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1.
J Immunol Methods ; 487: 112872, 2020 12.
Article in English | MEDLINE | ID: mdl-33007318

ABSTRACT

The presence of (1 â†’ 3)-ß-D-glucan in human plasma is a marker for fungal infections. Currently, the Limulus amebocyte lysate (LAL)-based assay is widely used for the quantification of plasma (1 â†’ 3)-ß-D-glucan. However, it has limitations in clinical use, such as an unstable supply of natural resources, complicated manufacturing process, and low-throughput of the reagents. Alternative assays exploiting specific antibodies against (1 â†’ 3)-ß-D-glucan have been developed to overcome these challenges. However, these methods are associated with low sensitivity and poorly correlate with the data obtained by the LAL-based assay. The aim of this study is to develop a novel enzyme immunoassay that is as sensitive and accurate in determining plasma (1 â†’ 3)-ß-D-glucan levels as compared to that obtained with the LAL-based assay. We generated specific monoclonal antibodies against (1 â†’ 3)-ß-D-glucan that recognizes four-unit glucose oligomers with (1 â†’ 3)-ß-D-linkages, and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) using these antibodies. The newly developed ELISA showed proportional increase in absorbance with the volume of (1 â†’ 3)-ß-D-glucan added. The limit of detection of the assay was 4 pg/ml of plasma (1 â†’ 3)-ß-D-glucan that was equivalent to the LAL-based assay and the working range was 4-500 pg/ml. The intra-assay coefficient of variation was 2.2-5.4% using three different concentrations of plasma samples. We observed strong correlation (R = 0.941, slope = 0.986) between the measurements obtained by our ELISA and Fungitec G test ES Nissui, a commonly used LAL-based assay, using 26 types of plasma samples. This could be attributed to the epitopes of the antibodies. Both antibodies could inhibit the LAL-based assay, suggesting that the antibodies recognize the identical regions in ß-D-glucan, thereby inactivating factor G, an initiation zymogen for coagulation cascade, in the LAL-based assay. Thus, the ELISA developed in this study can detect fungal infections in clinical settings with similar efficiency as the LAL-based assay. This assay is characterized by good performance, stable supply of materials, and simple manufacturing process and is more suitable for the high-throughput diagnosis of fungal infections.


Subject(s)
Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay , Limulus Test , Mycoses/diagnosis , beta-Glucans/blood , Antibody Affinity , Antibody Specificity , Biomarkers/blood , Epitopes , Humans , Mycoses/blood , Mycoses/immunology , Mycoses/microbiology , Predictive Value of Tests , Reproducibility of Results , beta-Glucans/immunology
2.
Clin Chim Acta ; 339(1-2): 97-103, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14687899

ABSTRACT

BACKGROUND: Drug therapy is considered essential to the clinical prevention of atherosclerotic lesions in patients with diabetes mellitus (DM). METHODS: To confirm the effects of fibrate therapy, we determined low-density lipoprotein (LDL) size by gradient gel electrophoresis and malondialdehyde-modified LDL (MDA-LDL) concentrations by enzyme-linked immunosolvent assay (ELISA) and clarified the association between apolipoprotein B (apo B) and MDA-LDL during the fibrate therapy. RESULTS: Mean MDA-LDL concentrations were higher in healthy men than in healthy women. There were no significant differences in mean MDA-LDL concentrations between age groups for males or females. According to the regression equation (y = 0.063x + 10.9) obtained for apo B and MDA-LDL concentrations with fibrate treatment, the apo B concentration in those may need to be decreased to 1260 mg/l to restore the MDA-LDL concentration to the control concentration (65 +/- 25 units/l). This slope of the apoB/MDA-LDL regression line was approximately half of that with no-drug treatment (y = 0.109x - 10.8). CONCLUSIONS: Fibrate therapy had an effect on reducing serum MDA-LDL concentration in diabetic patients.


Subject(s)
Bezafibrate/pharmacology , Diabetes Mellitus/blood , Diabetes Mellitus/metabolism , Lipoproteins, LDL/blood , Lipoproteins, LDL/metabolism , Malondialdehyde/chemistry , Adult , Aging , Apolipoproteins B/blood , Arteriosclerosis/blood , Arteriosclerosis/complications , Arteriosclerosis/metabolism , Arteriosclerosis/prevention & control , Diabetes Complications , Diabetes Mellitus/therapy , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lipoproteins, LDL/chemistry , Lipoproteins, LDL/drug effects , Male , Middle Aged , Sex Characteristics
3.
J Atheroscler Thromb ; 10(2): 72-8, 2003.
Article in English | MEDLINE | ID: mdl-12740480

