Immunoassay system automation using

Automated chemistry analyzers have traditionally relied on photometers and spectrophotometers for measurement of absorbance. Alternative approaches now being incorporated into analyzers include reflectance photometry and fluorom-etry. Immunoassay systems have used fluorescence (IMX), chemiluminescence (Centaur and Immulite), and electrochemiluminescence (ELECSYS) to enhance sensitivity. Ion-selective electrodes and other electrochemical teclmiques are also widely used. Principles of these measurement techniques have been discussed previously (see Chapter 4). This section reviews the special features and application of the various approaches to automated analysis. 

Commercial application of the dendrimer-based reagent technology has been demonstrated by the successful development of The Stratus CS STAT fluorometric analyzer [5] marketed by Dade Behring Inc. This rapid automated point of care immunoassay system provides quantitative analysis of whole blood or preprocessed plasma samples via unit use assay test packs. Up to four test packs can be introduced for each sample. All reagents [5-9] required for specimen analyses are contained within the test packs. 

Figure 3.29.A shows a flow-cell of 20 iL inner volume used to hold immobilized anti-mouse IgG bound to a rigid beaded support (activated Pierce trisacryl GF-2000). The cell was used to develop a two-site immunoassay for mouse IgG by consecutive injection of the sample, acridinium ester-labelled antibody and alkaline hydrogen peroxide to initiate the chemiluminescence, which started the reaction sequence shown in Fig. 3.29.B. Regenerating the sensor entailed subsequent injection of an acid solution, which resulted in a determination time of ca. 12 min (this varied as a fimction of the flow-rate used, which also determined the detection limit achieved, viz. 50 amol for an overall analysis time of 18 min) [218]. The sensor was used for at least one week with an inter-assay RSD of 5.9%. Attempts at automating the hydrodynamic system for use in routine analyses are currently under way.

Matsunaga, T., F. Ueki, K. Obata, et al. 2003. Fully automated immunoassay system of endocrine disrupting chemicals using monoclonal antibodies chemically conjugated to bacterial magnetic particles. Anal. Chim. Acta 475 75-83. 

The plate washing can be conducted automatically if a microplate washing system is available. We used the Perkin Elmer-Getus Pro/Pette instrument with a Pro/Wash head and the Beckman Biomek 1000 Automated Laboratory Workstation in our enzyme immunoassays. If automated instrumentation is not available, liquid transfers and plate washing can be carried out by a 12-channel micropipet. 

However, in the long term, ELISA is an ephemeral format. Even when streamlined and automated, it has too many steps. Certainly we should realize that it will be replaced by other systems, the most exciting of which will be biosensors. Also, other formats offer a proprietary edge in the market place which will be very important in the maturation of immunoassay systems in the environmental field. Finally, different formats will lend themselves to different environmental problems. We should continually emphasize that the same reagents can be used in many formats. Possibly in small letters we also should caution that certain antibody characteristics may be more important in one format than another, that some formats are more resistant to matrix effects, and that relative cross reactivities of compounds can change as one changes the subtle principles upon which an immunoassay works. For this reason a clear choice of formats should be made before initiating validation studies. 

Photodiodes are used as detectors in many automated systems either as individual components or in multiples as an array. Photomultiplier tubes are required in many immunoassay systems to provide adequate sensitivity and fast detector response times for fluorescent and chemiluminescent measurements. Several approaches have been used for the electrooptical integration and packaging in different analyzers. A logarithmic amplifier or microprocessor and/or computer software converts transmittance to absorbance. Low-cost analog-to-digital converters with conversion times... 

Immunoassays are used to measure PSA and are commercially available. Most of them use nonisotopic labels, such as enzyme, fluorescence, or chemiluminescence. The majority of these assays are automated on an immunoassay system. Different assays and even the same assay with different lots of reagent may produce different results. The reasons for such differences are due to changes in assay calibration, production lot variation, assay reaction time, reagent matrices, assay sensitivity, and imprecision. Antibodies react with different PSA epitopes therefore, some antibodies react dissimilarly with the various molecular forms of PSA. Assays are classified as equimolar if they bind to free and cPSA equally and nonequimolar if they bind to free or cPSA differently. Examples of equunolar assays are the ACCESS... 

