Automated immunoassay systems

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. 

Based on solid phase technologies numerous fully automated immunoassay systems which allow the determination of a broad spectrum of analytes are nowadays commercially available. While very successful in the case of nonisotopic methods, attempts for automation in the RIA field more or less failed due to the properties of the radioactive label. 

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... 

Chen I, Sperling M. Estimation of free thyroxine by automated immunoassay systems. Endocrinology and Metabolism In-Service Training and Continuing Education Program. Washington, DC AACC Press 1992 10 3-5. 

Keller C, Novotny M, Ramp S, Brashear J, Brandt D, Abbott K, A rapid, sensitive assay for human thyroid-stimulating hormone (hTSH) on the Abbott IMx automated immunoassay system. CHn Chem 1988 34 1208. 

Novotny M, Bogacz J, Peters TK, Mallas T, LaFoe M, Isom L, et al. Development of an automated random/continuous access microparticle enzyme immunoassay (MEIA) for the determination of hTSH on the Abbott AxSYM automated immunoassay system. CHn Chem 1996 42 S180. 

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. ... 

Baker HN, Massei MK, Ramp SK, Trach P, Strarup I, Garner W, et al. Development of a fully automated immunoassay for estradiol on the Abbott IMx automated immunoassay system. Clin Chem 1991 37 1036. 

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. 

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. 

OP13 Omand, K., Bowen, T.P., and Kost, G.J. (1991). Performance evaluation of the automated, random access Opus immunoassay system. Clin. Chem. 37, 1037, Abstr. 606. 

Comparison of the ES 600 automated enzyme immunoassay system with Stratus immunofluorometry and manual radioimmunoassay. Clin. Chem. 33, 947, Abstr. 327. 

STN15 Plaut, D.S. and McLellan, W.N. (1991). The Baxter Diagnostics Inc., Dade Stratus II automated fluorometric immunoassay system. J. Clin. Immunoassay 14, 120 125. 

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. 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

As its name implies, the chemical reaction phase occurs when aliquots of specimen and reagents are allowed to chemically react. Concerns related to this operation and the measurement of the reaction are addressed in the design of every automated analyzer. Design issues to be considered include (1) the vessel in which the reaction occurs and the cuvet in which the reaction is monitored, (2) the timing of the reaction(s), (3) the mixing and transport of reactants, and (4) the thermal conditioning of fluids. Separation of bound and unbound fractions is a fifth issue for immunoassay systems, as described in Chapter 9.. ... 

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. 

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... 

Letellier M, Levesque A> Daigle F, Grant A. Performance evaluation of automated immunoassays on the Technicon Immuno 1 system. Clin Chem 1996 42 1695-701. 

Enebo DJ, Rossow KL, Werness PG, An improved automated immuno-chemiluminometric third generation hTSH assay for the ACCESS immunoassay system. Clin Chem 1996 42 S170. 

Products/technologies These include Microtiter Plastics (plates, strips, accessories) Microtiter plate coating systems Microtiter detection reader systems, automated Microtiter plate immunoassay processing systems (such as the Dias immunoassay system) Microtiter plate washers and Revelation software. 

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