Low light level in vitro monitoring of cellular and antigen-antibody reactions using a photon detection camera system — New perspectives for clinical diagnosis and research

Summary This article briefly describes the use of a photon counting system (ARGUS-100) in the detection of low levels of light. The ARGUS-100 was used in determining ATP in cell sections from tumor tissues and in measuring a luminescenceenhanced immunoluminometric assay, using ferritin as the analyte, based on the luminol-peroxide-4-iodophenol reaction with peroxidase as the enzyme. The aim is not so much the presentation of data, but rather to show the potentials of the photon counting camera in increasing our knowledge of the cellular and subcellular levels, as well as lowering the detection limits in already sensitive systems, such as immunoassays.Supported by the Bundesministerium für Forschung und Technologie (DFVLR grant No. 01Z08801)     

Plasmodium falciparum immunodetection in bone remains of members of the Renaissance Medici family (Florence, Italy, sixteenth century)

Medical accounts and ancient autopsy reports imply that tertian malarial fevers caused the death of four members of the Medici family of Florence: Eleonora of Toledo (1522-1562), Cardinal Giovanni (1543-1562), don Garzia (1547-1562) and Grand Duke Francesco I (1531-1587).All members of the Medici family hunted in the endemic malarial areas of Tuscany, such as the marshy areas surrounding their villas and along the swampy Maremma and were, therefore, highly exposed to the risk of being infected by Falciparum malaria. To determine if the original death certificates issued by the court physicians were correct, we carried out immunological investigations and then compared the biological results to the historical sources.Bone samples were examined for the presence of Plasmodium falciparum histidine-rich- protein-2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH) using two different qualitative double-antibody immunoassays.Our findings provide the first modern laboratory evidence of the presence of P. falciparum ancient proteins in the skeletal remains of four members of the Medici family. We confirm the clinical diagnosis of the court physicians, using modern methods.Finally, this study demonstrates that immunodetection can be successfully applied not only to mummified tissues but also to skeletal remains, thus opening new paths of investigation for large archaeological series.     

Highly-sensitive troponin I is increased in patients with gynecological cancers

Objectives

To investigate troponin I (TnI) in patients with gynecological cancers.

Methods

Highly-sensitive (HS) and conventional TnI were measured in 25 patients with untreated ovarian cancer, 25 with endometriosis and 25 with benign masses.

Results

Both HS and conventional TnI were increase in cancer patients. Values above the cut-off were found in 44% and 16% cancer patients using HS and conventional TnI methods, respectively.

Conclusions

Cardiac involvement is frequent in patients with gynecological cancers and should be preferably assessed using HS troponin immunoassays.     

利用Excel电子表格制作质控图在标记免疫室内质控中的应用

目的探讨利用Excel电子表格制作标记免疫室内质控图的方法。方法利用Excel的表格制作与函数计算功能,在单元格中输入室内质控数据,自动计算平均值(x)、标准差(s)和变异系数(CV%),利用图表制作功能,设计制作出包括失控点在内的单值多点的定量质控图。结果将质控数据输入数据表中,计算机可将所有的测定点绘制在一张质控图上,图中的失控点与在控点区分明显。结论对于没有实验室信息系统的基层实验室,及一些需要手工操作的放射免疫项目的实验室,室内质控图对保证检验结果的可靠性具有重要作用。     

Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems

Cross-reacting material 197 (CRM₁₉₇), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM₁₉₇ proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM₁₉₇ molecules demonstrate that recombinant CRM₁₉₇'s expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM₁₉₇ expressed in alternative sources have the potential to replace CRM₁₉₇ expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM₁₉₇ should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM₁₉₇ carrier proteins for use in various conjugate vaccines.     

True Chromogranin A concentrations in plasma from patients with small intestinal neuroendocrine tumours

Abstract

Objective: The incidence of enteropancreatic neuroendocrine tumours (NET) is increasing. Chromogranin A (CgA) in plasma is a marker in patients suspected of NET tumours. CgA, however, is a precursor protein subjected to cellular processing that challenges quantitation and hence the use of CgA in diagnostics.

Materials and methods: CgA concentrations in plasma sampled from 130 well-characterized patients with small intestinal NETs and from 30 healthy subjects were measured with eight commercial CgA kits, an in-house radioimmunoassay (RIA) and a processing-independent assay (PIA). For the evaluation of diagnostic accuracy, we performed regression analyses and plotted receiver-operating characteristic curves (ROC). The specificity was further assessed by size chromatography.

Results: Five commercial assays (Thermo-Fisher, DRG Diagnostics, Eurodiagnostica (RIA and ELISA), and Phoenix), displayed a diagnostic accuracy with area under the curve (AUC) values >0.90, whereas three immunoassays (Yanaihara, CisBio RIA, and CisBio ELISA) discriminated poorly between disease stages (AUC: 0.60–0.78). Compared with the in-house assays, however, even the most accurate commercial immunoassay still missed patients with metastatic disease. Chromatography showed non-uniform patterns of large and small CgA fragments in plasma.

Conclusion: Available commercial immunoassays measure CgA in plasma with gross variability. Three commercial CgA immunoassays discriminate so poorly between health and disease that they should not be used. The highest diagnostic accuracy was obtained with processing-independent measurement of total CgA concentrations in plasma.