteins in individuals with neutralizing antibodies. An efficient in vitro immuni-zation method that induces the differentiation of memory B cells into plasmablasts has recently been developed13. We could apply this in vitro immunization method to detect and retrieve antigen-specific ASCs in ISAAC. Second, the ISAAC method can also be used to detect cytokine-secreting cells. In addition to monitoring B-cell immunity, monitoring T cell effector functions is critical in understanding various diseases and for monitoring the effect of immune therapies. In this context, ISAAC provides a suitable method for analyzing a variety of cytokine secretions from individual T cells. Moreover, cytokine-secreting T cells can be retrieved from the chip for further analyses, such as amplifying the T cell receptor cDNA and analyzing T cell repertoire.

Limitations of the methodThe crucial steps that determine the throughput for detecting and isolating ASCs are the steps involving the preparation of single-cell

using cell-surface marker and randomly isolated plasmablasts using a cell sorter. They obtained influenza-specific IgG from sorted blasts without analyzing antigen specificity. However, we were not able to find such a high responder to influenza vaccination. Therefore, when analyzing the low responders to vaccination, we found it more effec-tive to detect antigen-specific ASCs by analyzing antigen specificity using ISAAC. The ELISPOT assay can specifically detect secreted molecules from cells at the single-cell level. However, target antigen–specific ASCs cannot be retrieved using ELISPOT analysis because the cells are washed away during the detection of secreted antibodies. The ISAAC method allows the retrieval of target cells, which remain in the wells after the detection of secreted antibodies. However, there are some limitations to the ISAAC method compared with ELISPOT. Some cells are lost during the process of arraying the cells in micro-wells in ISAAC, whereas all the cells applied onto wells in ELISPOT can be analyzed. Nevertheless, the advantages of the ISAAC method in allowing not only accurate, high-throughput analysis but also the retrieval of antigen-specific ASCs outweigh its limitations.

Recently, another group reported a microengraving method for the selection of ASCs using an array of wells that were 50 µm (or 100 µm) in diameter and depth, improving the efficiency of

ImmunizedIymphocytesfrom mice orvolunteers(Steps 1–21)

Cells arrayed into a chip(Steps 22–25)

Microarray chip(230,000 wells)

Cells cultured for 3 h(Steps 26,27)

Detection andretrieval of ASCs

(Steps 28–39)

Detection of ASCs

Expanded Beforeretrieval

Afterretrieval

Antigen-specific ASCs formed spotsobserved under a FMS (left of top)

Scanning electron micrograph imagesWide view (left) and single-well view (right)

Cells stained byOregon Green

Figure 1 | A flow chart summarizing the protocol. Lymphocytes are prepared from immunized mice or vaccinated volunteers (Steps 1–21) and then applied to the chip (Steps 22–25). Cells are cultured in wells of a chip whose surface is coated with anti-IgG (Steps 26,27). Antigen-specific IgG-secreting cells are detected on the chip and retrieved using a microcapillary (Steps 28–39). Antigen-specific antibody-secreting cells can be detected and retrieved within 7 h. The protocol requires a microwell array chip and a relatively experienced researcher. Parts of the flow chart were derived from our previously published paper6.

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arrays with lymphocytes on microwell array chips and the cell-retrieval step. The number of cells that can be analyzed in ISAAC is limited by the number of wells on a chip and the number of chips that can be analyzed by a researcher. Because applying cells onto the chip must be done manually, at present it is difficult to analyze more than 5 million cells. Microwell array chips that have a large number of wells (≥106 wells per chip) and an automatic manipula-tion system for ISAAC (cell application, washing and staining steps) should be developed in the future. Currently, cells are retrieved from the well manually using a micromanipulator. It is difficult for unskilled persons to retrieve cells from the chip, although it takes approximately a minute for skilled persons to do. Thus, the cell-retrieval step limits the throughput of ISAAC. In this context, we have a prototypic semi-automatic cell-analysis and cell-retrieval system, which should be improved for practical use of ISAAC.

Concerning the specificity and the sensitivity of the ISAAC method, the spots of secreted antibodies formed in ISAAC are very clear, uniform and easily distinguishable from the nonspecific binding of the fluorescently labeled probe (antigen, in this case). Therefore, if the interaction between the antigen and the Ab is very specific, we can identify very rare cells (even 1 in 10,000 or less). However, the amplification of Ab cDNA is not so easy. Currently, we can amplify Ab cDNA pairs from, on average, 50% to 80% of retrieved single cells. If a more efficient cDNA amplification proce-dure were available, the sensitivity and efficiency of ISAAC would be greatly improved.

