A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood

Transcription

1 Supplementary information A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood Aishun Jin 1,2,6, Tatsuhiko Ozawa 1,6, Kazuto Tajiri 1,3,6, Tsutomu Obata 4, Sachiko Kondo 1, Koshi Kinoshita 1, Shinichi Kadowaki 1, Kazuo Takahashi 5, Toshiro Sugiyama 3, Hiroyuki Kishi 1 and Atsushi Muraguchi 1 1 Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama SC World Inc., 529 Takata, Toyama , Japan. 3 The Third Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama , Japan. 4 Central Research Institute, Toyama Industrial Technology Center, 150 Futagami-machi, Takaoka, Toyama , Japan. 5 Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Nakamichi, Higashinari-ku, Osaka , Japan. 6 These authors contributed equally to this work. Correspondence should be addressed to H. K. (immkishi@med.u-toyama.ac.jp).

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5 Supplementary Table 1. Antigen-specific antibodies generated by ISAAC in mouse spleen and human peripheral blood Sample source HEL-immunized mouse spleen a HBs-antigenvaccinated human peripheral blood b Antigen HEL HBs-antigen Influenzavaccinated human peripheral blood b Inactivated influenza viruses Analyzed cells 10, ,238 35,178 Spot-forming cells Retrieved cells Obtained V H +V L cdnas (pairs) Antibody expression Antigen-specific antibody a Average data of three independent experiments. b A representative data with similar results of at least three volunteers.

6 Supplementary methods Coating antibodies on a chip. We coated catching antibodies on the surface of the chip but not inside the wells. This was achieved by adding the catching antibody solution onto the well area. Because the wells are so small, the air in the wells remained and the antibody solution did not enter the wells. We usually used 10 μg ml 1 antibodies for coating them maximally on the chip. Blocking a chip. After coating the chip with the first antibody, we added the blocking solution onto the chip. We then removed the air from the wells by briefly putting the chip under a vacuum to cause the solution to enter the well. After 15 min incubation at room temperature, we washed the chip with PBS and used it in the following experiment. Deposition of cells in microwells. The cell suspension was applied onto the chip and the chip was allowed to remain still for about 10 min. During this time, the cells were spontaneously deposited into the wells with gravity. The cell suspension was removed from the chip and the chip's surface was washed gently with PBS to remove the cells outside the wells. The cells inside the wells did not move out of the wells with the gentle washing. Culture duration for antibody secretion. With regard to the relationship between the number of ISAAC signals (spots) and incubation time, the number of antigen-specific antibody-secreting cells (ASCs) that was detected with ISAAC increased with an

7 increase in incubation time for the first 3 h. Overnight incubation and 3 h incubation did not make significant difference in the number of spots (data not shown). Since overnight incubation results in stronger background signals, it is not practical to prolong cell incubation time beyond 3 h. Identification of an ASC in a spot. Secreted antibodies from a single well formed a distinct circular signal on the chip surface around the well. We regarded irregular fluorescent signals as noise. When a cell produced a large amount of antibodies, fluorescent signals extended to cover several wells. In that case, we regarded the cell in the well at the center of the circular signals as an ASC. The fluorescence intensity was the maximum around the well of the ASC. Detection of cells secreting antibodies with high affinities. Either CHO cells secreting HB0116 ( = M), HB1089 ( = M), HB4-22 ( = M), HB4-44 ( = M), HB4-53 ( = M), or HB3-68 ( = M) antibodies were cultured on human IgG-specific antibody-coated chips. The secreted antibodies were detected with FITC-antibody to human IgG. Binding of HBs-antigen to the secreted antibodies was detected with biotinylated HBs-antigen/Cy3-streptavidin. Fluorescence intensities of FITC and Cy3 of ISAAC spots for each type of antibodies were measured with ImageJ software (NIH). The following shows the theoretical assumption for detecting cells secreting antibodies with high affinity with ISAAC. Ag + Ab AgAb [Ag]T = [Ag]+ [AgAb] (1)

8 [Ab]T = [Ab]+ [AgAb] (2) If we assume that the amount of antigen that bound to the secreted antibody on the chip is negligible compared to the applied antigen, [Ag]T >> [AgAb]. Therefore, [Ag] [Ag]T. = [Ag][Ab] [AgAb] [AgAb] [Ab]T [Ag]T +[Ag]T When [Ag]T =, [AgAb] [Ab]T [Ag]T +[Ag]T = + = 1 2 When [Ag]T >> KD, [AgAb] [Ab]T [Ag]T +[Ag]T [Ag]T =1 [Ag]T That is, when we use 2 μg ml 1 HBs-antigen, which is equal to about 0.1 μm (10 7 M), for antibody of =10 6 M, [AgAb] [Ab]T for antibody of =10 7 M, [AgAb] 10 7 [Ab]T = for antibody of =10 8 M, [AgAb] [Ab]T = = [Ab] T corresponds to the amount of secreted antibody that was detected with FITC-antibody to IgG; [AgAb] corresponds to the amount of antigen bound to the secreted antibody on a chip, which was detected with Cy3-antigen. Consequently, we may be able to distinguish antibodies with higher affinity ( 10 8 M) and those with lower affinity.

9 Analysis of hepatitis B virus-neutralizing activity. Hepatitis B virus-neutralizing activity was investigated using HepaRG cells (supplied from BIOPREDIC) copies of hepatitis B virus and 1 μg of each mab were preincubated together for 1 h, and then added to HepaRG cells in William s E medium supplemented with 10% FCS, 100 units ml 1 penicillin, 100 μg ml 1 streptomycin, 5 μg ml 1 insulin, M hydrocortisone hemisuccinate, 2% DMSO, and 4% PEG8000. After incubation overnight, the HepaRG cells were gently washed and then cultured with fresh medium. On day 6 after the infection, the culture supernatant was harvested and HBs-antigen in the supernatant was quantified using Chemiluminescent Immunoassay. Influenza-neutralization test. A neutralization test for influenza virus was performed according to the protocol described by Okuno et al. with slight modification 2. Briefly, 60 μl serially diluted test antibodies and 60 μl influenza virus (480 focus-forming units) were mixed and incubated for 30 min at 37 C. After incubation, 50 μl of each mixture was transferred to wells containing MDCK cell monolayer in 96-well flat-bottom plates and adsorbed for 30 min at 37 C. The virus inocula were removed and washed with PBS. The cells were cultured for an additional 16 h with MEM supplemented with 2% FCS. The medium was removed, and the cells were washed with PBS, fixed with absolute ethanol for 10 min, and dried. Focus staining was done by successive treatment of the cells with mouse mabs to nuclear protein of either influenza A or B, and with peroxidase-conjugates of mouse IgG-specific antibody. After washing, the peroxidase reaction was developed with 3, 3'-diaminobenzidine tetrahydrochloride in the presence

10 of 0.01% H 2 O 2. The cells were then rinsed with tap water and dried. The stained foci were counted under a light microscope. The neutralizing antibody titer was expressed as the lowest concentration that reduced the number of foci to 50% or less of the control value. Supplementary References 1. Gripon, P., et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99, (2002). 2. Okuno, Y., et al. Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system. Journal of clinical microbiology 28, (1990).