The genesis and evolution of bead-based multiplexing.

Multiplexed analysis has the advantage of allowing for simultaneous detection of multiple analytes in a single reaction vessel which reduces time, labor, and cost as compared to single-reaction-based detection methods. Microsphere-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of both protein and nucleic acid targets in multiple assay chemistries. After Luminex's founding in 1995, it quickly became the leader in bead-based multiplexing solutions. Today, xMAP Technology is the most widely adopted bead-based multiplexing platform with over 35,000 peer-reviewed publications, an installed base of approximately 15,500 instruments, and over 70 Luminex Partners offering more than 1300 research use kits as well as custom assay solutions. Because of the open architecture of the xMAP platform it has been implemented in a variety of applications that range from transplant medicine, biomarker discovery and validation, pathogen detection, drug discovery, vaccine development, personalized medicine, neurodegeneration, and cancer research.

Autoantibodies in systemic lupus erythematosus: comparison of historical and current assessment of seropositivity.

Systemic lupus erythematosus (SLE) is characterized by multiple autoantibodies and complement activation. Recent studies have suggested that anti-nuclear antibody (ANA) positivity may disappear over time in some SLE patients. Anti-double-stranded DNA (dsDNA) antibody titers and complement levels may vary with time and immunosuppressive treatment, while the behavior of anti-extractable nuclear antigen (ENA) over time is less well understood. This study sought to determine the correlation between historical autoantibody tests and current testing in patients with SLE. Three hundred and two SLE patients from the ACR Reclassification of SLE (AROSE) database with both historical and current laboratory data were selected for analysis. The historical laboratory data were compared with the current autoantibody tests done at the reference laboratory and tested for agreement using percent agreement and Kappa statistic. Serologic tests included ANA, anti-dsDNA, anti-Smith, anti-ribonucleoprotein (RNP), anti-Ro, anti-La, rheumatoid factor (RF), C3 and C4. Among those historically negative for immunologic markers, a current assessment of the markers by the reference laboratory generally yielded a low percentage of additional positives (3-13%). However, 6/11 (55%) of those historically negative for ANA were positive by the reference laboratory, and the reference laboratory test also identified 20% more patients with anti-RNP and 18% more with RF. Among those historically positive for immunologic markers, the reference laboratory results were generally positive on the same laboratory test (range 57% to 97%). However, among those with a history of low C3 or C4, the current reference laboratory results indicated low C3 or C4 a low percentage of the time (18% and 39%, respectively). ANA positivity remained positive over time, in contrast to previous studies. Anti-Ro, La, RNP, Smith and anti-dsDNA antibodies had substantial agreement over time, while complement had less agreement. This variation could partially be explained by variability of the historical assays, which were done by local laboratories over varying periods of time. Variation in the results for complement, however, is more likely to be explained by response to treatment. These findings deserve consideration in the context of diagnosis and enrolment in clinical trials.

An overview of various labeled assays used in medical laboratory diagnosis. Immune and non-immune assays.

In this review, some light is thrown on various labeled immunoassays that depend on antigen-antibody (Ag-Ab) reactions, including immunofluorescence, radioimmunoassay, and enzyme immunoassay (EIA or ELISA). Their definitions, principles, and applications are described, then they are discussed chronologically to show their stepwise development that led finally to full automation. Enzyme labeled immunoblot assays (Western blot, blot spot, and recombinant immunoblot assay), and luminescence (bioluminescence and chemiluminescence) are also discussed chronologically. Labeled assays, that do not involve Ag-Ab reaction but rather, utilizing biotin-streptavidin (BS) interaction and probe-target DNA interaction, are described, together with their applications for DNA/RNA detection and genotyping. Finally, included in the discussion were some luminescent labeled techniques that utilize the immune Ag-Ab reaction together with non-immune BS reaction, such as the luminescent oxygen channeling immunoassay, and its commercialized AlphaLISA, both eliminate the washing steps without sacrificing high sensitivity, or wide dynamic range.

A selected history and future of immunoassay development and applications in clinical chemistry.

BACKGROUND: The first immunoassay was described by Berson and Yalow in 1959. Their work resulted in their receipt of the Nobel Prize in Medicine in 1977. Since this introduction, immunoassays have evolved considerably. METHODS: There have been several milestones that have led to the proliferation of modern immunoassays. The development of monoclonal antibodies from mouse hydridoma cells by Millstein and Kohler (Nobel Prize in 1984) enabled the production of high quantities of antibodies with well characterized epitope specificity. The first homogenous immunoassay (no separation step required) was the Enzyme Multiplied Immunoassay Technique (EMIT), which enabled adaptation of this assay onto automated chemistry platforms. EMIT was also one of the first immunoassay that made use of non-isotopic labels. Other non-isotopic labels became available such as chemiluminescence to improve the analytical sensitivity of immunoassays. The advantages of high-sensitivity immunoassays have created expanded diagnostic roles for some existing assays such as thyroid stimulating hormone for hyperthyroidism, C-reactive protein for cardiovascular risk assessment, and other applications. The development of instrumentation capable of automated heterogeneous immunoassays (separation step to improve sensitivity) has enabled movement of this technology from the "special chemistry" sections of a clinical laboratory into the "core" laboratory with other high-volume testing. CONCLUSION: Today, immunoassays play a prominent role in the analysis of many clinical laboratory analytes such as proteins, hormones, drugs, and nucleic acids. The future involves development of assays with higher sensitivities which will enable the discovery of new biomarkers for disease diagnosis, and technology that will enable simultaneous multimarker analysis of tests whose needs are naturally grouped together (e.g., cytokines and allergens).

