Quantitative Mass Spectrometry Evaluation of Human Retinol Binding Protein 4 and Related Variants

BACKGROUND Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker. METHODS RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants. RESULTS The linear range of the assay was 7.81-500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 - 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA  = 1.05× ELISA - 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%. CONCLUSION The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker.