A Novel ELISPOT Assay to Quantify HLA-Specific B Cells in HLA-Immunized Individuals

Sebastiaan Heidt*, D L Roelen, Y J H de Vaal, M G D Kester, C Eijsink, S Thomas, N M van Besouw, H D Volk, W Weimar, F H J Claas, A Mulder

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

62 Citations (Scopus)


Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B-cell hybridomas producing HLA antibodies. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non-HLA-immunized individuals and transplant patients with serum HLA antibodies. B-cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA-immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while nonimmunized individuals had none. Patients who were immunized by an HLA-A2-mismatched graft had up to 143 HLA-A2-specific B cells per million total B cells. In conclusion, we have developed and validated a highly specific and sensitive HLA-specific B-cell ELISPOT assay, which needs further validation in a larger series of transplant patients. This technique constitutes a new tool for quantifying humoral immune responses.

Original languageEnglish
Pages (from-to)1469-1478
Number of pages10
JournalAmerican Journal of Transplantation
Issue number6
Publication statusPublished - Jun 2012

Bibliographical note

© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

Research programs

  • EMC MM-04-39-05


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