PMC3625552_01
Flow diagram of the AMS-ELISA shown for the detection of HLA class I molecules. (a) Binding of the donor's solubilized HLA class I molecules by monoclonal capture antibodies recognizing a monomorphic epitope on HLA class I molecules. (b) Binding of the donor-specific anti-HLA antibodies out of the recipient's serum to the HLA molecules of the donor. (c) Binding of alkaline phosphatase-conjugated secondary antibodies to the recipient's bound donor-specific anti-HLA class I antibodies and subsequent colour reaction. The original protocol was modified by substituting the human IgG-specific by a human IgG/M/A-specific secondary antibody. (d) Lysate control using an alkaline phosphatase-conjugated monoclonal detection antibody directed against a second monomorphic epitope to confirm the immobilization of a sufficient amount of HLA molecules by the solid-phase-bound capture antibody. The AMS-ELISA variant for the identification of donor-specific antibodies directed against HLA class II molecules is correspondingly designed.
CRIM.TRANSPLANTATION2013-746395.001.jpg
PMC3625552_02_CRIM.TRANSPLANTATION2013-746395.001.jpg
fig1
In accordance with a prior agreement with the transplant centre the obligatory CDC-based pretransplantation crossmatch was performed in parallel to the alternative AMS-crossmatch ELISA (Figure 1) which led to an unequivocally negative outcome.
PMC3625552_02
Flow diagram of the AMS-ELISA shown for the detection of HLA class I molecules. (a) Binding of the donor's solubilized HLA class I molecules by monoclonal capture antibodies recognizing a monomorphic epitope on HLA class I molecules. (b) Binding of the donor-specific anti-HLA antibodies out of the recipient's serum to the HLA molecules of the donor. (c) Binding of alkaline phosphatase-conjugated secondary antibodies to the recipient's bound donor-specific anti-HLA class I antibodies and subsequent colour reaction. The original protocol was modified by substituting the human IgG-specific by a human IgG/M/A-specific secondary antibody. (d) Lysate control using an alkaline phosphatase-conjugated monoclonal detection antibody directed against a second monomorphic epitope to confirm the immobilization of a sufficient amount of HLA molecules by the solid-phase-bound capture antibody. The AMS-ELISA variant for the identification of donor-specific antibodies directed against HLA class II molecules is correspondingly designed.
CRIM.TRANSPLANTATION2013-746395.001.jpg
PMC3625552_02_CRIM.TRANSPLANTATION2013-746395.001.jpg
fig1
In accordance with a prior agreement with the transplant centre the obligatory CDC-based pretransplantation crossmatch was performed in parallel to the alternative AMS-crossmatch ELISA (Figure 1) which led to an unequivocally negative outcome.