The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes.

BACKGROUND: A multicenter study conducted in Southeast Asia to derive reference intervals (RIs) for 72 commonly measured analytes (general chemistry, inflammatory markers, hormones, etc.) featured centralized measurement to clearly detect regionality in test results. The results of 31 standardized analytes are reported, with the remaining analytes presented in the next report. METHOD: The study included 63 clinical laboratories from South Korea, China, Vietnam, Malaysia, Indonesia, and seven areas in Japan. A total of 3541 healthy individuals aged 20-65 years (Japan 2082, others 1459) were recruited mostly from hospital workers using a well-defined common protocol. All serum specimens were transported to Tokyo at -80°C and collectively measured using reagents from four manufacturers. Three-level nested ANOVA was used to quantitate variation (SD) of test results due to region, sex, and age. A ratio of SD for a given factor over residual SD (representing net between-individual variations) (SDR) exceeding 0.3 was considered significant. Traceability of RIs was ensured by recalibration using value-assigned reference materials. RIs were derived parametrically. RESULTS: SDRs for sex and age were significant for 19 and 16 analytes, respectively. Regional difference was significant for 11 analytes, including high density lipoprotein (HDL)-cholesterol and inflammatory markers. However, when the data were limited to those from Japan, regionality was not observed in any of the analytes. Accordingly, RIs were derived with or without partition by sex and region. CONCLUSIONS: RIs applicable to a wide area in Asia were established for the majority of analytes with traceability to reference measuring systems, whereas regional partitioning was required for RIs of the other analytes.

Automation of biochip array technology for quality results.

BACKGROUND: Proteomics' requirement for simultaneous measurement of multiple markers is now possible with biochip array technology. Many laboratories utilise in-house, manual procedures for biochip fabrication and sample testing. Reproducibility and standardisation of biochip processes is vital to ensure quality of results and offer the best tool for elucidation of complex relationships between multiple proteins in diseased conditions. METHODS: Various novel control checks have been implemented in biochip fabrication, reagent manufacture, automation and imaging processes for the Evidence analyser. Reference spots enable location of discrete test regions on the surface of the biochip and simultaneous quantification of multiple markers. Performance and standardisation methods are presented. RESULTS: Formulation of dispense solution for discrete test regions had a direct effect on their shape, stability and integrity on the biochip surface. Assays for fertility hormones and drugs of abuse demonstrated excellent precision, stability and comparison with other commercial methods. CONCLUSION: Control processes employed in the manufacture and analysis of Evidence components ensure reproducibility of assays for a range of routine and novel markers.

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue.

The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 microl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

A time-resolved fluorescence immunoassay (DELFIA) increases the sensitivity of antigen-driven cytokine detection.

In an effort to improve the quantification of the low levels of cytokines released in response to antigenic stimulation of T cells, a sandwich dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) was developed and compared to a standard sandwich ELISA. The DELFIA enhanced the sensitivity of a mouse IL-2 assay 8- to 27-fold, and a human GM-CSF assay 10-fold, as compared to colorimetric ELISA. The increase in sensitivity allows for the use of lower sample volumes per well, and the ability to run more assays per supernatant sample. This sensitive, nonisotopic alternative to other cytokine detection methods will be useful for those researchers wanting to quantitate low levels of antigen-driven cytokine production.

Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method.

Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection.

The development of non-animal-based bioassays for cytokines and growth factors.

Cytokines (including growth factors) exist as a family of proteins that possess a vast array of pleiotropic biological activities and therapeutic potential. As with any biological therapeutic, appropriate assessment of biological potency is vital to the development of cytokines as medicinal products. In early investigations of cytokine activity, in vivo bioassays were used to assess the whole body effects of cytokines in animals. The identification of the cellular targets of each cytokine allowed the extraction of tissue and the use of cells to produce in vitro bioassays, with the drastic reduction in the number of animals required. The discovery of cytokine-responsive tumours introduced a range of immortal, homogeneous tumour cell lines that are dependent on cytokines for proliferation. This allows cell lines to be made available for general distribution to laboratories internationally for bioassays, without the need for animals. More recently, the use of recombinant DNA technology to clone the specific receptor for the cytokine required for bioassay into an unresponsive cell line has led to a highly specific 'receptor-transduced' cell line. The most recent advances include the development of receptor signalling-based biochemical bioassays for cytokines.

Cytokine bioassays: an overview.

Although eclipsed in recent years by immunoassays and molecular biology techniques, bioassays remain a vital research tool for cytokine biology. Like any type of biological system, cytokine bioassays require meticulous technique to obtain accurate and reproducible results. However, these minor difficulties are more than compensated for by their exclusive detection of biologically active molecules, a feature as yet unmatched by other assay methods.

Analytical characterisation of cytokines and growth factors.

