Development of a mouse IgA monoclonal antibody-based enzyme-linked immunosorbent sandwich assay for the analyses of RBP4

Nam Seok Lee,Han Soo Kim,Se Eun Park,Cheol-Young Park,Matthias Blüher,Byung-Soo Youn

doi:10.1038/s41598-018-20762-x

Abstract

Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R2 = 0.859). After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.

Highlights

Insulin resistance contributes to the progression from normal glucose tolerance (NGT) to impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2D)[1]
The cDNA sequence corresponding to the J chain was verified. These findings confirm that AG102 is a monoclonal immunoglobulin A (IgA) that recognizes an N-terminus epitope of Retinol-binding protein 4 (RBP4) that is likely to be exposed on the surface of RBP4 and unlikely to be affected by either high-affinity RBP4·TTR interactions or RBP4 C-terminal proteolysis, factors proposed to contribute to variability and technical limitations in other assays[14,15]
We demonstrated a newly developed IgA mAb-based enzyme-linked immunosorbent sandwich assay (ELISA), which is a semi-quantitative tool for measuring the major serum protein, RBP4, upon a proper single dilution

Summary

Introduction

Insulin resistance contributes to the progression from normal glucose tolerance (NGT) to impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2D)[1]. It is likely that the relative concentrations of RBP4 and TTR in circulation, the affinity of RBP4·TTR binding, and the variable presence of proteolyzed RBP4 can influence the ability of immunoassays to accurately quantify RBP4. In addition to this diagnostic pitfall, RBP4 serum concentration is typically higher than a few hundred μg/ml, resulting in the so-called “hook effect” even after significant dilution[19]. The distribution features of serum recovery via this IgA-based ELISA were comparable to those of q-Western blotting We propose that this novel IgA-based immunoassay should be used to define the relationships between urinary RBP4 level and clinical measures of insulin-glucose homeostasis and urinary albumin excretion

Methods

Results

Conclusion