The immuassay handbook parte64


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The immuassay handbook parte64

  1. 1. 585© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. The VITROS® 350 Chemistry System, VITROS® 4600 Chemistry System, and VITROS® 5600 Integrated Sys- tem are automated, random access clinical chemistry sys- tems that have the capability to perform immunoassays using Immuno-Rate technology developed by Ortho Clinical Diagnostics (OCD), part of the Johnson & John- son Family of Companies. The Immuno-Rate and clinical chemistry assays use dry slide technology (MicroSlides™), which was introduced in 1978 by the Eastman Kodak Company as an extension of its photographic technology. These coated, multilayer thin-film elements greatly sim- plify the number of operations performed by the cus- tomer since all the reagents required for an assay are contained within a postage stamp-sized MicroSlide™. The VITROS® 4600 Chemistry System offers extended immunoassay and special chemistry capability using MicroTip™ technology, while the VITROS 5600® Inte- grated System provides a truly integrated testing platform by combining MicroSlide and MicroTip technologies with VITROS Microwell™ heterogeneous immunoassay technology. The VITROS analyzers are floor-model instruments (Figs. 1–3) with high throughput and sample capacity. The VITROS 350 can perform any of 45 MicroSlide assays currently available, while the VITROS 4600 Chemistry System supports an additional 35+ currently available assays in MicroTip format. The VITROS 5600 Integrated System supports 120+ currently available assays in MicroSlide, MicroTip, and Microwell formats. The VIT- ROS analyzers use direct (primary) tube sampling and positive sample identification, and can be incorporated into totally automated laboratory instrumentation systems (with results sent to central laboratory information sys- tems). Microslide reagents are provided in a dry, chemis- try-specific thin-film format. Only sample addition is required to initiate reactions for conventional clinical chemistry analytes, while Microslide-based immunoassays (Immuno-Rate assays) use an additional, automated wash step for bound-free separations prior to analyte detection; potentiometric assays represent a third category of Microslide assays and require the addition of both sample and a reference fluid. In addition to the dry MicroSlides, the VITROS 4600 Chemistry System and VITROS 5600 Integrated System also have ready-to-use liquid reagent packs supplied by OCD to provide an extended MicroTip assay menu. Immuno-Rate immunoassays use either a competitive or immunometric mechanism. Competitive assays include those for digoxin, phenytoin, phenobarbital, and carbam- azepine, while C-reactive protein is measured using an immunometric assay. MicroTip immunoassays are liquid homogeneous assays which use either enzyme multiplied immunoassay technique (EMIT™) or turbidimetric technology for therapeutic drugs, drugs of abuse, serum proteins, and a number of other analytes, including high sensitivity C-reactive protein (hsCRP), hemoglobin A1c (d%A1c), and rheumatoid factor (RF). These assays can be randomly ordered among the normal clinical assays, thus giving a laboratory the capability of running chem- istries using either potentiometric, colorimetric, rate, Immuno-Rate, MicroTip technology (on the VITROS 4600 Chemistry System and VITROS 5600 Integrated System), or MicroWell technology (on the VITROS 5600 Integrated System)—in any order, at any time. VITROS® Immuno-Rate and MicroTip™ Assays John W. Backus ( Susan J. Danielson David A. Hilborn C H A P T E R 7.14 FIGURE 1 VITROS® 350 Chemistry System (The color version of this figure may be viewed at FIGURE 2 VITROS® 4600 Chemistry System (The color version of this figure may be viewed at