Cytokines (including growth factors) are playing an ever-increasing role in the therapy of human disease. The rapidity by which new cytokines are discovered, cloned and enter the clinics has placed a burden on control authorities and manufacturers to ensure their safety, quality and efficacy. The appropriate characterisation of these proteins plays a vital role in ensuring the development of cytokines as useful therapeutic agents. Highly sophisticated physicochemical techniques exist that can produce specific information about the structure and composition of cytokines including methods such as nuclear magnetic resonance (NMR) and mass spectrometry. However, even a bank of such techniques cannot yet predict the biological activity of cytokines. Biological assays are essential to assess the potency of cytokines and can take several forms, from in vivo assays to in vitro cell-line bioassays and more recently biochemically-based assays. Bioassays require a biological reference standard to define an appropriate unit that can be used to assess the potency of a biological therapeutic. However, bioassay-derived potency is a quality issue and should not have an impact on the perceived efficacy of a biological therapeutic.

The immunoassay of cytokines and growth factors in biological fluids.

There are a number of problems associated with the development of standards suitable for use in the most commonly used assays to detect cytokines in biological fluids. These problems include: (i) the failure of some MoAbs used in immunoassays to detect all different <> of recombinant or natural material; (ii) the use of many different MoAbs, with different specificities, in different immunoassay kits, and (iii) the detection of non-active cytokines (fragments, inhibitors, receptor antagonists, etc.) in these immunoassays. As a result, it is possible to have biologically active material which is not detected in these immunoassays. Alternatively, biologically inactive material can be detected in these assays and is indistinguishable from biologically active material. In addition, the use of different antibodies with different specificities, affinities and avidities in different kits designed to detect the same biological materials results in markedly different sensitivities and specificities. Many of these same concerns can be raised for the use of bioassays for detection of molecules in biological fluids. The solution will not be simple (if possible at all). In most cases, the immunoassay kits are designed to detect <> material in biological fluids, but are made with MoAbs against recombinant material. Because of the markedly different specificities, affinities, etc. of the MoAbs in these kits, their standardization is possible only with a highly purified preparation of natural material. For the assay of recombinant materials, immunoassays should be specifically designed with the recombinant material in mind (i.e. the MoAbs made specifically against the recombinant material to be detected or shown to bind effectively with the recombinant material). Importantly, it should be made clear to investigators using different immunoassays that: (i) the reporting of biological material detected using immunoassays can only be made in units of weight (i.e. ng/ml); (ii) because of the detection of biologically active and inactive material using immunoassay kits these assays cannot be directly compared to bioassays or their results represented as <>; (iii) because of the difference in specificity and sensitivity of the different reagents used in different immunoassays, the results from different assays cannot be directly compared, and (iv) because of these same considerations, comparison of different > of materials within a single immunoassay is also not possible. The use of specific immunoassays for recombinant material in combination with bioassays and the use of cytokine standards, made from highly purified natural material, would help to standardize the results in this field.

Spectroscopic characterization of chemokines: relevance for quality control and standardization.

Chemokines are mediators of inflammation and trafficking of cells of the immune system including a pivotal role in the recruitment and activation of leukocytes. Due to their involvement in a variety of disease processes, chemokines are potential therapeutic targets. The use of chemokines as pharmaceuticals will require that the folded state and the association properties of the protein are well characterized. In this report, we describe the utility of nuclear magnetic resonance spectroscopy as a tool to study these aspects of chemokine structural properties.

Bioassays for the characterisation and control of therapeutic cytokines; determination of potency.

The primary value of bioassays is that they alone directly assess the biological activity of bioactive substances and products like cytokines. Appropriately designed bioassays reflect the fundamental aspects of the biological activity of a cytokine molecule, including ligand-receptor binding, signal transduction processes (often poorly understood) and the final observed biological effects. Biological assays therefore complement physicochemical and biochemical procedures which normally only assess precise molecular structural features of cytokines. Bioassays provide valuable information concerning the potency of cytokine products. This is essential for evaluating batch-to-batch consistency, appropriate formulations and stability. Bioassay data are crucial at all stages in the development of cytokine products, from early research to final quality control of finished product. However, the type and design of bioassays may differ according to the information required and its intended use. The assays may or may not directly relate to the clinical use of the product. Bioassays can be difficult to perform and time consuming, although this often reflects bad assay choice and/or design. Correct analysis of the assay results is essential if valid data are to be obtained. Standardisation, using correctly calibrated primary and secondary standards, is essential. In vivo bioassays are normally more unreliable than in vitro procedures, but in some cases in vitro systems are either not available or do not address important biological characteristics of a product. Bioassays must be validated for their intended purpose and for the types of samples to be measured. Appropriate statistical analysis should be used to derive the significance and specifications of results. This needs to address both variability in samples and assay performance. Specifications (limits) for product acceptability need to be derived from real data using several batches of cytokine product.