A specific and sensitive assay for blood levels of glycated CD59: a novel biomarker for diabetes

Abstract

Increasing evidence links the complement system with complications of human diabetes. The complement regulatory protein CD59, an inhibitor of formation of membrane attack complex (MAC), is inhibited by hyperglycemia-induced glycation fostering increased deposition of MAC, a major effector of complement-mediated tissue damage. CD59, an ubiquitous GPI-anchored membrane protein, is shed from cell membranes by phospholipases generating a soluble form present in blood and urine. We established an enzyme-linked immunosorbent assay (ELISA) to measure serum/plasma glycated human CD59 (hCD59) (GCD59) and evaluated its potential as a diabetes biomarker. We used a synthetic peptide strategy to generate (a) a mouse monoclonal antibody to capture hCD59, (b) a rabbit monoclonal antibody to detect GCD59, and (c) a GCD59 surrogate for assay standardization. ELISA conditions were optimized for precision, reproducibility, and clinical sensitivity. The clinical utility of the assay was initially evaluated in 24 subjects with or without diabetes and further validated in a study that included 100 subjects with and 90 subjects without a diagnosis of diabetes. GCD59 (a) was significantly higher in individuals with than in individual without diabetes, (b) was independently associated with HbA1c, and (c) identified individuals with diabetes with high specificity and sensitivity. We report the development and standardization of a novel, sensitive, and specific ELISA for measuring GCD59 in blood. The assay distinguished individuals with diabetes from those without, and showed strong correlation between GCD59 and HbA1c. Because GCD59 likely contributes to the pathogenesis of diabetes complications, measurement of blood levels of GCD59 may be useful in the diagnosis and management of diabetes.

Figure 1. Specificity of the anti-glycated hCD59 rabbit monoclonal antibody (MABGlyCD59)

A. GCD59 (G) and NGCD59 (NG) affinity purified from a urine pool individuals with poorly controlled diabetes, separated by SDS-PAGE and immuno-blotted with goat polyclonal anti-hCD59 antibody before (non-reduced) and after (reduced) treatment with Na-borohydride (left panels in the Reduced and Non-reduced frames depicting red fluorescent bands). The GCD59 band is also recognized by the MABGlyCD59 antibody but only after Na-borohydride reduction (green fluorescent band in the G lane of the Reduced frame). The full-length blotted membranes used to create this Figure are shown in Supplemental Figure 1. B. Affinity purified hCD59 from a group of individuals with and without diabetes separated by SDS-PAGE and immunoblotted first with goat polyclonal anti-CD59 antibody (Goat Poly Anti-CD59; red fluorescence) and then with MABGlyCD59 antibody (green fluorescence). The bar graph at the bottom of Figure 1B depicts the ratio of green (GCD59) over red (total CD59) fluorescence (quantified using an infrared Odyssey scanner) for each individual sample. The inset shows the mean ± SEM of the ratios for the individuals with (D) and without diabetes (N).

Figure 2. Specificity of the ELISA for GCD59

Sandwich ELISA of affinity purified GCD59 before and after immunoprecipitation (IP) with increasing concentrations of goat polyclonal anti-CD59 antibody. The insets show the western blot (WB) analysis for total (red fluorescence) and GCD59 (green fluorescence) of the sample before (inset at 0 antibody concentration) and after immunopreciptiation (IP) with 12 μg/ml antibody (S= WB of the supernatant after IP; E = WB of eluate from agarose beads carrying the antibody-CD59 complex).

Figure 3. Synthetic Surrogate GCD59 for assay standardization

A. Diagram of synthetic surrogate standard in which both antigen peptides, hCD59[44-66] and (K41(N^ε-glucitol)hCD59[37-50] (hCD59[37-50]), respectively used to raise the capture mouse monoclonal antibody MABTotCD59 and the detection MABGlyCD59 antibodies, were coupled by a PEG linker. Represents the pre-formed N^ε-glucitollysine residue used to synthesize the (K41(N^ε-glucitol)hCD59[37-50] peptide for the position equivalent to K41 in the native protein. The detailed structure of GCD59 synthetic surrogate is shown in Supplemental Figure 3. B. Characteristic surrogate GCD59 standard peptide dose-response curve in the here described sandwich ELISA that utilizes MABTotCD59 as capture and MABGlyCD59 as detection antibodies.

Figure 4. Higher levels of plasma GCD59 in individuals with as compared with individuals without diabetes

A. Mean ± SEM of plasma GCD59 10 non-diabetic and 14 diabetic individuals. B. Bar graph representation of the plasma GCD59 value in each study subject; the numbers above the bars represent fasting plasma glucose (top value) and HbA1c (bottom value) measured simultaneously with GCD59. In this cohort selected without any reference as to duration, treatment or control level of diabetes, a cut-off value of 1 SPU differentiates the cohorts without and with diabetes with 100 % specificity and 93% sensitivity.