  2. 2. 586 The Immunoassay Handbook Typical Assay Protocols For Immuno-Rate assays, both competitive and immuno- metric assays are performed automatically starting with the application of 11µL of a serum or plasma sample to a dry chemistry slide into which reagents specific for each assay have been incorporated. The slides are then incu- bated for 5min at 37°C to allow for equilibration of binding reactions. After incubation, 12µL of a wash solu- tion is applied to the slide. This wash solution serves a dual purpose. It separates bound reagents from free reagents using the flow of the fluid to move mobile (non- bound) reagents out of the detection area of the slide and also initiates the enzymatic reaction required for the detection reaction because it contains the enzyme sub- strate. Following the wash step, there is a second 2.5min incubation at 37°C during which reflectance densitome- try measurements are made at regular intervals. The rates of these reactions can be correlated to the concentration of analyte in the original serum sample. From the appli- cation of the first sample to its prediction requires approximately 8–9min. MicroTip assays are performed automatically with the application of 2–16.7µL of a serum or plasma sample to a disposable cuvette into which reagents specific for each test have been added. The cuvette is then incubated at 37°C to allow for thermal equilibration. After this time, a second reagent is added to the cuvette. Following the addi- tion of the second reagent, there is a second incubation at 37°C during which spectrophotometric measurements are made at regular intervals. The rates or endpoints of these reactions can be correlated to the concentration of analyte in the original serum sample. From the application of the first sample to its prediction requires 8–16min, depending on the assay requirements. Product Features G VITROS systems are self-contained and fully auto- mated. They are capable of performing both immuno- assays and standard clinical chemistry tests in batch or random access fashion. G Less than 17µL of sample is required for each MicroSlide- or MicroTip-based immunoassay. G For Immuno-Rate assays, MicroSlide reagents (other than a standard wash solution) are stored on the instrument for up to one week. G For MicroTip immunoassays, reagents are stored in ready-to-use liquid reagent packs on the instrument for up to four weeks. G There is no risk of carryover from sample to sample or reagent to reagent. Disposable tips are used for indi- vidual metering steps for both Immuno-Rate and MicroTip assays. G The integrity of patient results is maintained, and sample repeats and redraws are minimized by the presence of Intellicheck™ Technology which includes liquid level sensing, sample clot and bubble detection, sample and reagent aspirate, and dispense verification features on the analyzers. G VITROS systems provide on-board dilution capability for improved labor optimization and error reduction. G VITROS systems do not require any plumbing to a water source or drain to execute testing and manage processing waste. G VITROS systems allow for bidirectional interfacing with hospital laboratory information processing sys- tems, supporting Broadcast Download, (and Host Query on the VITROS 4600 Chemistry System and VITROS 5600 Integrated System), allowing for increased laboratory productivity. In addition, the VITROS 4600 Chemistry System and VITROS 5600 Integrated System: G Utilize MicroSensor™ technology to measure sample quality indices for hemolysis, turbidity, and icterus without using additional samples, reagents, disposables, or slowing system throughput. G Support remote, real-time system diagnostics using e-Connectivity which can reduce time spent on maintenance. G Do not require the use of fixed probes or reagent mix- ing assemblies. Assay Principle Immuno-Rate assays are based on heterogeneous competi- tive and immunometric methods adapted to thin-film for- mats (Fig. 4). In general, the layers are formed as follows. Buffering agents are placed in a cross-linked gelatin layer that comprises the lowest layer of the thin-film format. When sample is applied, the buffer is rehydrated and helps control the pH at which the immunoassay binding reactions and subsequent enzyme reactions occur. Over the gelatin layer is an isotropically porous polymeric bead-spreading layer whose function is to accept the serum or plasma sample and assure that it spreads to an area so that the surface den- sity of analyte is constant. The non-porous beads that com- prise this layer are about 30µ in diameter, leaving ample interstitial capillary space so the sample is accepted rapidly (a few seconds). This layer also may contain buffer, stabilizers for enzymes and antibodies, dyes that can be developed by the enzyme label, and other reactants as needed. Antibodies that have been covalently immobilized on polymeric beads (about 1µ in diameter) may also be placed in this layer or FIGURE 3 VITROS® 5600 Integrated System (The color version of this figure may be viewed at