Experimental designA flow chart briefly describing all stages of the protocol is shown in Figure 1.

An array of single live cells is prepared by applying ASCs onto a microwell array chip. The chip surface is coated with antibodies against immunoglobulin, and the antibodies secreted by an ASC

are trapped on the surface around the well. Before detecting anti-gen-specific ASCs, it is best to examine whether the antigen can specifically detect antigen-specific Ab using ELISA. When the fluo-rescently labeled antigen is added onto the chip, the antigen binds to specific antibodies and forms distinct circular or doughnut-like spots, which are easily distinguishable from nonspecific signals out-side the wells under a fluorescence microscope. The cells in the spots are considered to be antigen-specific ASCs. As a positive control experiment, we can detect IgG-secreting cells using fluorescently labeled IgG-specific Ab. The analyzed cells are stained on the chip with Oregon Green (Molecular Probes), which penetrates live cells and produces green fluorescence. Thus, antigen-specific ASCs can be analyzed among human PBLs.

To detect ASCs specific for HBs antigen (HBsAg) or influenza viruses, we immunized healthy volunteers6. At 7–10 d after the final boost, PBLs were isolated using the standard Ficoll-Hypaque method with Lymphosepal (IBL). We purified plasmablasts from human CD138-specific mAb-conjugated microbeads using mag-netic-activated cell sorting (MACS). Antigen-specific ASCs were then detected among the CD138-positive cells. To this end, we first arrayed live CD138 + cells containing ASCs onto a chip coated with human IgG-specific antibodies. The cells were cultured on the chip for 3 h at 37 °C, and secreted antibodies were captured and bound to the human IgG-specific antibodies on the chip surface. Biotinylated antigen was then added to the chip, followed by Cy3-conjugated streptavidin (Sigma-Aldrich). Finally, the cells were stained with Oregon Green. Antigen-specific antibodies released from single cells can be observed using a fluorescence microscope or cell scanner. A cell scanner can also be used to calculate the number of cells on the chip. For subsequent analysis of ASCs, antigen-specific ASCs can be retrieved from individual wells using a micromanipulator fitted with capillaries under a fluorescence microscope (Fig. 1). The retrieved cell can be used to amplify Ab cDNA.

MaterIalsREAGENTS

Lymphosepal I (Immuno-Biological Laboratories, cat. no. 23010)CD138 MicroBeads (human; Miltenyi Biotec, cat. no. 130-051-301)AutoMACS running buffer (Miltenyi Biotec, cat. no. 130-091-221)RPMI 1640 (Invitrogen, cat. no. 31800-089)FBS (Biowest, cat. no. S1820)Penicillin (Meiji Seika)Streptomycin (Meiji Seika)Biotin-AC

5 sulfo-OSc (Dojindo, cat. no. 348-06811)

Biolipidure (NOF Corporation, cat. no. BL-203) crItIcal This reagent can rapidly and completely block the nonspecific binding of secreted antibodies or antigen to the chip surface.Recombinant hepatitis B surface antigen (recombinant HBsAg; Kaketsuken) Inactivated influenza antigens (A/NewCaledonia/20/99(H1N1); HyTest, cat. no. 8IN73-3)Inactivated influenza antigens (A/Panama/2007/99 (H3N2); HyTest, cat. no. 8IN74-2)Inactivated influenza antigens (B/Tokio/53/99; HyTest, cat. no. 8IN75-2)Donkey affinity-purified Ab to goat IgG (MP Biomedicals LLC, cat. no. 55071)Goat affinity-purified Ab to human IgG Fc (MP Biomedicals LLC, cat. no. 55071)Cy3-conjugated streptavidin (Sigma-Aldrich, cat. no. S6402)Cy3-conjugated anti-human IgG (Fc) (Sigma-Aldrich, cat. no. C2571)Oregon Green (Invitrogen, cat. no. O6809)

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EQUIPMENTAutoMACS Pro Separator (Miltenyi Biotec, cat. no. 130-092-545)Hemocytometer (Erma, Burker-Turk line)Microwell array chip (10-µm, 90,000 wells; SC World; this chip is not freely available. Please contact SC World; see Fig. 1)Pipetman P200 (Gilson, cat. no. F123601)Cell scanner (SC@Scanner, SC World)DecompressorHumidified dark box (handmade)Kimwipes (Kimberly-Clark, cat. no. S-200)Humidified tissue culture incubator (37 °C, 5% CO

