Immunoassay optimization

As with any analytical technique, generation of a reproducible standard curve with minimal error is critical. An assay calibration consists of several steps during which the value of the primary standard is transferred to the calibrators used in the final assay [22]. Immunoassay optimization is usually difficult due to protein heterogeneity and matrix effects and these factors, heterogeneity and matrix effects, will also affect MIP based assays [22]. 

Khosraviani, M., A.R. Pavlov, G.C. Flowers, et al. 1998. Detection of heavy metals by immunoassay Optimization and validation of a rapid, portable assay for ionic cadmium. Environ. Sci. Technol. 32 137-142. 

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. 

Optimization of the immunoassay was performed with respect to tracer and antibody concentrations to obtain the required sensitivity. These conditions differed depending on the detection system used photographic detection required higher antibody and tracer concentrations than when the plate luminometer was used. A further complication arose from the very low affinity of the tracer for the antibody when using an antibody dilution of 1 3000 and a tracer dilution of 1 4000 less than 1% of the tracer was bound after a 2-h incubation. This means that the antibody, in the absence of clenbuterol, binds less than 10 pg of the... 

Optimization and applications of CL detection in flow injection and liquid chromatographic analysis and the relatively new use of CL in capillary electrophoresis are extensively described. Particular interest is attached to the universally applied peroxyoxalate CL reactions, as well as to the applications of new acridan esters in immunoassay. Obviously, the related applications of BL and CL imaging techniques in analytical chemistry, and the increasing importance of these techniques in DNA analysis—including the recent strategies in the development of CL sensors—are also presented. 

Mire-Sluis, A.R. et al., Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products, J. Immunol. Methods, 289, 1, 2004. 

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

Recently, a novel immunoassay has been developed for the quantitative determination of polybrominated biphenyls using indirect competitive format. The new method was optimized concenung the coating conjugate and antibody concentration, incubation time and temperature, the tolerance to organic solvents and so on. Under optimized conditions, PBB15 can be determined in the concentration range of 0.01-100 pg/L with a detection hmit of 0.02 pg/L. The cross-reactivities of the assays were below 8%. While water samples could be analyzed directly [94]. 

Ezan, E. and Grassi, J., Optimization, in Immunoassays A Practical Approach, Gosling, J.R, Ed., Oxford University Rress, Oxford, 2000, chap. 7. 

We start with examples of the sequential approach. With this approach, you begin with more routine experiments, ones that are reasonably likely to succeed (e.g., calibration or optimization procedures). The initial set of experiments can also serve as a test case and/or show that you can reproduce literature values. For example, Aga (P6) proposes first to explore conditions that will optimize immunoassay sensitivity, and Spain (P7) proposes to begin with a study of topography, using published methods and a self-assembled monolayer with a known structure. 

Of course, a successful proposal must also forge ahead into less familiar territory. It is not enough to conduct the easy experiments you must approach the cutting edge or forefront of your field. For this reason, Aga goes on to describe how the optimized immunoassay will eventually be used to test for analytes in more complex environmental samples, and Spain proposes a sequence of experiments that will culminate in the deposition of translationally hot metal atoms on a self-assembled monolayer system. The important point in these examples is how authors develop a clear and logical order for their proposed work. 

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