Table 1.
HLA-DR immunoaffinity enrichment methods comparison.
| Donor | No. of DC (x 106) | Format | Bead Amt (mg) | Ab Clone | Ab Amt (µg) | Target Conjugation Ratio | No. of HC Peptides | No. of LC Peptides | No. of Total Peptides | mAb/Total Peptides (%) | Ab-Bead Chemistry | Comments on Experience | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1^ | A | 1 | Pierce NHS | 3 | G46-6 | 150 | NA | ND | ND | ND | ND | NHS | -insufficient sensitivity |
| 2# | A | 1 | FG | 1.6 | G46-6 | 50 | NA | 1 | 0 | 15,826 | 0.0063 | NHS | -difficult to acquire beads -beads challenging to use |
| 3 | B | 2 | Dynabeads M-280 | 3 | G46-6 | 50 | 20:1 | ND | ND | ND | ND | Biotin-Streptavidin | -insufficient sensitivity |
| 4 | B | 2 | Sera Mag | 1 | G46-6 | 50 | 20:1 | 3 | 8 | 25,725 | 0.042 | Biotin-Streptavidin | |
| 5 | A | 2 | Sera Mag | 2 | G46-6 | 75 | 20:1 | 8 | 3 | 12,416 | 0.089 | Biotin-Streptavidin | -easily adapted for automation on the KingFisher -reagents are easier to acquire -reagents arrive in intended condition |
| 6 | A | 2 | Sera Mag | 4 | G46-6 | 150 | 20:1 | 5 | 2 | 12,150 | 0.058 | Biotin-Streptavidin | |
| 7 | C | 2 | Sera Mag | 2 | G46-6 | 100 | 10:1 | 12 | 8 | 29,428 | 0.068 | Biotin-Streptavidin | -G46-6 and GNE generated L243 are comparable |
| 8* | C | 2 | Sera Mag | 2 | L243 (GNE) | 100 | 10:1 | 13 | 6 | 28,803 | 0.066 | Biotin-Streptavidin | -automated, consistent biotin incorporation, optimized bead, antibody, & DC amount, consistent peptide & cluster identification |
| 9 | C | 2 | Sera Mag | 2 | L243 (GNE) | 100 | 20:1 | 14 | 5 | 29,050 | 0.065 | Biotin-Streptavidin |
^ Manufacturer recommendations # Published method (13) * Current optimized conditions PSM, Peptide Spectral Matches. NA, Not Applicable; ND, Not Detected.
The immunoaffinity enrichment of HLA-DR bound peptides was compared using different beads and resin with varying chemistries and sizes. Format number 8 is the optimum automated condition with consistent biotin incorporation, optimized streptavidin bead, capture antibody, and DCs results in consistent biotherapeutic peptide and cluster identification.