The exciting story of cardiac biomarkers: from retrospective detection to gold diagnostic standard for acute myocardial infarction and more.

This paper reviews the history of the contribution of the laboratory medicine to clinical cardiology and discusses the most important steps in this field. Until 20 years ago, the clinical laboratory only placed at the cardiologist's disposal a few assays for the retrospective detection of cardiac tissue necrosis, such as enzymatic methods for creatine kinase and lactate dehydrogenase activities. However, in the latter part of the 20th century, highly sensitive and specific assays, such as cardiac troponins, as well as assays for markers of myocardial function, such as cardiac natriuretic peptides, rapidly changed the scenario of clinical management of patients with cardiac diseases, assigning to the laboratory a pivotal role in the overall diagnostic flow. This is witnessed by the recent incorporation of these markers into international guidelines and in the redefinition of myocardial infarction. For the foreseeable future, new serum markers of myocardial ischemic, i.e. reversible, injury or related to coronary plaque instability and disruption are expected.

Inventions leading to the development of the diagnostic test kit industry--from the modern pregnancy test to the sandwich assays.

The universities are encouraged by the government nowadays to stimulate innovations and also to provide the proper machinery for assisting the protection and commercialisation of innovations. A better understanding of the innovation process may help to create an atmosphere suitable for inventions at the university. Examples can be taken from successful innovations previously made at the university. During the 1960's I made a series of inventions, which ultimately led to the development of the diagnostic test kit industry. The first, which I made as an undergraduate, was a simple and reliable test kit for diagnosis of pregnancy. This was followed by the solid phase radioimmunoassay and a solid phase assay for vitamin B12; next, the dual specific non-competitive sandwich assay and the in-vitro test for diagnosis of allergy, called RAST (Radioallergosorbent test). Organon in Holland with the pregnancy test kit, and Pharmacia in Sweden with test kits for radioimmunoassay, became pioneers among the diagnostic test kit industries. Pharmacia Diagnostics later became one of the leading diagnostic test kit companies in the world and has continued to be so in the field of allergy diagnosis. Each one of these inventions started with a few unique observations leading to a technical development. The pregnancy test as well as the allergy test emerged from the development of assay methods with unique qualities with the subsequent search for appropriate applications. The foreseeing of a commercial value on a future market was a very important step. This was followed by the search for a suitable industry interested to exploit the invention with its new business opportunity i.e. apply for a patent, produce and market the products, which in my case consisted of the necessary reagents and equipments for particular diagnostic tests. Finally, an agreement had to be settled between the entrepreneur and the inventors. This report describes these inventions and particularly discusses some crucial steps of the innovation processes.

Typing for human platelet alloantigens.

Antibodies to platelet alloantigens, and sometimes to isoantigens, induce severe clinical problems such as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP) and refractoriness to platelet transfusions (PTR). For example, NAIT affects approximately 1 in 5,000 live births. It is essential, therefore, to screen pregnant women for platelet antibodies in order to save babies' lives. Almost 40 years ago, two platelet alloantigen systems were discovered using relatively simple methods, namely the platelet agglutination test and the complement fixation test. However, these methods were not sensitive enough to identify all antibodies in mothers and patients, even in those with severe clinical problems. Tremendous effort has been devoted to establish more sensitive and reliable methods. In recent years, excellent new serological and immunochemical methods have been established and several new platelet antigen systems have been discovered. Simultaneously, newly developed molecular genetic techniques have been introduced for the typing and analysis of human platelet alloantigen systems. These methods allow DNA typing for cases in which serological typing is not available. In this article, the history of studies on human platelet alloantigen systems and isoantigens, the nomenclature of platelet alloantigen systems and their alleles, the present status of antibody detection and typing techniques and, finally, ethnic variations in platelet antigen profiles are reviewed.

Ligand assays: from electrophoresis to miniaturized microarrays.

The main developments in the "ligand assay" field in which I have been involved are traced. These include the original development of "first generation" competitive assays relying on radiolabeled analyte markers; the development of the first "second generation", noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first "third generation" miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter--applicable both to immunoassay and to DNA/RNA analysis--are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.

[Methods of saturation analysis in endocrinology]. / Metody saturacní analýzy v endokrinologii.

The article is a brief review of the development of the method of saturation analysis (MSA) from its beginning to nowdays. Our clinic participated at some stages on this development. Srámková introduced the RIA of GH in 1968, one year later RIA for prolactin and in 1973 RIA for insulin. The extention of the RIA principle to MSA was after its foundation applicated by Justová et al. for the determination of metabolites of vitamin D. Calcitriol, hormon of vitamin D-metabolites was analysed by radioreceptorassay (RRA). The same principle of RRA was applicated at the determination of receptors for insulin by Hilgertová. The application of MSA at our clinic is summarized in table 1. and 2. The home made methods and the choice of commercial kits were verified not only from the point of view of the demands on the quantitative parameters of the method but also from the point of view of the cost of a kit. At our clinic we follow the development of MSA of specific binding proteins (BP), trying to introduce or modify the described techniques f. e. the analyses of BP of GH (GHBP), BP for IGF-I (IGFBP-3, IGFBP-1). The extention of the analysed compounds leads to the precision in the diagnosis and more effective monitoring in the process of treatment. Generally, the article describes the utilization of MSA in laboratories interested not only in the routine work, but in the research activity as well.