The development of immunochemical techniques is one of the most rapidly emerging fields of study for biological and medical research. The recent parallel establishment of reliable immunological methods for producing good quality monoclonal and polyclonal antibodies, and of techniques for complexing proteins onto gold particles to form immunological probes, now allows researchers and medical workers to investigate a vast range of antigens in cells and tissues. Virtually every biological and microscopical laboratory has the ability to perform these investigations simply, effectively, and safely through the availability of high quality protein-gold complexes.

With increasing incidences of infectious and chronic devices, development of novel assays in various autoimmune diseases as oncology and cardiology, and applications in diagnosing drug monitoring tests, toxicology testing, infectious diseases, and detection of cancer biomarkers, immunochemistry instruments are expected to grow further in adoption and install base. There has been a string of favorable trends that have fueled the growth in this segment, including a rising demand for non-invasive diagnostics techniques, a growing need for reagents, analytical techniques and sensitive assays, a wide range of accessible reagents, and identification and introduction of novel biomarkers.

Technology Trends

Chemiluminescence immunoassay. In terms of technology trends, there has been an upsurge in the adoption of chemiluminescence immunoassay (CLIA) systems, which are replacing older versions. Smaller labs still opt for fluorescent immunoassay (FIA) systems or semi-automated CLIA systems. The future will see CLIA as a growing analyzer segment with tremendous focus on areas such as infectious disease testing and autoimmune and oncology segments. CLIA provides a sensitive, high throughputw and economical alternative to conventional colorimetric methodologies, such as ELISA. The non-radioactive nature of the technique enables it to replace RIA in several immunoassay applications in varied disciplines, including microbiology, clinical laboratory, and therapeutic medicine.

Industry Speak
Advances in HbA1c Testing

HbA1c is an important test recommended by the American Diabetes Association and other diabetes organizations worldwide, for diagnosis and management of patients with diabetes mellitus. Currently, there are several methods available for measuring HbA1c including HPLC, immunoassays, and enzymatic assay.

HPLC is considered a gold standard method but has low-throughput and high-cost. Many studies and NGSP reported that the HPLC results show interference with various hemoglobin variants; sometimes, no results or abnormally high results are obtained in cases with abnormal hemoglobin. In such cases, the chromatograms have to be scanned by experts before commenting on the results.

All available immunoassays require prior hemolysate preparation manually before it can be analyzed by chemistry analyzer. They use dual channel testing which means separate GHb, THb analysis. Results are achieved by running both assays in parallel with chemistry analyzers. Immunoassays highly specific for antigen-antibody reactions are still found to be affected by the presence of Hb variants and high HbF.

Enzymatic HbA1c assay is a single channel test for THb and GHb and reports %HbA1c values directly, without the need for a separate THb test. HbA1c assay shows good correlation with HPLC and immunoassays. The enzymatic HbA1c test uses two ready-to-use liquid stable reagents and do not contain latex particles; hence do not coat analyzer cuvettes and lines which is a common problem seen with immunoassays.

Sysmex HbA1c enzymatic assay is unique in itself. Since it uses a single channel, it has on-board hemolysis and shows no interference with the labile forms of GHb, acetylated or carbamylated Hb. It has an excellent correllation of HPLC. An uique feature of Sysmex that HbA1c and plasma glucose can be performed simultaeneously with same fluoride vial. So it can be conlude that, Enzymatic HbA1c has all the advantages of both the HPLC and immunoassay methods in the areas of accuracy, specificity, applicability to chemistry analyzers and yet is cost effective, simpler, and faster and has less interference.

Shobhit Jain
Assistant Manager-Biochemistry,
Sysmex India Private Limited

Automated CLIAs are useful for the detection of hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) antigens/antibodies in blood donor screening. Chemiluminescence detection methods have provided clinicians and researchers interested in oncology with efficient tools for early detection and accurate diagnosis of cancer.

Enzyme-linked immunosorbent assay. ELISA systems and open systems are witnessing an adoption with their open platform capabilities and larger test menu and affordability. FIA and CLIA systems tend to be more expensive, which has worked in favor of ELISA systems. However, with consolidation, most laboratories are converting to high-throughput systems over ELISA systems which lack sensitivity and specificity. This has spurred vendors to offer less-expensive immunoassay or standalone clinical chemistry systems.

Rapid immunodiagnostic tests for Ebola virus have been developed that use lateral flow immunochromatographic technology, and antigen/antibody detection through ELISA. Rapid antigen tests for influenza A and B, Clostridium difficile, and selected other microbes have also been adopted widely. Vendors are including upgrades, adding automation capabilities to existing modules and offering long reagent rental contracts to retain the install base.

Multi-parametric assays. With the complexity of biological systems and the wide variance in individual genetic and metabolic backgrounds, tests based on single biomarkers may have low sensitivity and/or specificity. The increasing awareness of the multifactorial nature of various diseases and pathological states calls for simultaneous, time-saving and cost-effective measurement of multiple analytes. The current trend is moving toward multi-parametric assays, and a large number of companies are exploring the use of multiplex technologies using protein and peptide arrays for autoimmune diagnostics. The trend toward the development of tests based on the simultaneous measurement of multiple biomarkers has shown significant improvement in assay performance and clinical utility. A number of multiplex technologies are currently available for biomarker testing, including suspension microspheres, planar microarrays, and microplate-based microarrays. Application of multiplex ELISA in cancer diagnosis has enabled simultaneous analysis of multiple cancer biomarkers.

This multi-marker strategy could be then translated into more robust diagnostic algorithms, better-fitting prognostic models and more effective population screening. Of note, multiplex immunoanalysis is perfectly suited for diagnostics of immune system disorders. A broad selection of assays for both the clinical chemistry and immunoassay allow these analyzers to screen the sample for multiple biomarkers. The number of tests offered for an instrument promotes the installed base and future reagent sales. The competitive advantage lies with companies that offer a broad testing menu on an integrated and automated system. Advances in microfluidic systems enable the sensitive quantification of proteins in a small sample volume by using parallel detection systems. In contrast to multiplexing, this parallel approach prevents cross-reactivities of detection antibodies and negative effects on sensitivity of the assays.

Every year, in vitro diagnostics (IVD) manufacturers launch updated products specially designed for use in clinical laboratories. There is a critical need for sensitive and highly specific immunoassays of clinical samples using minimal sample volume. A wide range of immunochemistry assays are now used to measure proteins (including antibodies), hormones, drugs, and other analytes. Fully automated immunoassay systems are now found in clinical laboratories. These automated analyzers perform batch or continuous access and fast processing of specimens containing both large and small molecular weight analytes. Companies are now adding immunoassay components or clinical chemistry systems to existing analyzers in order to make it an integrated analyzer. Offering large discounts on the existing list price of these analyzers has worked in favor of the laboratories that have stringent budgets.

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