#59
HLA SPECIFIC ANTIBODY DETECTED IN SOLID PHASE BUT NOT CELLULAR ASSAYS: LINEAR VS CONFORMATIONAL EPITOPES.
W. Shumway BS 1, L. Wright MT 1 and W.M. LeFor PhD 1. 1 LifeLink Foundation, Inc., Tampa, FL .
When manufacturing solid phase assays using purified HLA, 
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dissociates from some Class-I Ags. Antibody to these denatured HLA molecules can give clear specificity in solid phase assays but do not react to cells. Two patients, AE and DC, with Class-I PRA by EIA and flow beads, show nearly identical reactivity with Class-I single antigen beads (SAB-I). AE reacts to normal cells but DC does not. Cells treated at pH3 for 10 minutes (to dissociate 2 and destabilize Class-I 3D structure) become positive with DC and negative with AE. SAB-I results: both AE 
DC react with B8 37 41 42; additional positives are B18 59 for AE and B44 45 for DC. Review of published Class-I sequences shows B8 37 41 42 44 45 (DC
s pattern) share the sequence around the unique residue aspartic acid (D)-155. This area folds into the 
2 helix and is accessible to direct Ab binding. It is likely that AE reactivity with B8 37 41 42 is to the D-155, 3D epitope, while DC recognizes the linear epitope. This would require that intact B44 45 have conformational changes in the D-155 region relative to other, intact D-155 Ags. Predicted 3D structure provides evidence that this may be true. Residue 116 is on the floor of the peptide binding groove, centered between the two helices, right across from D-155. In B8 41 42, residue 116 is tyrosine and in B37 it is phenylalanine. Position 116 is aspartic acid in B44 and leucine in B45. With an aromatic ring projecting from residue 116 (B8 37 41 42), the binding groove should contain either different types of peptides or significantly different orientations of the same peptides compared to antigens without the aromatic ring (B44 45). As the peptide is in direct contact with the D-155 region, this would explain a D-155 Ab not reacting to B44 45.