Purpose: Human soluble HLA-E (sHLA-E) is potentially an important biomarker in cancer immunotherapy. Originally, we developed chemiluminesent HLA-E ELISA, but most of the samples were below LLOQ. A more sensitive sHLA-E assay was required and developed by using Quanterix platform, which is based on isolating single beads in microarray wells.
Methods: Simoa HD-1 Analyzer is a fully automated ultra-sensitive immunoassay platform utilizing digital readout of individual beads in microarray wells. The capture antibody was coupled to paramagnetic beads, and amount of bound biotinylated detection antibody was quantified by reporter enzyme, streptavidin β-galactosidase (SβG), and its fluorescence substrate, resorufin β-D-galactopyranoside. We optimized the concentrations of capture and detection antibodies in both platforms. For LCMS method, sHLA-E was captured from human plasma using anti-sHLA-E antibody conjugated to Dynabeads M-280 Tosylactivated, followed by trypsin digestion and LCMS using multiple reaction monitoring (MRM).
Results: Under the optimal conditions, sHLA-E capture mAb was conjugated to the beads at 0.5 mg/ml with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide at 0.5 mg/ml for 2 hours at room temperature, and 0.16 mg/ml of the mAb was coated with 32 % conjugation efficiency. Standard curve was constructed with recombinant human HLA-E protein in homebrew sample diluent containing 2% mouse serum (assay buffer). With the biotinylated HLA-E detection mAb at 2 ug/ml and SβG at 165 pM, we were able to achieve an LOD of 3 pg/mL, LLOQ of 11 pg/ml, CV% <15 %, spike-recovery of 90 to 110%, and dilution linearity at 2 to 16-fold dilutions. The assay dynamic range was 3-8000 pg/ml. High endogenous sHLA-E plasma samples were used to assess parallelism at 2 to 8-fold dilutions in assay buffer and the CV% of back calculated concentrations were <20%. The specificity of sHLA-E immunoasay was confirmed by LC-MS/MS in which three human HLA-E unique peptides were selected to be monitored by using multiple reaction monitoring (MRM) method by LCMS. All three peptides were identified in a HLA-E positive human plasma sample, but not in HLA-E negative sample. The Quanterix assay indicated 10-fold increase in sensitivity as compared to Chemiluminescent ELISA. Blood samples from 200 normal healthy individuals and patients with different types of cancers were analyzed at MRD 4 in the assay buffer and the results showed that the assay can quantify sHLA-E concentrations in 89% of healthy and cancer individuals.
Conclusion: Highly sensitive homebrew Simoa-based sHLA-E assay was developed with the specificity being confirmed by LCMS and with additional benefits of automation in sample analysis.
Zhe Cheng– Postdoctoral fellow, BMS
Rong Liu– BMS
Hao Jiang– BMS
Surendran Rajendran– BMS
Laurence Menard– BMS
Yan Zhang– Director, Analytical and Bioanalytical Operations, Bristol-Myers Squibb Company, Princeton, New Jersey
Alexander Kozhich– Senior Principal Scientist, Bristol-Myers Squibb Company, Lawrenceville, New Jersey