2)

Variable-speed centrifuge with a fixed-angle rotorFluorescence microscope (Olympus, cat. no. BX51WI)Micromanipulator (Eppendorf, TransferMan NK2, cat. no. 5188 000.047)Microaspirator (Eppendorf, CellTram Vario, cat. no. 5176 000.033)Capillaries (12-µm; Primetech, cat. no. PINS12-00BT45)

REAGENT SETUPOregon Green Dilute 1 µl of Oregon Green (1 mM stock, kept at − 20 °C) in 1 ml of PBS before use. It should be freshly prepared.Biolipidure Dilute 5% (wt/vol) Biolipidure (stock) to 0.01% (wt/vol) in PBS before use. It should be freshly prepared.Culture medium RPMI 1640 supplemented with 10% (vol/vol) FBS, 100 U ml − 1 penicillin and 100 µg ml − 1 streptomycin. It can be stored at 4 oC for 2–3 months.

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proceDurelymphoprep and cD138 + cell enrichment ● tIMInG 2 h1| Collect heparinized peripheral blood into syringes at 7 d post-vaccination. Divide 5 ml of whole blood into 15-ml tubes (50 ml blood total). ! cautIon Human blood should be considered infectious and appropriate safety precautions should be taken. Studies using human subjects should be approved by the appropriate institutional committees.

2| Dilute the blood with an equal volume of PBS.

3| Carefully layer the diluted blood over the Lymphosepal by adding 4 ml of Lymphosepal to the bottom of the tube containing diluted whole blood through a Pasteur pipette inserted into the blood. This is done by first inserting a Pasteur pipette into the diluted whole blood; next, from the top of the pipette, 4 ml of Lymphosepal is added to the bottom of the tube through the pipette.

4| Centrifuge for 30 min at 800g at room temperature (20–25 °C) with the brake off.

5| After centrifugation, the enriched PBLs will form a band at the interface between the plasma and the Lymphosepal. Collect the cells in this band with a Pasteur pipette and transfer them to a new 50-ml centrifuge tube.

6| Rinse the enriched PBLs by diluting them to a volume of 50 ml with PBS. Centrifuge the diluted PBLs for 5–10 min at 800g at room temperature.

7| Remove the supernatant and discard without disturbing the cell pellet.

8| If more than one tube is used, combine the cells by suspending them in a small volume (~1 ml) of PBS and transferring them into one 15-ml centrifuge tube.

9| Add PBS to make up the volume to 10 ml and then count the cells with a hemocytometer.

10| Centrifuge the cell suspension at 360g for 5–10 min at room temperature. Afterward, the brake may be used.

11| Remove the supernatant and discard without disturbing the cell pellet.

12| Resuspend approximately 5 × 107 cells in 450 µl of autoMACS running buffer and add 50 µl of anti-human CD138- conjugated microbeads.

13| Incubate the cells for 15 min at 4 °C.

14| To wash the cells, dilute the cells with autoMACS running buffer to a volume of 10 ml and centrifuge the cell suspension at 360g for 5 min at room temperature. Afterward, apply the brake.

15| Remove the supernatant and discard without disturbing the cell pellet. Repeat Step 14.

16| Remove the supernatant and discard without disturbing the cell pellet, and then resuspend the cells in 500 µl of autoMACS running buffer.

17| Separate CD138 + cells using an autoMACS Pro Separator according to the manufacturer’s instructions, as detailed in Box 1.

18| Count the cells with a hemocytometer.

19| To wash CD138 + cells, dilute the CD138 + cells with PBS to a volume of 10 ml and centrifuge the cell suspension at 360g for 5 min at room temperature. Afterward, apply the brake.

20| Remove the supernatant and discard without disturbing the cell pellet. Repeat Step 19.

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21| Remove the supernatant and discard without disturbing the cell pellet, and then resuspend the cells in the culture medium to prepare the cell array. crItIcal step The volume of the culture medium should be adjusted so that the cell concentration is from 105 to 5 × 105 cells per 100 µl.

