Association of Serum Antibody Levels against TUBB2C with Diabetes and Cerebral Infarction

Abstract

Cerebral infarction (CI), cardiovascular disease (CVD), diabetes mellitus (DM) and chronic kidney disease (CKD) are atherosclerosis-related diseases, which are major causes of health damage. For early and sensitive diagnosis, development of novel biomarkers is expected and of significant practical importance. First screening was carried out by phage expression cloning to identify antigen proteins recognized by serum IgG antibodies in patients with atherosclerosis. RPA2, LRPAP1, EEF1A1, SPOCK1, LOC729260, tubulin beta 2C (TUBB2C) and KIAA0020 markers were identified. We then compared the serum antibody levels against the candidate proteins between healthy donors (HD) and patients with CI, CVD, DM, or CKD by Alpha (amplified luminescent proximity homogeneous assay)-LISA method. The results showed that the serum TUBB2C antibody levels were significantly higher in patients with CI, DM, or CKD than those in HD. Using the average + 2SD of HD as the cut-off value, the positive thresholds of TUBB2C antibody markers were 14.8% in CI, 25.8% in DM, and 18.3% in CKD. TUBB2C antibody levels were well correlated with artery stenosis degrees such as plaque score, maximum intima-media thickness and cardio ankle vascular index. Consequently, TUBB2C antibody markers are useful to diagnose atherosclerosis, DM, and CKD, and can be applied to the prediction of the onset of CI. The serum anti-TUBB2C antibody markers are useful for the diagnosis of DM and CKD.

Elevated Levels of Autoantibodies against ATP2B4 and BMP-1 in Sera of Patients with Atherosclerosis-related Diseases

“…Phage expression cloning, also called SEREX, is effective for the screening of tumor antigens [15][16][17][18] and biomarkers of stroke [19] and DM [20]. Through the expression cloning method, we have identified two antigens that were recognized by serum IgG antibodies in patients with ATS.…”

“…Positive reactions were developed using 100 mM Tris-HCl (pH 9.5) containing 100 mM NaCl, 5 mM MgCl 2 , 0.15 mg/ml of 5-bromo-4-chloro-3-indolylphosphate, and 0.3 mg/ml of nitro blue tetrazolium (Wako Pure Chemicals). Positive clones were re-cloned twice until obtaining monoclonality as previously described [17,19,20].…”

“…The reaction mixture was incubated at room temperature for 6-8 h. Next, anti-human IgG-conjugated acceptor beads (2.5 µl of 40 µg/ml) and glutathione-conjugated donor beads (2.5 µl of 40 µg/ml) were added and incubated further for 7-14 days at room temperature in the dark. The chemical emission was read on an EnSpire Alpha microplate reader (PerkinElmer) as previously described [20,23]. Specific reactions were calculated by subtracting Alpha values of GST control from the values of GST-fusion proteins.…”

“…Blood test data, such as data on total protein, albumin/globulin ratios, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, total bilirubin, γ-glutamyl transpeptidase, albumin, blood urea nitrogen, creatinin, estimated glomerular filtration rates, uric acid, amylase, total cholesterol, HDL-cholesterol, LDLcholesterol, triglyceride, Na, K, Cl, Ca, P, Fe, C-reactive protein, blood sugar, and HbA1c were also included as described [20,23]. A total of 741 specimens from the Sawara Hospital including 139 specimens from HD, 79 from patients with deep and subcortical white matter hyperintensity, 15 patients with asymptomatic CI, 29 patients with TIA, 227 with aCI, 58 with chronic CI, and 194 from disease controls.…”

“…The SEREX (serological identification of antigen by recombinant cDNA expression cloning) [15] method is effective and convenient for comprehensive screening of tumor antigens [16][17][18]. We also introduced SEREX into the screening of autoantigens and autoantibodies responsible for ATS-related diseases and identified RPA2 for stroke [19] and TUBB2C for DM and CI [20]. Here, we report anti-ATP2B4 and anti-BMP-1 antibodies as general ATS markers related to CI, CVD, DM, and CKD.…”