  3. 3. 587CHAPTER 7.14 VITROS® Immuno-Rate and MicroTip™ Assays alternatively in a thin receptor layer which lies between the gelatin layer and the spreading layer. For competitive assays, the enzyme-labeled drug (horseradish peroxidase) is coated in a thin layer at the top of the bead-spread layer in order to prevent binding to the antibody before the serum sample is applied. For immunometric assays, where binding cannot occur in the absence of the analyte, the enzyme-label is incorporated into a receptor layer. Binding reactions are initiated upon sample addition. Because of the small spaces between the beads of the spread layer, diffusional distances are very small (a few microns) so that equilibrium is reached rapidly (within a few min- utes) with no agitation necessary. After 5min incubation at 37°C, a wash solution is applied. The wash fluid flows by capillary action from the point of application through the read region of the slide to the periphery of the slide. Wash is efficient, a result of bulk fluid movement being the primary mode of molecular movement within the porous spreading layer and the small capillary structure within the spreading layer which directs fluid flow away from the point of application. Unbound molecules are carried away in the direction of fluid flow from their immobilized coun- terparts. There is little or no difference in movement between high and low molecular weight materials. Thus, a 12µL volume is sufficient for the bound-free separation required by the heterogeneous assay format. The wash removes unbound material and initiates the enzyme reac- tion used for detection. The bound horseradish peroxidase produces a blue color. Rate measurements are made using reflectance densitometry at 670nm during a 2.5min incu- bation (at 37°C) following the wash solution application. Movement of an inert magenta dye in the slide is auto- matically monitored to ensure that the wash step has been completed. MicroTip immunoassays are based on homogeneous competitive and turbidimetric methods using traditional liquid reagents. In general, two liquid reagents in an inte- grated reagent pack are available for each of the Micro- Tip immunoassays. The reagent packs are stored refrigerated on the analyzer. The analyzer automatically uncaps and recaps the reagents when required. The assays themselves are carried out in disposable spectrophoto- metric cuvettes that are incubated at 37°C. A typical assay protocol uses a disposable pipette tip to add about 100µL of the first liquid reagent to a cuvette. The sample is pipetted (2–16.7µL) using a specially designed disposable MicroTip, which provides the necessary metering preci- sion. This tip also is used to mix the reagents and sample. After a typical 5min incubation time to allow for thermal equilibration, the second reagent is added and mixed using a disposable tip. Incubation continues at 37°C while spectrophotometric or turbidimetric readings are taken at prescribed times. Therapeutic drugs are measured using EMIT™ technology while serum protein and other assays (e.g. high sensitivity CRP, %A1c, and rheumatoid factor) are measured turbidimetrically. Samples are automati- cally diluted if required by the specific assay protocol. Because MicroTip assays are processed using disposable tips, cuvettes, and reagents, the maintenance, cost, and carryover associated with the use of reusable cuvettes, fixed probes, water, wash reagents, plumbing, drains, and mixing assemblies are eliminated. MicroTip technology also eliminates sample and reagent carryover that may occur with sample probes, mixing devices, and reusable cuvettes. Calibration Immuno-Rate assays use a stored, lot-specific calibration function that has been factory determined and delivered to the instrument using a calibration disk supplied by OCD. Wet calibrations are performed on the analyzers using a 3-level multianalyte calibration set available from OCD. A new lot of MicroSlides is calibrated when it is first used on an instrument. Subsequent re-calibrations are done every 3–6 months. Assays can be purchased in 18 or 50/60 slide cartridges. Unopened cartridges are stored refrigerated or frozen for up to 18 months. FIGURE 4 Principle of competitive binding VITROS® Immuno-Rate assays.