Detection and retrieval of antigen-specific ascs ● tIMInG 6 h22| Coat the chip with 100 µl of 10 µg ml − 1 donkey anti-goat IgG in PBS at room temperature for 1 h in a humidified atmosphere. This step can be done up to 2 h before the experiments. pause poInt Coated chips may be stored at 4 °C for 1 week in a humidified atmosphere. crItIcal step Incubate the chip in a humidified atmosphere. All subsequent incubation steps are performed in the same manner to prevent the chip surface from becoming dry. The buffers or cell suspensions are added using a Pipetman P200 in the following steps.

23| After removing the Ab solution, add 100 µl of 0.01% (wt/vol) Biolipidure in PBS to the chip, and remove the air from the wells by briefly placing the chip under vacuum to allow the solution to enter the wells. Incubate the chip for 15 min at room temperature to block the remaining binding sites. crItIcal step This step is best performed immediately before arraying the cells.

24| After washing the chip three times with culture medium, array the cells (100 µl) suspended in culture medium in Step 21 on the surface of the chip at a density of 1–5 × 103 cells per µl. Allow the cells to settle into the wells under gravity for 10 min. ! cautIon Washing the chip entails gently removing or adding buffer (100 µl) to the surface of the chip using a Pipetman P200.

25| Remove residual cells outside the wells by gently washing with culture medium several times. ! cautIon After adding cells onto the chip, the washing steps should be gentle. Do not add the buffer directly onto the cells. We usually aspirate the buffer with a Kimwipe and then add the buffer (100 µl) with a Pipetman P200 from outside the well area. Repeat the process of aspirating and adding buffer four or five times.

26| Add 100 µl of 10 µg ml − 1 goat anti-human IgG (Fc) in culture medium to the chip containing cells to be analyzed. Incubate the chip for 40 min at room temperature and then wash the chip gently with culture medium four times. crItIcal step We add goat anti-human IgG after adding CD138 + cells onto the chip because a fraction of the CD138 + cell preparation contains cell-surface IgG-positive cells that adhere to the chip surface if anti-human IgG is coated on the chip before adding the cells.

Box 1 | CELL SEPARATIoN USING THE autoMACS Pro Separator priming the autoMacs pro separator1. Switch on the autoMACS Pro Separator and wait for the instrument to complete initialization.2. After initialization, verify the status of the instrument.3. Ensure that the symbols for the fluid containers are green.4. Ensure that the symbol for the columns is green.5. Ensure that the MACS MiniSampler is installed correctly.6. The autoMACS Pro Separator is ready for priming. Select Separation and Wash only from the lower navigation bar.7. Select Rinse and Run.cell separation1. Select a Possel separation strategy and a Sleep wash protocol in the separation program.2. Label two empty 15-ml tubes as ‘positive’ and ‘negative’.3. Place the tube containing the labeled cells at position A.4. Place the ‘negative’ tube for recovering CD138 − cells at position B and the ‘positive’ tube for recovering CD138 + cells at position C.5. Select Run to start.6. CD138 − cells are separated into the ‘negative’ tube at position B and CD138 + cells are separated into the ‘positive’ tube at position C.7. After the cell separation, autoMACS Pro Separator automatically performs the Sleep wash program.8. Upon completion of the Sleep program, switch off the autoMACS Pro Separator with the main power switch.

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27| Incubate the cells for 3 h at 37 °C in a humidified atmosphere containing 5% CO2. Secreted antibodies will be bound to anti-IgG on the chip surface during this incubation.

28| Wash the chip four times with culture medium.

29| Add 100 µl of 2 µg ml − 1 biotinylated antigen to the chip and incubate the chip for 30 min at room temperature.

30| Wash away the biotinylated antigen with PBS and add 100 µl of Cy3-conjugated streptavidin to the chip. Incubate the chip for 30 min at room temperature.

31| Wash the chip four times with PBS.

32| Observe the spots on the chip under a fluorescence microscope. Secreted antibodies from a single well form a distinct circular or doughnut-like signal on the chip surface around the well.? trouBlesHootInG

33| Finally, stain the cells by adding 1 µM Oregon Green (100 µl) to the chip for 3 min at room temperature.

34| Wash the chip four times with PBS and observe the cells with a fluorescence microscope on the chip.? trouBlesHootInG

35| (Optional) Calculate the number of cells on the chip using a cell scanner. This step can be omitted unless it is necessary for the analysis of ASCs.

cell retrieval ● tIMInG 1 min36| Observe the target cells with an orange circular signal under a fluorescence microscope (capillary and micromanipulator is at ‘ejection position,’ as in Fig. 2, panel c). ! cautIon When a cell produces a large amount of antibodies, the fluorescent signal will extend to cover several wells. When this occurred, we took the cell in the well at the center of the circular signals to be an ASC. Retrieving irrelevant cells can affect the efficiency of cell analysis. ! cautIon Be careful not to puncture fingers or eyes with the capillary. Wear glasses to protect eyes.

