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Enzyme-linked immunospot assay for detecting cells secreting antibodies against human blood group A epitopes
Enzyme-Linked Immunospot Assay for Detecting Cells Secreting Antibodies Against Human Blood Group A Epitopes Y. Tanaka, H. Ohdan, W. Zhou, T. Onoe, H. Hara, D. Tokita, K. Mizunuma, H. Tashiro, and T. Asahara
T
HE USE OF ABO-incompatible donor organs is a possible solution to the shortage of donor organs for transplantation, but antibodies (Abs) against blood group A/B epitopes in sera is a major impediment to its success.1 Despite the significance of these Abs, a method for identifying cells producing anti-A/B Abs has not been established. Since its initial description,2 the enzyme-linked immunospot (ELISPOT) assay has become a popular alternative to the classical hemolytic plaque-forming cell assay to detect specific antibody-secreting cells.3–5 We have used the ELISPOT assay to detect cells secreting anti-A Abs. Because normal Balb/c mice have naturally occurring Abs specific for the human blood group A antigens, we tested the potential of the ELISPOT assay using lymphocytes obtained from various mouse tissues. MATERIALS AND METHODS Serial dilutions of spleen, lymph node, peripheral blood lymphocyte, bone marrow, or peritoneal cavity cell suspension taken from Balb/c mice (Clea Japan Inc., Osaka, Japan) were placed in triplicate wells. Some Balb/c mice were previously immunized with human blood group A erythrocytes (8 ⫻ 105 cells/mouse). Nitrocellulose membranes in a 96- well filtration plate (Millipore, Bedford, Mass, USA) were precoated with 5 g/mL of synthetic GalNAc␣1-3,Ful␣1-2Gal conjugated to BSA (A-BSA) (Dextra Laboratories Ltd, Reading, UK) for incubation at 4°C overnight. Nonspecific binding sites were blocked with 0.4% BSA in Iscove’s modified Dulbecco’s medium (IMDM) (Sigma, St. Louis, Mo, USA). Serial dilutions of cell suspension in IMDM supplemented with 0.4% BSA, 5 g/mL insulin (Sigma), 5 g/mL transferrin (Sigma), 5 ng/mL sodium selenite (Sigma), 50 mol/L 2-mercaptoethanol, and 1 g/mL gentamicin were added to the wells. Plates
Fig 1. ELISPOT assay for determining the frequency of anti-A Ab-producing cells. were incubated at 37°C, 5% CO2, and 99% humidity for 24 hours. After incubation, the membranes were vigorously washed with PBS and PBS-Tween to remove cells. Then bound Abs were detected using horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig)M plus IgG Abs (Southern Biotechnology Associates,
From the Department of Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Supported in part by Grant-in-Aid for Research on Human Genome, Tissue Engineering, and Food Biotechnology, Health Sciences Research Grants, Ministry of Health, Labour and Welfare of Japan. Address reprint requests to Hideki Ohdan, MD, Department of Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Fig 2. ELISPOT detection of anti-A Ab-producing cells. The representive pictures of ELISPOT wells are shown. 106 cells were seeded per well. SPL, spleen; BM, bone marrow; PC, peritoneal cavity; MLN, mesenteric lymph node; and CLN, cervical lymph node. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 555–556 (2003)
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03883-6 555
556 Birmingham, Al, USA) as second Abs, followed by color development with 3-amino-9-ethyl carbazole (Sigma) (Fig 1). After the membranes were washed with tap water and dried, the red spots in each well were counted using a stereomicroscope. The tiny spots without halo formed from Ab output were excluded as false spots caused by cell debris or shed materials.
TANAKA, OHDAN, ZHOU ET AL
this frequency and the serum levels of anti-A Abs. These findings indicate the ability of the ELISPOT assay to quantify anti-A Ab-producing cells.
