The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: 1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), 2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), 3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), 4) recombinant M2BP produced by HEK293 cells (rM2BP), and 5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that, reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc, and in part of recombinant, depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.

Background: Assays to screen for and confirm the presence of the antibody for human T-lymphotropic virus type I (HTLV-I) are currently performed with serum or plasma. We developed and evaluated a new counting immunoassay (CIA) for the detection of HTLV-I antibody in whole blood, using recombinant and synthetic peptide antigens. Methods: We assessed the CIA for detection of HTLV-I antibody in whole blood and plasma. The CIA is an immunity-measuring method that combines latex agglutination with particle-counting technology. The numbers of agglutinated latex particles, single latex particles, and blood cells in a sample are measured based on differences in particle size between latex particles and blood cells. Results: The CIA and ELISA methods were in agreement for all 24 plasma samples tested, including those from 6 patients with HTLV-I-associated diseases, 6 HTLV-I carriers, and 12 HTLV-I antibody-negative individuals. The concordance between the ELISA (plasma) and the CIA (whole blood) for samples from 24 patients was 100%. The concordance between a particle agglutination method (plasma) and the CIA (plasma or whole blood) for 1065 patients was 99.5%. The concordance between results obtained for 1065 pairs of plasma and whole blood samples with the CIA method was 100%. HTLV-I antibody in whole blood was stable for 3 days after blood collection. With this CIA method, results were available within 15 min.

Four electrophoretic variants of acid phosphatase have been already reported in the silkworm hemolymph. We found an electrophoretic variant (type E) which had been designated 0 (null) type since the distinct band had not been detected.The band E was undiscernible when the substrate (a-naphthyl phosphate) and chromogen (Fast blue B) were poured simultaneously on the gel after electrophoresis according to the former procedure.However, if the gel was incubated with a-NP then Fast blue B was added, a distinct band E appeared. Quantitative analyses showed that Fast blue B inhibited the isozyme E competitively but other isozymes noncompetitively.Several lines of experimental evidence suggest the isozyme E is not only a charge isomer but also an isozyme showing different kinetic properties with other isozymes. By crossing experiments isozymes A-E were considered to be controlled by codominant alleles, BphA-BphE, respectively.