37| Bring the capillary and the micromanipulator to ‘retrieval position,’ as in Figure 2b. Adjust the tip of the capillary to the position of the target cell under a microscope using a Cell TransferMan NK2 (Fig. 2a). Aspirate cells with a microaspirator (CellTram Vario).? trouBlesHootInG

38| Bring the capillary to the ‘ejection position,’ and eject cells from the capillary by forming a tiny drop (~0.2 µl) of buffer.

39| Transfer a drop containing a cell to the RT-PCR buffer (Fig. 2d).! cautIon Be careful not to puncture your fingers or eyes with the capillary. Wear glasses to protect eyes.! cautIon Tubes containing RT-PCR buffer should be kept on ice.

40| Tubes containing single cells may be stored for months if they are immediately flash frozen at − 80 °C after collection. Retrieved cells can be used for subsequent experiments as required.

? trouBlesHootInGTroubleshooting advice can be found in table 1.

a b

c d

Capillary

Chip

Figure 2 | Cell retrieval using a micromanipulator. (a) Position of capillary for cell retrieval. (b) Position of capillary and micromanipulator for cell retrieval (retrieval position). (c) Position of capillary and micromanipulator for cell ejection (ejection position). (d) Transfer of a cell into a PCR tube.

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● tIMInGSteps 1–21, Lymphoprep and CD138 + cell enrichment: ~2 hSteps 22–35, Detection of antigen-specific ASCs: ~6 hSteps 36–40, Cell retrieval: ~1 min per cell (for skilled persons)

antIcIpateD resultsThis protocol has a wide range of applications, similar to ELISPOT, for the analysis of single cells. In addition, it can retrieve cells of interest for the development of candidate immunotherapeutic agents. In a separate report, we described HBsAg- specific and influenza virus-specific mAbs obtained using the ISAAC method6. To detect ASCs specific to HBsAg, we used human IgG (Fc)-specific Abs as a coating reagent to capture secreted Abs and then detected antigen-specific Abs using biotinylated HBsAg, followed by Cy3-conjugated streptavidin. For influenza virus-specific mAbs, we used a mixture of inactivated influenza viruses (H1N1, H3N2 and type B) as coating antigens to capture secreted Abs. As was the case with HBsAg, we successfully detected ASCs for influenza viruses using the ISAAC method. In the influenza experiments, we occasionally observed nonspecific binding of CD138 + cells to the chip surface coated with influenza viruses, which may have affected the results of our cell analysis. Nonspecific cell adhesion tends to occur when the concentration of the coating antigen is too high. This might be due to the binding of influenza virus hemagglutinin to sialic acid receptors on human B cells14. Although there are some limitations to the ISAAC method, it still shows very high efficiency in the detection and retrieval of antigen-specific ASCs.

We confirmed that the ISAAC method could detect antigen-specific ASCs among primary lymphocytes at the single-cell level with high efficiency and sensitivity (table 2 and Fig. 3). To verify the specificity of this method, we amplified Ab VH and VL cDNAs from the retrieved cells using single-cell-based 5′-RACE15. We amplified 17 pairs of IgH and IgL chains from 22 retrieved cells (77%). We inserted these pairs of cDNAs into expression vectors and transfected each pair into HEK 293T cells or Chinese hamster ovary cells. As shown in table 2, most of the pairs (16 pairs) of IgH/IgL cDNAs produced detectable amounts of IgG, and ten pairs produced antigen-specific IgG when analyzed by ELISA (Fig. 3). In our experiments, if Ab cDNA could be successfully amplified from single cells, 60–80% of the Ab cDNA pairs produced antigen-specific anti-bodies, demonstrating that our method can detect antigen-specific ASCs with high specificity. Notably, all the procedures to detect antigen-specific ASCs can be completed in < 1 d. When we compared the number of spots in ISAAC with that of the ELISPOT assay, we found a comparable number of antigen-specific ASCs detected in ISAAC and ELISPOT (table 3), although we detected more spots of antigen-specific ASCs in ISAAC compared with ELISPOT.

taBle 1 | Troubleshooting table.

step problem possible reason solution

32 No doughnut-like signals No cells remained in wells Wash the chip more gently (Steps 25–32)

Inadequate amount of IgG-specific Ab (or antigen; Steps 22, 26)

