Development of an Ultra-Sensitive Digital Immunoassay for Human Soluble HLA-E Biomarker

2018 AAPS
|

Yanshan Dai, Zhe Cheng, Rong Liu, Hao Jiang, Surendran Rajendran, Laurence Menard, Yan Zhang, Alexander Kozhich

PURPOSE

Human soluble HLA-E (sHLA-E) is potentially important biomarker in cancer immunotherapy. Originally, we developed chemiluminescent HLA-E ELISA, but most of the samples were below LLOQ. A more sensitive sHLA-E assay was required and was developed using Quanterix platform, which is based on digital form of ELISA, trapping and sealing individual immunocomplexes on paramagnetic beads in thousands of femtoliter wells.

METHODS

• Simoa HD-1 Analyzer is a fully automated ultrasensitive immunoassay platform utilizing digital readout of individual beads in microarray wells.
• The capture antibody was coupled to paramagnetic beads
• Amount of bound biotinylated detection antibody was quantified using reporter enzyme, streptavidin βgalactosidase (SβG), and its fluorescence substrate, resorufin β-D-galactopyranoside.
• We optimized capture and detection antibodies concentrations in both platforms.
• For LCMS sHLA-E was captured from human plasma using anti-sHLA-E anibody conjugated to Dynabeads M-280 Tosylactivated,
• Followed by trypsin digestion and LCMS using multiple reaction monitoring (MRM).

RESULTS

• sHLA-E capture mAb was conjugated to the beads at 0.5 mg/ml with 1- Ethyl-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.
• With the biotinylated HLA-E detection mAb at 2 µg/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 immunoassay 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. 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 MRD4 in the assay buffer and the results showed that the assay can quantify sHLA-E concentrations in 89% of healthy and cancer individuals.

CONCLUSIONS

• The endogenous plasma human HLA-E protein was enriched by HLA-E capture antibody conjugated to Dynabeads M-280 Tosylactivated
• Three human HLA-E unique peptides were selected to be monitored using multiple reaction monitoring (MRM) method by LCMS
• Sensitive homebrew Simoa-based sHLA-E assay was developed, with the specificity confirmed by LCMS, and with additional benefits of automation in sample analysis.