Influence of antibody and complement components on phagocytosis and chemiluminescence of macrophages

Macrophages are known to release reactive oxygen species (O₂^?, ¹O₂, H₂O₂, OH·) in response to various membrane stimuliHowever, our studies show that phagocytic stimulation of macrophages is not necessarily accompanied by a stimulation of the oxidative burstWhereas IgG-opsonized erythrocytes were capable to induce phagocytosis and a chemiluminescence response, both being dependent on the number of IgG bound per erythrocyte, C3b-bearing erythrocytes were well ingested but failed to induce any chemiluminescence reactionFurthermore, stimulation of macrophages, via the Fc-receptors, seems to alter their functional state in regard to the activation of a receptor, which enables them to recognize membrane lesions on the target erythrocyteThe presence of IgG and membrane lesions, e.gthe C5b-9-complex of complement, induced a marked increase in chemiluminescence compared with stimulation by IgG-bearing particles aloneThe augmented response of macrophages was at least in part due to an additional release of H₂O₂, which was not liberated in response to IgG-bearing erythrocytesThis «Alesion recognizing receptor» in the macrophage membrane could not be activated by stimulation of C3b-receptors, indicating its functional linkage to the Fc-receptors.     

Dexamethasone and clenbuterol detection by enzyme immunoassay in Bovine Liver Tissue: A new multiresidue extraction procedure

A fast and simple extraction procedure was developed for simultaneous determination in bovine liver of two veterinary drugs, widely used as growth promoters in meat production: dexamethasone (a synthetic corticosteroid drug) and clenbuterol (a beta2‐adrenergic agonist drug). Liver samples were extracted by acetonitrile, without any clean‐up step. Two different ELISAs, specific for the two classes of drugs, were used to determine the residue concentration in the extracts. The intra‐ and inter‐extraction variability was determined at different concentrations: the intra‐extraction coefficients of variation (CVs) were between 2.5 and 17.7% for dexamethasone and between 0.9 and 9.8% for clenbuterol; the inter‐extraction CVs were between 2.0 and 16.8% for dexamethasone and between 0.5 and 10.8% for clenbuterol. Recovery ranged from 92 to 154% for dexamethasone and from 78 to 105% for clenbuterol. The limit of detection was 1.43 ng g^?1 and 0.43 ng g^?1, respectively. The limit of quantification for dexamethasone was 2.09 ng g^?1 and for clenbuterol was 0.72 ng g^?1. The combination of the new extraction procedure with an ELISA detection permitted the rapid semi‐quantitative determination of both dexamethasone at its maximum residue level (MRL: 2.5 ng g^?1 in liver tissue), and clenbuterol at low concentration level.     

Lucigenin-dependent chemiluminescence as a new assay for NAD(P)H-oxidase activity in particulate fractions of human polymorphonuclear leukocytes

The enzyme responsible for the respiratory burst in human neutrophils is an oxidase that catalyzes the reduction of oxygen to superoxide anion (O-2). Superoxide anion production may be measured by chemiluminescence (CL) in the presence of lucigenin (10,10'-dimethyl-9,9'- biacridinium dinitrate). We established an assay of the oxidase, by measuring the CL of particulate fractions of PMN in the presence of lucigenin . This CL required the addition of NAD(P)H and was very low in fractions of resting cells. In particulate fractions of PMNs stimulated with PMA selectively, the NADPH-dependent CL was found to be increased. CL was linear with protein concentrations up to 100 micrograms and was shown to be at least 10 times more sensitive for the detection of O-2 than the assay based on the spectrophotometric determination of superoxide mediated cytochrome c reduction. CL was abolished by inactivating the enzyme at 56 degrees C.     

Competitive inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM

Competitive-inhibition enzyme immunoassays for the measurement of human IgG, IgA and IgM are described. These assays can be readily performed with commercial antisera and a recently developed method for purifying human IgA and IgM with high yield. The assays described are specific, with undetectable (<0.5%) cross-reactivity between the immunoglobulin classes in all systems, except with purified IgM, which cross-reacted to 1.9% with the IgG enzyme immunoassay.Minimal detectable concentrations of 2.5±0.8 ng/ml for IgG 4.2±0.9 ng/ml for IgA and 7.2±1.4 ng/ml for IgM were recorded, indicating that these assays are particularly sensitive. There is little within-assay variation (mean coefficient of variation = 3.9%), although the between-assay variation was substantially greater (mean coefficient of variation = 23.5%). These assay systems appear to be particularly suited to the measurement of immunoglobulin production by lymphocytes in culture. In such studies the assay must be specific, sensitive and be capable of discriminating between levels of immunoglobulin produced in response to various experimental treatments.     

Development of a reversible bienzymatic system in immunoenzymatic technique

The use of specific captors solubilized with a ligand on to proteic membranes together with an automatic-computerized system is proposed for the determination of various haptens or antigens in biological and industrial fluids by an enzyme-linked immunoassay test. Two enzymes are used in this technique: the first enzyme for linking reversibly the immunocomplex to the insoluble matrix, the substrate of this enzyme being immobilized on the matrix; the second (beta-d-glucose oxidase) for labelling the antigen. Its activity is measured by fixing the immunocomplex gelatin membrane on to a pO(2) sensor. After incubation of the antigen labelled with glucose oxidase and the free antigen with specific antibodies linked with the first enzyme in a pre-established concentration, the reaction medium is introduced inside the continuous flow cell. O(2) consumption due to the enzyme reaction is measured in actual time when the electrode is in contact with the glucose standard solution. The signal is correlated by an injection of urea solution. The signal is processed with an automated microcomputer system.     

