Anti-kelch-like 12 and anti-hexokinase 1: novel autoantibodies in primary biliary cirrhosis

Abstract

Background & aims: Using high-density human recombinant protein microarrays, we identified two potential biomarkers, kelch-like 12 (KLHL12) and hexokinase-1 (HK1), in primary biliary cirrhosis (PBC). The objective of this study was to determine the diagnostic value of anti-KLHL12/HK1 autoantibodies in PBC. Initial discovery used sera from 22 patients with PBC and 62 non-PBC controls. KLHL12 and HK1 proteins were then analysed for immunoglobulin reactivity by immunoblot and enzyme-linked immunosorbent assay (ELISA) in two independent cohorts of PBC and disease/healthy control patients.

Methods: Serum samples from 100 patients with PBC and 165 non-PBC disease controls were analysed by immunoblot and samples from 366 patients with PBC, 174 disease controls, and 80 healthy donors were tested by ELISA.

Results: Anti-KLHL12 and anti-HK1 antibodies were each detected more frequently in PBC compared with non-PBC disease controls (P < 0.001). Not only are both markers highly specific for PBC (≥95%) but they also yielded higher sensitivity than anti-gp210 and anti-sp100 antibodies. Combining anti-HK1 and anti-KLHL12 with available markers (MIT3, gp210 and sp100), increased the diagnostic sensitivity for PBC. Most importantly, anti-KLHL12 and anti-HK1 antibodies were present in 10-35% of anti-mitochondrial antibody (AMA)-negative PBC patients and adding these two biomarkers to conventional PBC assays dramatically improved the serological sensitivity in AMA-negative PBC from 55% to 75% in immunoblot and 48.3% to 68.5% in ELISA.

Conclusions: The addition of tests for highly specific anti-KLHL12 and anti-HK1 antibodies to AMA and ANA serological assays significantly improves efficacy in the clinical detection and diagnosis of PBC, especially for AMA-negative subjects.

Keywords: diagnostics; liver; serology.

Figure 1

Quantile normalized protein microarray autoantibody data for (A) HK1 and (B) KLHL12 for 80 distinct serum samples.

Figure 2

Serological immunoglobulin reactivity of patients with PBC against (A) KLHL12 and (B) HK1 by immunoblot. Representative results from 10 anti-KLHL12/HK1 positive patients in PBC (lane 1~10) and 16 anti-KLHL12/HK1 negative patients in disease controls (lane 11~26) are shown. Healthy and HRP-conjugated anti-human secondary antibody only (2^nd Ab Only) controls were analyzed in parallel.

Figure 3

Distribution of (A) anti-KLHL12 and (B) anti-HK1 antibodies in different clinical groups by ELISA. Total of 366 PBC, including 277 AMA-IFA positive and 89 AMA-IFA negative, as well as 174 non-PBC disease controls, and 80 healthy controls are shown.

Figure 4

Receiver operating characteristics (ROC) analysis for serological detection of PBC biomarkers by ELISA.

Figure 5

Overlap of autoantibodies in PBC. Anti-KLHL12, anti-HK1, AMA, and ANA were determined by (A) the combination of immunoblot and IFA (n = 100, including 80 AMA IFA positive and 20 AMA IFA negative) or by (B) ELISA (n = 366, including 277 AMA IFA positive and 89 AMA IFA negative). Majority of patients with PBC had AMA and/or ANA. Interestingly, 5~20.4% of the aforementioned PBC patients had concurrent anti-KLHL12 and anti-HK1 antibodies. The cohort tested by immunoblot and IFA was distinct from that tested by ELISA.

Figure 6

Increasing sensitivity of serological biomarkers in (A) all PBC (n = 100, including 80 AMA IFA-positive and 20 AMA IFA-negative) and (B) AMA IFA-negative PBC (n = 20) patients by the combination of immunoblot and IFA, and in (C) all PBC (n = 366, including 277 AMA IFA-positive and 89 AMA IFA-negative) and (D) AMA IFA-negative PBC (n = 89) patients by ELISA. The “all PBC” cohorts were selectively enriched in AMA-negative PBC patients and the cohorts in (A) and (B) were distinct from those in (C) and (D).