Serum AFP can be determined by immunometric assay using either a radioactive or enzyme label (see Chapter 54). Automated immunoassay systems to measure AFP using these techniques are available. The detection limit of AFP immunoassay is about 1 to 2pg/L. 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

Considerable effort was expended in the development of alternative technologies that did not require the use and measurement of radioactivity. A number of nonisotopic assays for T4 were subsequently developed commercially for use on fuUy automated immunoassay systems or for use with existing chemistry analyzers. According to a 2002 College of American Pathologists Ligand Assay Survey, more than 95% of laboratories now use a nonisotopic T4 method. A variety of different labels were used to construct these nonisotopic assays. Enzymes such as horseradish peroxidase, alkaline phosphatase, and [3-D-galactosidase were the most... 

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

To solve the problem, we have developed a highly specific and sensitive assay for C-peptide in serum and plasma using specific monoclonal antibody (MoAb) to the N-terminal of the C-peptide molecule. In this report, we describe the assay performance of C-peptide on the LUMIPULSE system. The system is a fully automated chemiluminescent enzyme immunoassay (CLEIA) system that uses AMPPD as a substrate for alkaline phosphatase and ferrite micro-particles as a solid phase. ... 

Tanaka, T. Takeda, H. Ueki, F. Obata, K Tajima, H. Takeyama, H. Goda, Y. Fujimoto, S. Matsunaga T. (2004), Rapad and sensitive detection of 17 P-estradiol in environmental water using automated immunoassay system with bacterial magnetic particles. J. Biotechnol, 108 153-159. 

This immunoassay system was engineered into a laboratory prototype device which includes all system components. The prototype is a compact (20 x 8 x 25 cm), self-standing device and has the potential for complete automation allowing its use in laboratory settings as well as in field conditions. In this work the prototype was used in a bench-top experimental set-up where the bacteria containing sample is fed into the immunoassay system device and the output is recprded on a PC-based data acquisition system. 

Immunosensors promise to become principal players ia chemical, diagnostic, and environmental analyses by the latter 1990s. Given the practical limits of immunosensors (low ppb or ng/mL to mid-pptr or pg/mL) and their portabiUty, the primary appHcation is expected to be as rapid screening devices ia noncentralized clinical laboratories, ia iatensive care faciUties, and as bedside monitors, ia physicians offices, and ia environmental and iadustrial settings (49—52). Industrial appHcations for immunosensors will also include use as the basis for automated on-line or flow-injection analysis systems to analyze and control pharmaceutical, food, and chemical processing lines (53). Immunosensors are not expected to replace laboratory-based immunoassays, but to open up new appHcations for immunoassay-based technology. 

Enzyme immunosensors are used in flow injection systems and Hquid chromatography to provide automated on-line analyses (71—73). These systems are capable of continuously executing the steps involved in the immunoassays, including the binding reactions, washing, and the enzyme reaction, in about 10 minutes. 

Table 1.10 Typical immunoassay procedure using Zymate robotic laboratory automation system... 

This author and coworkers at Beckman Coulter first described the use of a low form 96-well plastic microplate for automated micro-ELISA immunoassays (Matson et al., 2001). The polypropylene plate was first modified by a radiofrequency plasma amination process (Matson et al., 1995) followed by conversion to an acyl fluoride surface chemistry for rapid covalent attachment of biomolecules. Proteins (1 to 2 mg/mL) were prepared in 50 mM carbonate buffer, pH 9, containing 4% sodium sulfate (to improve spot uniformity) and printed using a conventional arrayer system. Approximately 200-pL droplets of monoclonal antibodies (anti-cytokine) were deposited into the bottom of the microwells using a Cartesian PS7200 system equipped... 

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