PMC4655030_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 color reaction. The original protocol was modified by substituting the human IgG-specific antibody 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.
CRIT2015-763157.001.jpg
PMC4655030_02_CRIT2015-763157.001.jpg
fig1
Because strong reactions of both positive controls (LCRI/II) were demonstrable (Figure 1(d)) there was no doubt about the validity of the negative reaction using the patient's serum.
PMC4655030_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 color reaction. The original protocol was modified by substituting the human IgG-specific antibody 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.
CRIT2015-763157.001.jpg
PMC4655030_02_CRIT2015-763157.001.jpg
fig1
Because strong reactions of both positive controls (LCRI/II) were demonstrable (Figure 1(d)) there was no doubt about the validity of the negative reaction using the patient's serum.
PMC4655030_03