Immunoassay system automation using injection

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.

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. 

Abdel-Hamid et al. [122] used a flow-injection amperometric immunofll-tration assay system for the rapid detection of total E. coli and Salmonella. Disposable porous nylon membranes served as a support for the immobilization of anti- ]. coli or anti-Salmonella antibodies. The assay system consists of a flow-injection system, a disposable filter-membrane, and an amperometric sensor. A sandwich immunoassay specifically and directly detected 50 cells ml total E. coli or 50 cells ml Salmonella. The immunosensor can be used as a highly sensitive and automated bioanalytical device for the rapid quantitative detection of bacteria in food and water. 

The future use of coated piezoelectric devices as immunochemical sensors, even directly in the liquid phase, is very promising and could be considered a very competitive alternative to other types of immunoassay. Only this technique and that of surface plasmon resonance provide labelless methods for the direct study of antigen-antibody reactions, and their analytical possibilities. The devices can be easily automated or combined with flow injection systems, extending their capability of continuous and repeated assays. This raises an exciting possibility of using crystal arrays to assay for different analytes in complex samples with an on-line display of the results. 

In this study two different flow-injection immunoassays are presented as well as the flexible automation system CAFCA (Computer Assisted Flow Control Analysis), which has been used for their control, uptake measurement, evaluation and visualization. Both immunoassays (a heterogeneous and a homogeneous assay) are based on the principles of flow-injection analysis and were developed for reliable, fast monitoring of relevant proteins in animal cell cultivation processes. Off-line applications of measurements of medium samples as well as online application during a mammalian cell cultivation are presented. All results are compared to results obtained with ELISA (Enzyme Linked Immunosorbend Assay). The requirements of the automation of flow-injection immunoassays with respect to their flexible control are discussed. 

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