Immunoassay performance

For pesticide residue immunoassays, matrices may include surface or groundwater, soil, sediment and plant or animal tissue or fluids. Aqueous samples may not require preparation prior to analysis, other than concentration. For other matrices, extractions or other cleanup steps are needed and these steps require the integration of the extracting solvent with the immunoassay. When solvent extraction is required, solvent effects on the assay are determined during assay optimization. Another option is to extract in the desired solvent, then conduct a solvent exchange into a more miscible solvent. Immunoassays perform best with water-miscible solvents when solvent concentrations are below 20%. Our experience has been that nearly every matrix requires a complete validation. Various soil types and even urine samples from different animals within a species may cause enough variation that validation in only a few samples is not sufficient. 

Figure 2 An illustration of matrix effects on immunoassay performance. Calibration curves of atrazine were run in buffer ( ), in skim milk (O) and in whole milk (A). Reprinted from M. Franek, V. Kolarand S. A. Evemin,Analyh ca Chimica Acta, 311,349-356, Copyright 1995, with permission from Excerpta Medica Inc...

Y. Zhou, J. V. Magill, R. M. De La Rue. P. J. R. Laybourn, and W. Cushley, Evanescent fluorescence immunoassays performed with a disposable ion-exchanged patterned waveguide, Sensors and... 

Figure 13.8 (a) Pictorial of competitive immunoassay performed on a capillary elec-... 

Since immunoassays are primarily analytical techniques, in addition to studies for a better understanding of the nature of antibody-antigen interaction, there are continuous efforts to improve immunoassay performance (e.g., sensitivity, selectivity, precision and accuracy) in terms of robustness and reliability when analysing complex samples. The present chapter attempts to summarize the most commonly used immunoassay concepts, as well as the main approaches employed for the improvement of immunoassay sensitivity, selectivity and precision. The discussion is focussed aroimd the main thermodynamic and kinetic principles governing the antibody-antigen interaction, and the effect of diverse factors, such as assay design, concentration of reactants, incubation time, temperature and sample matrix, is reviewed in relation to these principles. Finally, particular aspects on inummoassay standardization are discussed as well as the main benefits and limitations on screening vs. quantification of analytes in real samples. 

Matrix effects have traditionally been dealt with by standard addition, i.e., addition of known amounts of standard analyte in the sample and extrapolating the result to zero added standard. However, this method is diflicult to apply for immimoassay because of the non-linearity of the concentration-response relationship. An alternative approach is presented as a modality to optimize the standards and buffer, so that it reproduces as closely as possible the effects of a specific matrix on immunoassay performances [42]. 

Fig. 54. Schematic representation of the various immunoassays performed with the Stratus system.

Some experimental parameters (coating and blocking conditions, Tween-20) were studied with two aims (1) to improve immunoassay sensitivity, (2) to study immunoassay performance under the optimal conditions. These experiments were carried out using the proposed method described above. Criteria used to evaluate the optimization were RLU and RLUmax so. 

The next step is the application of the microcantilever as biosensor in the environmental control field, by following an immunological detection scheme. For this purpose, we present the inhibition immunoassay performed to detect the highly toxic organochlorine insecticide compound dichlorodiphenyltrichloroethane (DDT). 

The first immunoassay performed in a capillary driven system was reported in 1978 [67]. Based on this technique, the commonly known over-the-counter pregnancy test was introduced into the market in the middle of the 80 s. Today, this microfluidic platform is commonly designated as a lateral flow test (LAT) [14]. Other terms are test strip , immunochromatographic strip , immunocapillary tests or sol particle immunoassay (SPIA) [68]. Astonishingly, hardly any publications from a microfluidic point of view or in terns of material classification exist, and apparently many company secrets are kept unpublished [69]. 

To ensure the reliability of analytical techniques, they need to be validated. Validation provides information on the overall performance of the assay as well as on individual parameters and factors that can be used to estimate the degree of uncertainty associated with an assay (Ellison et al., 2000). An adequate validation procedure assesses, and therefore ensures, that the immunoassay performs within an acceptable range of established criteria. Parameters used to evaluate the performance of the assays may be affected by (1) factors inherent to the analytical technique, such as antibody specificity and antibody cross-reactivity, and (2) external factors such as environmental conditions (temperature) and type of sample (matrix, processed food vs. raw ingredients). A... 

Nowadays, whole blood microfiuidic immunoassay performs an important role in medical applications. It requires low cost, fast analysis, portability, and sensitivity. With the rapid progress of the new materials, immobilization methods, surface modification methods, microfahtication technology, and detection approaches, better whole blood microfiuidic immunoassay technology will be developed in the future. 

Several reviews devoted to or including immunoassays performed in HPLC or CE systems have been published [4-12]. These methods have been used for analysis of small and large molecules. Besides the application to detect the presence and concentration of an analyte in a sample, immunochromatographic and immunocapillary electrophoretic systems have a wide application in the determination of antibody-antigen binding constants. 

This chapter is focused on methods that use antibody-antigen interactions in HPLC or CE systems for the detection of proteins. Although immunoassays performed in LC and CE systems are similar, they vary in many practical aspects. For this reason, immunochromatographic and immunocapillary electrophoretic detection will be described separately. 

The borderline between immunoassays performed in the flow injection analysis format and those achieved in the immunochromatographic format is sometimes difficult to establish. Flow injection renewable surface immunoassay (FIRSl) avoids the desorption step of immunochromatographic assays by using a layer of antibody-coated beads held in a jet ring cell instead of the chromatographic column. The coated beads are discarded after each analysis. To ensure assay reproducibility, antibody-coated beads with constant antigen capacity should be obtained [129,130]. 

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