– Research Technologist III, Northwestern University, United States
Aim: While single antigen bead (SAB) assays provide insight into the patients’ HLA antibody profile, they do not allow identification of the specific epitopes targeted within the polyclonal serum. This study aimed to optimize Adsorption Elution (Ads/Elu) assays.
Methods: Serum samples from highly sensitized patients were selected based on high levels of HLA-DQ antibodies. Antibody strength was evaluated prior to Ads/Elu using 4-fold serial dilutions. Multiple steps of the Ads/Elu assay were optimized including HLA antigen targets: attached to microparticle beads (250,000 to 1x10^6) compared to homozygous B-LCLs (1x10^6 to 5x10^6), time of incubation for adsorption and elution steps (3-30 mins), multiple sources of eluate solution, number of washes at each step, and importantly the ratio between HLA antibodies (based on baseline titer) and the HLA antigen targets.
Results: While in some cases results obtained using beads or cells as source of HLA antigen targets yielded similar results, this was not always the case. Notably, we observed that in many cases neither antigen source succeeded in demonstrating complete adsorption of the corresponding antibody. Increasing the number of targets was somewhat successful in mitigating this issue, but a sufficient resolution was not achieved. We therefore hypothesized that HLA-DQ molecules serving as targets for antibodies were saturated and proceeded to perform Ads/Elu using multiple dilutions of the serum sample to a range in which antibodies were not above saturation. This approach proved to achieve complete adsorption. In fact, we observed a correlation between baseline antibody titer and the dilution required to obtain successful complete adsorption. We further tested the other variables, as listed in the methods section, to further optimize the assay, which yielded minor revisions to the baseline protocol.
Conclusion: Optimizing Ads/Elu is a critical step in the ability to identify individual targets within a polyclonal serum. The results of this study found that cells are the best source of targets for adsorption. They maintain physiologically relevant conformation and are easily replenishable. We further demonstrated that it is vital to dilute the serum to a range in which a fixed quantity of cells can achieve complete adsorption, allowing for results that can be compared between assays. This approach creates opportunities to investigate polyclonal sera with reproducible results.
