ABSTRACT
Aberrant glycosylation of IgA1 is involved in the development of IgA nephropathy (IgAN). There are many reports of IgAN markers focusing on the glycoform of IgA1. None have been clinically applied as a routine test. In this study, we established an automated sandwich immunoassay system for detecting aberrant glycosylated IgA1, using Wisteria floribunda agglutinin (WFA) and anti-IgA1 monoclonal antibody. The diagnostic performance as an IgAN marker was evaluated. The usefulness of WFA for immunoassays was investigated by lectin microarray. A reliable standard for quantitative immunoassay measurements was designed by modifying a purified IgA1 substrate. A validation study using multiple serum specimens was performed using the established WFA-antibody sandwich automated immunoassay. Lectin microarray results showed that WFA specifically recognized N-glycans of agglutinated IgA1 in IgAN patients. The constructed IgA1 standard exhibited a wide dynamic range and high reactivity. In the validation study, serum WFA-reactive IgA1 (WFA+-IgA1) differed significantly between healthy control subjects and IgAN patients. The findings indicate that WFA is a suitable lectin that specifically targets abnormal agglutinated IgA1 in serum. We also describe an automated immunoassay system for detecting WFA+-IgA1, focusing on N-glycans.
Subject(s)
Glomerulonephritis, IGA , Biomarkers , Female , Glomerulonephritis, IGA/diagnosis , Humans , Immunoassay , Immunoglobulin A , Lectins , Male , Plant Lectins , Polysaccharides , Receptors, N-AcetylglucosamineABSTRACT
BACKGROUND: Aberrant glycosylation has been reported to play important roles in progression of cholangiocarcinoma (CCA) and hence the aberrant expressed glycans are beneficial markers for diagnosis and prognostic prediction of CCA. METHODS: Five CCA-associated glycobiomarkers-carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen-S27 (CA-S27), CCA-associated carbohydrate antigen (CCA-CA), WFA-positive MUC1 (WFA+-MUC1), and WFA-positive M2BP (WFA+-M2BP), in the sera from CCA patients (N = 138) were determined in comparison with non-CCA control subjects (N = 246). RESULTS: Receiver operating characteristic analysis suggested the significance of each glycobiomarker in discriminating CCA from non-CCA with area under curve of 0.580-0.777. High levels of CA19-9, CCA-CA, CA-S27, or WFA+-MUC1 were associated with poor prognosis and poor survival of CCA patients. Combination of these glycobiomarkers and graded as a GlycoBiomarker (GB)-score could increase the power of the tests in diagnosis than an individual marker with 81% of sensitivity, specificity and accuracy. CONCLUSIONS: According to the GB-score, these glycobiomarkers not only increased diagnostic power but also discriminated survival of patients indicating the diagnostic and prognostic values of GB-score.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Bile Duct Neoplasms/diagnosis , Bile Ducts, Intrahepatic , Biomarkers, Tumor , CA-19-9 Antigen , Cholangiocarcinoma/diagnosis , Humans , PrognosisABSTRACT
This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.