  4. 4. 588 The Immunoassay Handbook MicroTip immunoassays use multiple level calibrators to generate a calibration curve on the analyzer. Calibra- tions are good for at least 4 weeks or until there is a change in the lot of assay reagents. Calibrator kits have a shelf-life of greater than 1 year. The liquid reagent packs contain fluids for 50 assays. The typical shelf-life of an unopened refrigerated reagent pack is greater than 1 year. Opened reagent packs can be stored on analyzer for typically 4 weeks. Antibodies All VITROS Immuno-Rate assays use mouse monoclonal antibodies that have been covalently immobilized to small (1µ), uniform-sized copolymeric latex beads. The C-reactive protein (CRP) assay contains both a monoclonal anti-CRP antibody conjugated to horseradish peroxidase and a deriva- tive of phosphorylcholine covalently bound to the 1µ latex beads in place of a second monoclonal antibody. MicroTip immunoassays use either monoclonal or poly- clonal antibodies. Turbidimetric (agglutination) assays use uncomplexed antibodies or antibodies coupled to latex particles. EMIT assays use uncomplexed antibodies that have been selected for their ability to inhibit specific drug- enzyme conjugates in the absence of free drug. Separation In Immuno-Rate assays, separation of bound enzyme-label from free enzyme-label is accomplished by application of a wash solution as described above. No separation is required in the homogeneous MicroTip assays. Signal Generation and Detection The Immuno-Rate assays use horseradish peroxidase as the enzyme label. Its reaction is initiated by the addition of the wash fluid, which contains hydrogen peroxide and an electron transfer agent, 4-hydroxyacetanilide. The reaction results in a blue color due to the oxidation of a leuco dye that is incorporated in one of the thin-film layers. Detection of the rate of blue color formation is by periodic reflectance densitometry reads at 670nm. Only the color development at the center of the slide (washed region) is read. The MicroTip immunoassays for therapeutic drugs (EMIT technology) use drug conjugates of glucose-6- phosphate dehydrogenase (G6P-DH). The reaction rate is monitored as 340nm to detect the formation of NADH (NAD is a cofactor of G6P-DH). The turbidimetric MicroTip assays measure the agglutination reactions at a variety of wavelengths. Data Processing Once a rate or end-point has been calculated by internal algorithms, it is transformed into a predicted concentra- tion using the on-board calibrations described above. If the predicted concentration is outside the pre-determined calibration range either at the low or high end, the opera- tor is alerted through an error message (flag). If the sample is beyond the high end of the concentration range, the sample can be automatically diluted and re-processed by the analyzer. Patient reports are flagged if there is not enough sample volume for the assay, and, for Immuno- Rate assays, if the sample is not applied to the thin-film, or if the wash fluid is not applied. Quality control fluids for immunoassays are also available from OCD. They can be run daily with the results tabulated and/or plotted in a variety of customer-specified formats. Interfacing to Laboratory Information Systems An RS232-C serial interface with bidirectional capabil- ity, along with the necessary software, is available to link the VITROS chemistry systems with central laboratory information management systems. The instruments can be interfaced with lab automation consisting of a variety of transport systems that automate the pre- and post- analytical processes including centrifugation, aliquoting, sample integrity checking, stopper removal and recap- ping, sample sorting, and sample storage. VITROS sys- tems can also be connected to a variety of automation systems, including the enGen™ Automation solution from OCD. The design of these systems is centered around a concept in which the metering track is extended so that the disposable sample tip can sample from a tube while it is on the automation system transport track, thus eliminating the need for robotics to move tubes to and from the sample supply and for extra aliquot tubes. The VITROS Systems retain the capability to process urgent STAT samples in a manual mode. Further Reading Blasutig, I.M., Jung, B., Kulasingam, V., et al. Analytical evaluation of the VITROS® 5600 Integrated System in a pediatric setting and determination of pediatric reference intervals. Clin. Biochem. 43, 1039–1044 (2010). Curme, H.G., Columbus, R.L., Dappen, G.M., et al. Multilayer film elements for clinical analysis: General concepts. Clin. Chem. 24, 1335–1342 (1978). Danielson, S.J. Thin-film immunoassays. In: Immunoassays (eds Diamandis, E.P. and Christopoulos, T.K.), 505–535 (Academic Press, San Diego, 1996). Danielson, S.J. and Hilborn, D.A. Single-layer and multilayer thin-film immunoas- says. In: Principles and Practice of Immunoassays (eds Price, C.P. and Newman, D.J.), 545–577 (Stockton Press, New York, 1997). Kwong, T., Meiklejohn, B., Bodman, V., et al. Performance of a new digoxin thin-film immunoassay in a hospital setting. Clin. Chem. 41, S196(A) (1995). Wu, A., Harmoinen, A., Chamber, D., et al. Comparison of a thin-film immunoas- say for C-Reactive Protein with a turbidimetric method. Clin. Chem. 40, 1018(A) (1994).