Methylglyoxal-derivative advanced glycation endproducts: detection by competitive immunofluorometric assay and quantifying in serum and urine.

BACKGROUND: Advanced glycation endproducts (AGE) are a family of heterogeneous chemical structures formed on the host protein in the conditions of carbonyl or oxidative stress. Among AGE precursors, methylglyoxal (MG) is considered one of the key intermediates. METHODS: In the current study, we describe and evaluate a solid phase time-resolved fluoroimmunoassay (DELFIA) based on the competitive reaction between MG-AGE antibody and competitive antigen for detecting MG-adducts in serum and urine. The fluorometry assay was validated by comparison with previously established competitive ELISA. RESULTS: The concentration of MG-adducts assayed by competitive DELFIA in the sera of diabetic patients (DM, n=66) were higher in comparison to non-diabetic controls (C, n=28); DM median=294 mgEq/L (10th and 90th percentile 158-564) vs. C median=224 mgEq/L (10th and 90th percentile 124-290) (p=0.0022). In diabetic subjects, urinary excretion of MG-adducts exceeded the mean level measured in controls; DM median=36.0 mgEq/L (10th and 90th percentile 1-210) vs. C median=11.5 mgEq/L (10th and 90th percentile 1-49) p=0.0036. MG-adducts in urine were low and undetectable in 8 of 28 control subjects and 2 of 66 diabetics. The percentage recovery of MG-adduct added in the same concentration to control and diabetic pool sera showed under-recovery in the latter. Comparison of total AGE level and the amount of MG-adducts revealed MG-derivatives to account for 37% of the heterogeneous structure commonly termed AGE. CONCLUSIONS: The fluoroimmunoassay for MG-derivative AGE evaluated can be utilized on biomonitoring of MG-adducts in serum and its urinary excretion. The competitive DELFIA assay was found to be substantially more sensitive than the standard ELISA having a wider dynamic range, while sharing similar quenching attributes.

A microassay for measurement of Fc function of human immunoglobulin preparations by using tetanus toxoid as antigen.

In order to minimize possible adverse reactions, the functional integrity of proteins in products derived from human plasma has to be unaffected by methods of preparation and storage conditions. Numerous biologically relevant functions of IgG, a major component of immunoglobulin for intravenous use preparations (IVIG), rely on the integrity of Fc fragments. Manufacturers are obliged to prove that Fc-mediated functions are maintained in IVIG preparations. The European Pharmacopoeia's monograph proposes a Rubella antigen-based test for Fc function of immunoglobulins. We present a modification of the proposed method achieved by using more convenient and readily available tetanus toxoid as an alternative antigen target and by adapting the procedure for the use on microtitre plates, thus greatly enhancing its feasibility and sample throughput. The test conditions were optimized so that batch-to-batch variability in tetanus antibody content did not influence the result. The precision of the test was within +/- 5%. By using this test, we compared Fc functionality of 9 commercial IVIG-7S preparations, which were prepared by using different virus inactivation/removal approaches. No significant differences between them have been found.

In vitro glycation of human immunoglobulin G.

BACKGROUND: Glucose can covalently bind to the proteins by nonenzymatic process often termed glycation. Glycation of IgG is of special interest due to its possible influence on the functionality of immunoglobulins and overall immunocompetence. METHODS: The glycation of IgG was studied using radioactive D-[U-14C]-glucose. RESULTS: The kinetics, concentration/temperature dependence and distribution of glycation binding sites of human IgG were studied under various conditions. (a) In average, 0.7 glucose molecules were found to be bound per IgG after 90 days of incubation at 37 degrees C with normal serum IgG and glucose concentrations, and 3.1 molecules after incubation with glucose in the concentration characteristic for diabetics. (b) Incubation of the same solutions for 270 days at 4-8 degrees C resulted in binding of 0.3 and 0.8 glucose molecules per IgG, respectively. (c) After 90 days at 37 degrees C in the presence of 0.56 mol/l of glucose, IgG was glycated with averagely 47 glucose molecules per IgG and with 9 after 270 days at 4-8 degrees C. (d) At very high glucose concentration (1.67 mol/l) in concentrated IgG solution (87 g/l), the molar ratio glucose/IgG reached 41 after incubation at 60 degrees C for 10 h. CONCLUSIONS: Glycation of IgG with glucose nearly resembled the first order reaction kinetics. No preferential glycation sites were found as bound glucose was equally distributed throughout F(ab)2 and Fc fragments as well as on the light and heavy immunoglobulin chains.

Cellular and humoral immunity to purified protein derivative (PPD) in PPD skin reactive and nonreactive patients with pulmonary tuberculosis: comparative analysis of antigen-specific lymphocyte proliferation and IgG antibodies.

AIM: To evaluate in vitro reactivity against tuberculin purified protein derivative (PPD) in patients with active pulmonary tuberculosis scoring either positive or negative upon intradermal PPD application (PPD-DTH). METHOD: Two groups of patients with pulmonary tuberculosis, 22 PPD+ and 22 PPD-, were studied. Peripheral blood mononuclear cells (PBMC) were assayed for in vitro proliferation to PPD antigen, phytohaemagglutin, concanavalin A, and pokeweed mitogens. In the proliferation assay PBMC were incubated in a medium supplemented with serum (20% concentration) from healthy donors, autologous serum, or allogenic serum. Anti-PPD IgG concentration in patients sera were analyzed by ELISA. CD3+ lymphocytes from 10 patients in each group were tested for the expression of surface activation markers (HLA-DR and CD25/IL-2 receptors) by flow cytometry. RESULTS: PPD- patients showed clinically and radiologically more advanced forms of pulmonary tuberculosis as compared with PPD+ patients. PBMC from both groups of patients proliferated in response to PPD effectively, but significantly higher de novo DNA synthesis was observed in PPD+ patients (p<0.001). Proliferative activity was not affected by the type of the serum supplement (autologous or allogenic) in the culture medium. Mitogen stimulation elicited similar proliferative responses in both groups. Similar percentages of T-lymphocytes and T-lymphocytes expressing CD25 activation markers were observed in both groups of patients. There was a borderline difference in the percentage of CD3+HLA-DR+ lymphocytes between these two groups of patients (p=0.05). At 1:1000 serum dilution a significant difference (p=0.002) in anti-PPD IgG concentrations was found between PPD- and PPD+ patients. CONCLUSION: Patients with active pulmonary tuberculosis with a more favorable clinical course have a more potent specific cell-mediated immunity to PPD (positive skin reactivity in vivo and significantly greater lymphocyte proliferative response in vitro) than patients with a clinically more severe form of the disease. The concentration of PPD specific IgG in the serum appears to be higher in patients with relatively more severe forms of the disease.