Comparison of Flow-cytometric Antibody Secreting Cell Assay and Mabtech Immunoglobulin ELISpot Assay

Background: The increase of donor-specific Antibodies (DSA) after transplantation could be a more important marker than the level of DSA in pre-transplantation sera. The assessment of sensitized cells that can secrete DSA is needed. We developed an assay for antibody-secreting cells (ASCs) measured with the use of flow cytometry and compared it with the Mabtech immunoglobulin (IgG) ELISpot assay.

Methods: Thirteen patients who were awaiting or received organ transplantation and 15 healthy control subjects were included. All subjects were positive for anti-cytomegalovirus (CMV) IgG. Peripheral blood mononuclear cells (PBMCs) were seeded with CpG 2006 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), 500 ng/mL human CD40 Ligand, and 50 ng/mL interleukin-21 in complete RPMI media. Eight micrograms of CMV pp28 antigen were added to test wells and compared with nonstimulated PBMCs. After 72 hours, analysis with the use of the human IgG ELISpot kit (Mabtech) and flow cytometry with anti-CD19-PE, CD27-PE-Cy7, CD38-APC, IgG-FITC Antibodies was performed.

Results: The flow-cytometric ASC assay was moderately correlated with Mabtech IgG ELISpot assay (r = 0.554; P < .001). The ASCs measured by means of flow cytometry were significantly higher in healthy control subjects compared with patients (P < .001). ASCs measured by means of flow cytometry in CMV antigen-stimulated PBMCs were significantly higher compared with nonstimulated PBMCs (P < .001). The IgG-secreting cells measured by means of Mabtech ELISpot assay was not different between healthy control subjects and patients nor between CMV antigen-stimulated and nonstimulated PBMCs.

Conclusions: Flow-cytometric ASC assay can differentiate ASCs for CMV antigen better than Mabtech IgG ELISpot assay. Flow-cytometric ASC assay might be useful for assessing sensitization status in patients awaiting organ transplantation.