Enzyme-amplified immunoassays

The sensitivity of enzyme immunoassays may be enhanced by the use of enzyme-amplification. This technique uses the enzyme label in the immunoassay to provide a trigger substance for a secondary system that can generate a large quantity of coloured product. Two examples of enzyme amplifiers are described, using either a substrate cycle with phosphorylated hexose sugars, or a redox cycle involving the coenzyme NAD⁺. The redox enzyme-amplifier has a detection limit of less than one attomole for the enzyme label, alkaline phosphatase.

The limited dynamic range of enzyme-amplified immunoassays may be overcome by kinetic analysis of the colour development in the enzyme-amplifier, to add at least a further order of magnitude to the range of directly measured analyte concentrations in the immunoassay. This is illustrated in an enzyme-amplified immunoassay for human thyroid stimulating hormone. Amperometric measurement of the enzyme-amplifier provides a method to extend the dynamic range still further and compares favourably with the performance of a gamma counter, a luminometer or a fluorimeter.     

探讨两种全自动化学发光免疫分析仪检测方法在梅毒检测中的应用价值

目的:探讨i2000和Centaur XP两种全自动化学发光免疫分析仪在梅毒检测中的应用价值。方法:选取在医院体检、门诊和住院行梅毒血清学筛查的120例患者的120份血清标本,分别使用i2000型全自动化学发光免疫分析仪化学发光微粒子免疫试验(CMIA)和Centaur XP型全自动化学发光免疫分析仪化学发光免疫试验(CLIA),对梅毒螺旋体(TP)抗体进行血清学检测。以样本吸光度与临界值的比值(S/CO)≥1.0为阳性,并与以TP明胶凝集试验(TPPA)梅毒特异性抗体确认方法平行测定的S/CO结果进行比较。结果:在120例受检者中,CMIA检测阳性结果94例,阳性率为78.33%;CLIA检测阳性结果72例,阳性率为60.00%;TPPA检测阳性结果74例,阳性率为61.67%。以TPPA试验作为确认方法,CMIA检测的灵敏度和特异度分别为97.30%和52.17%,CLIA分别为94.59%和95.65%。CMIA与TPPA检测的阳性率、符合率比较差异均有统计学意义(χ²=14.101,χ²=7.937;P<0.05)。两种全自动免疫化学发光分析法与TPPA检测的阳性符合率均呈正相关。结论:两种全自动化学发光免疫分析检测方法在梅毒S/CO值>10.00时均具有较高的灵敏度和特异度;当CMIA的S/CO值为1.00~10.00时可选用CLIA复核,缩短标本周转时间;当CLIA的S/CO值<4.70时可附加TPPA复核,以保证检测结果的准确性。     

2016—2019年常州地区无偿献血者酶免和核酸平行检测结果分析

目的分析ELISA和NAT平行的血液筛查模式对降低经输血感染病原体风险的有效性。方法收集常州市2016—2019年270215例无偿献血者血液标本,采用两次ELISA并行检测HBsAg、抗HCV、抗HIV和抗TP。268264例ELISA双阴性标本采用6人份混样(pool)NAT进行HBV DNA、HCV RNA和HIV RNA的检测,核酸阳性的pool进行拆分单检。结果270215例无偿献血者HBsAg、抗HCV、抗HIV和抗TP的阳性率分别为2.58‰(697例)、1.49‰(402例)、0.23‰(61例)和3.06‰(827例)。268264例酶免阴性无偿献血者HBV DNA、HCV RNA和HIV RNA阳性率分别为0.86‰(230例)、0.01‰(3例)和0.01‰(2例)。结论ELISA与NAT两种检测方法能相互补充,极大降低了输血感染病原体的残余风险,保障输血安全。NAT能进一步缩短血液传染性病毒的检测“窗口期”,检出隐匿性病毒。     

Immunoassays at a quartz-liquid interface: theory, instrumentation and preliminary application to the fluorescent immunoassay of human immunoglobulin G

The theoretical basis and instrumental requirements of an optical detection technique for monitoring antibody-antigen reactions at a quartz-liquid interface are described. The antibody is covalently immobilized on the optical surface of a planar, fused-quartz waveguide and reacted with antigen solution. A light beam is internally reflected within the waveguide and penetrates into the solution only a fraction of the wavelength of the incident light. This is the evanescent wave which interacts optically with the growing number of antigen-antibody complexes but minimally with the bulk solution. A two-site immunofluorescent assay for human IgG measurement is described using fluorescein as the label. The assay detection limit is approximately 0.8 micrograms/ml and individual fluorescence measurements are completed within 10 min. It is expected that this evanescent wave immunoassay should have wide applicability in both routine and research fields.