ABSTRACT

Several reports have suggested that HDL has anti-oxidative actions. We investigated the relationship between HDL-cholesterol (HDL-C) and malondialdehyde-modified LDL (MDA-LDL) concentrations using enzyme linked immunosolvent assay. We divided our study subjects into four groups on the basis of concentrations of triglyceride (TG) and HDL-C by the following lipid profiles: serum TG < or = 1.69 mmol/L and HDL-C > or = 1.16 mmol/L (control group, n = 26); TG >1.69 and HDL-C < or = 1.16 (high TG group, n = 22); TG >1.69 and HDL-C < or = 0.91 (high TG & low HDL group, n = 67); TG < or = 1.69 and HDL-C < or = 0.91 (low HDL group, n = 21). MDA-LDL concentrations, MDA-LDL/apolipoprotein B (apo B) ratio, and LDL size were different between subjects in high TG & low HDL and control groups. MDA-LDL concentrations in both high TG and low HDL groups did not differ significantly from those in the control. However, MDA-LDL/apo B ratio in low HDL group was significantly higher than that in the control (P < 0.05). The MDA-LDL/apo B ratio reflects the extent of MDA modification of apo B in LDL. Therefore, our data suggest that as HDL-C concentrations fall, the extent of MDA modification per one LDL particle increases. Moreover, accompanied by high TG concentration, LDL size in subjects with lower HDL-C concentrations became smaller.


Subject(s)
Cholesterol, HDL/metabolism , Cholesterol, LDL/metabolism , Malondialdehyde/metabolism , Oxidative Stress/physiology , Adult , Aged , Apolipoproteins B/blood , Apolipoproteins B/metabolism , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Female , Humans , Male , Malondialdehyde/blood , Middle Aged , Triglycerides/blood , Triglycerides/metabolism
4.
J Lipid Res ; 43(2): 325-34, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11861675

ABSTRACT

We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. These are mouse anti-human LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486, which react with the peptides corresponding to human LCAT amino acid residues R159-E179, M258-S273, S274-S294, D352-S376, and N415-E440, respectively. We also successfully used two of these antibodies to develop an ELISA, which uses a solid phase monoclonal antibody, 36486, that reacts with the C-terminus of LCAT, and a detection monoclonal antibody, 36487, that reacts with an epitope located in the center of the LCAT primary structure. We observed a significant positive correlation between the values of LCAT protein determined with ELISA and LCAT activity determined with liposome substrate (r = 0.871, P < 0.001) or the endogenous self-substrate method (r = 0.864, P < 0.001), and we obtained inter- and intra-assay coefficients of variation less than 6.1%, minimum detection limit of 0.1 microg/ml. Highly specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT. Therefore, this precise and sensitive LCAT assay will help clarify the role of this enzyme in the metabolism of HDLs, and can be used for diagnostic purposes in investigating liver function. We obtained five monoclonal antibodies that recognized different epitopes on LCAT and developed a sandwich-type ELISA. Highly specific monoclonal antibodies provide a sensitive and specific analytical system for measurements of LCAT protein.


Subject(s)
Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/immunology , Phosphatidylcholine-Sterol O-Acyltransferase/blood , Amino Acid Sequence , Binding Sites, Antibody/drug effects , Humans , Lipoproteins/blood , Lipoproteins/chemistry , Molecular Sequence Data , Oligopeptides/pharmacology , Phosphatidylcholine-Sterol O-Acyltransferase/chemistry , Phosphatidylcholine-Sterol O-Acyltransferase/immunology
5.
J Atheroscler Thromb ; 9(6): 280-7, 2002.
Article in English | MEDLINE | ID: mdl-12560588

ABSTRACT

In a study population, we compared the level of malondialdehyde-modified LDL (MDA-LDL) with the concentrations of lipid parameters in serum and found a strong correlation between MDA-LDL and apolipoprotein B (apo B) concentrations. Their interrelations had a turning point at an apo B concentration of 1,150 mg/l. In diabetic patients, the ratio of MDA-LDL/apo B increased at apo B concentrations above 1,150 mg/l. This ratio represents the extent of modification of apo B by MDA. In the control subjects, this ratio remained stable. When we divided the patients into medication groups (statins and insulin), we found that the 1,150 mg/l threshold disappeared. At apo B concentrations above 1,150 mg/l, the ratio of MDA-LDL/apo B in the statin group was as high as that in the non-drug group. In the insulin group, the means of MDA-LDL/apo B in all ranges of apo B levels decreased to an extent statistically indistinguishable from those of the control group. In conclusion, insulin therapy represses LDL oxidation even at apo B concentrations > 1,150 mg/l and should be noted for its anti-oxidation properties.


Subject(s)
Cholesterol, LDL/metabolism , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Hypoglycemic Agents/administration & dosage , Insulin/administration & dosage , Adult , Aged , Apolipoproteins B/metabolism , Cholesterol, HDL/metabolism , Electrophoresis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Hypolipidemic Agents/administration & dosage , Lipoproteins, LDL/metabolism , Male , Malondialdehyde/metabolism , Middle Aged , Triglycerides/metabolism , fas Receptor/metabolism
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