REFERENCES RESULTS AND DISCUSSION
Anti-A Ab-producing cells were localized exclusively to the spleen and bone marrow of mice (Fig 2). The frequency of anti-A Ab-producing significantly increased in both anatomical sites by sensitization with human blood group A erythrocytes. There was a significant co-correlation between
1. Rydberg L, et al: Transfus Med 11:325, 2001 2. Sedgwick JD, Holt PG: J Immunol Methods 57:301, 1989 3. Lakew M, Nordstrom I, Ceerkinsky C, et al: J Immunol 203:193, 1997 4. Slifka KM, Ahmed R: J Immunol Methods 199:37, 1996 5. Schielen P, van Rodijnen W, Tekstra J, et al: J Immunol Methods 188:33, 1995
T
HE USE OF ABO-incompatible donor organs is a possible solution to the shortage of donor organs for transplantation, but antibodies (Abs) against blood group A/B epitopes in sera is a major impediment to its success.1 Despite the significance of these Abs, a method for identifying cells producing anti-A/B Abs has not been established. Since its initial description,2 the enzyme-linked immunospot (ELISPOT) assay has become a popular alternative to the classical hemolytic plaque-forming cell assay to detect specific antibody-secreting cells.3–5 We have used the ELISPOT assay to detect cells secreting anti-A Abs. Because normal Balb/c mice have naturally occurring Abs specific for the human blood group A antigens, we tested the potential of the ELISPOT assay using lymphocytes obtained from various mouse tissues. MATERIALS AND METHODS Serial dilutions of spleen, lymph node, peripheral blood lymphocyte, bone marrow, or peritoneal cavity cell suspension taken from Balb/c mice (Clea Japan Inc., Osaka, Japan) were placed in triplicate wells. Some Balb/c mice were previously immunized with human blood group A erythrocytes (8 ⫻ 105 cells/mouse). Nitrocellulose membranes in a 96- well filtration plate (Millipore, Bedford, Mass, USA) were precoated with 5 g/mL of synthetic GalNAc␣1-3,Ful␣1-2Gal conjugated to BSA (A-BSA) (Dextra Laboratories Ltd, Reading, UK) for incubation at 4°C overnight. Nonspecific binding sites were blocked with 0.4% BSA in Iscove’s modified Dulbecco’s medium (IMDM) (Sigma, St. Louis, Mo, USA). Serial dilutions of cell suspension in IMDM supplemented with 0.4% BSA, 5 g/mL insulin (Sigma), 5 g/mL transferrin (Sigma), 5 ng/mL sodium selenite (Sigma), 50 mol/L 2-mercaptoethanol, and 1 g/mL gentamicin were added to the wells. Plates
Fig 1. ELISPOT assay for determining the frequency of anti-A Ab-producing cells. were incubated at 37°C, 5% CO2, and 99% humidity for 24 hours. After incubation, the membranes were vigorously washed with PBS and PBS-Tween to remove cells. Then bound Abs were detected using horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig)M plus IgG Abs (Southern Biotechnology Associates,
From the Department of Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Supported in part by Grant-in-Aid for Research on Human Genome, Tissue Engineering, and Food Biotechnology, Health Sciences Research Grants, Ministry of Health, Labour and Welfare of Japan. Address reprint requests to Hideki Ohdan, MD, Department of Surgery, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Fig 2. ELISPOT detection of anti-A Ab-producing cells. The representive pictures of ELISPOT wells are shown. 106 cells were seeded per well. SPL, spleen; BM, bone marrow; PC, peritoneal cavity; MLN, mesenteric lymph node; and CLN, cervical lymph node. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 555–556 (2003)
0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03883-6 555
556 Birmingham, Al, USA) as second Abs, followed by color development with 3-amino-9-ethyl carbazole (Sigma) (Fig 1). After the membranes were washed with tap water and dried, the red spots in each well were counted using a stereomicroscope. The tiny spots without halo formed from Ab output were excluded as false spots caused by cell debris or shed materials.
TANAKA, OHDAN, ZHOU ET AL
this frequency and the serum levels of anti-A Abs. These findings indicate the ability of the ELISPOT assay to quantify anti-A Ab-producing cells.
REFERENCES RESULTS AND DISCUSSION
Anti-A Ab-producing cells were localized exclusively to the spleen and bone marrow of mice (Fig 2). The frequency of anti-A Ab-producing significantly increased in both anatomical sites by sensitization with human blood group A erythrocytes. There was a significant co-correlation between
1. Rydberg L, et al: Transfus Med 11:325, 2001 2. Sedgwick JD, Holt PG: J Immunol Methods 57:301, 1989 3. Lakew M, Nordstrom I, Ceerkinsky C, et al: J Immunol 203:193, 1997 4. Slifka KM, Ahmed R: J Immunol Methods 199:37, 1996 5. Schielen P, van Rodijnen W, Tekstra J, et al: J Immunol Methods 188:33, 1995