Use a higher concentration of IgG-specific Abs to coat the chip

Too few antigen-specific ASCs Try to use lymphocytes ~1 week after vaccination. Timing should be determined in each case

34 Low number of cells on the chip

Inadequate air removal from wells (Step 23)

Try the air removal step several times

Low number of cells applied to the chip (Step 24)

Add more cells to the chip

The wash step was too strong (Steps 25–34)

Wash the chip more gently

37 Retrieve unwanted cells Adjustment of pressure of CellTram Vario is not appropriate

Try to adjust the pressure so as to not aspirate cells when a capillary is approaching the target cells (difficult)

Position of the capillary is not appropriate

Adjust the position of the capillary as it is brought near the target cell in retrieval position

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acknoWleDGMents We thank S. Hirota for technical assistance and K. Hata for secretarial work. We are very grateful to M. Isobe and N. Kurosawa for their helpful discussions. This research was supported by grants from the Hokuriku Innovation Cluster for Health Science Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

autHor contrIButIons H.K. conceived of the ISAAC method. H.K. and A.M. supervised the project. A.J. designed and performed experiments. T. Obata developed the chip and A.J. tested it. A.J., T. Ozawa and K.T. tested and showed the utility of the protocol.

coMpetInG FInancIal Interests The authors declare competing financial interests (see the HTML version of this article for details).

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2. Tajiri, K. et al. Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity. Cytometry A 71, 961–967 (2007).

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taBle 2 | Identification of TRAIL-R1-specific IgG with ISAAC.

cell / paira IgG (ng ml − 1)b Binding to agc

401 200

402 –

403 –

404 150

405 –

406 50 ×

407 50

408 150

409 250 ×

410 250 ×

411 150

412 250

413 –

414 –

415 250 ×

416 250

417 250

418 250 ×

419 250

420 ND

421 250 ×

422 250

Abbreviation: ND, not detected.a, both Igγ and Igκ were amplified; –, either Igγ or Igκ alone was amplified, or neither was amplified. b293T cells were transfected with a cDNA pair and the IgG production in the supernatant was measured using ELISA. cAntigen specificity of the produced IgGs was examined using ELISA. , bound to antigen; ×, did not bind to antigen.

OD

(40

5 nm

)

TR1-40

1

TR1-40

4

TR1-40

7

TR1-41

2

TR1-41

6

TR1-41

7

TR1-41

9

TR1-42

2

TR1-43

8

TR1-27

2PC

1.2

1.0

0.8

TRAIL-R1Uncoated

0.6

0.4

0.2

0

Figure 3 | Identification of antigen-specific ASCs using ISAAC. TransChromo Mice that have an artificial chromosome containing human Igγ and human Igκ genes were immunized with a recombinant human TRAIL-R1/Fc fusion protein, and TRAIL-R1 (TR1)-specific ASCs in CD138 + splenocytes were detected using ISAAC. Antibody cDNAs were recovered from individual cells and used for antibody production (table 3). The antigen specificity of the produced antibodies was analyzed using ELISA. x axis, antibodies analyzed; y axis, optical density (OD). PC, serum from a TRAIL-R1-immunized mouse; closed columns, optical densities from TRAIL-R1-coated wells; open columns, optical densities from uncoated wells. Error bars indicate s.e.m. (n = 3). The Committee on Animal Experiments at the University of Toyama approved the protocols for the animal experiments. The results were derived, with permission, from our published paper16.

taBle 3 | Comparison of ELISPOT and ISAAC.

ag elIspot (per 105 cells) Isaac (per 105 cells)

gp120 + gp41 38 40

HA 10 30Antigen-specific ASCs in the same samples were enumerated in ELISPOT and ISAAC.

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10. Czerkinsky, C. et al. Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J. Immunol. Methods 110, 29–36 (1988).

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13. Matsumoto, S.E. et al. In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells. Cytotechnology 52, 227–233 (2006).

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15. Ozawa, T., Kishi, H. & Muraguchi, A. Amplification and analysis of cDNA generated from a single cell by 5′-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells. Biotechniques 40, 469–470, 472, 474 passim (2006).

16. Jin, A. et al. Generation of TRAIL-receptor 1-specific human monoclonal Ab by a combination of immunospot array assay on a chip and human Ab-producing mice. Eur. J. Immunol. 40, 3591–3593 (2010).

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