Immunoassay sensitivity

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. 

Jl. Jackson, T. M., and Ekins, R. R, Theoretical limitations on immunoassay sensitivity. Current practice and potential advantages of fluorescent Eu + chelates as non-radioisotopic tracers. J. Immunol Methods 87, 13-20 (1986). 

Antibacterial Matrix Sample preparation Type of immunoassay Sensitivity (ppb) Ref. 

Immunoassay Sensitivity. Yalow (2) points out that as little as 0.1 picogram (0.05 picomolar) gastrin can easily be detected by immunoassay in a milliliter of incubation medium. Immunoassays to small lipophilic molecules are generally less... 

Radioimmunoassay. In urine benzoylecgonine (comparison with TLC, GLC and enzyme immunoassay), sensitivity 2 ng/ ml—S. J. Mule et al., Clin. Chem., 1977, 23, 796-801. In blood or urine benzoylecgonine (comparison with HPLC), limit of detection 20 ng/ml—K. Robinson and R. N. Smith, J. Pharm. Pharmac., 1984, 36, 157-162. 

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. 

Since immunoassay sensitivity is intimately related to assay precision, it is important to define and distinguish between some commonly used terms. 

Fig. 9.5. (a) Bias in estimation of immunoassay sensitivity due to erroneous curve fitting SI, calculated sensitivity S2, true sensitivity, (b) Precision profile (PP) variation of precision (CV in %) for the signal response and estimated concentration (translated from the signal response) within the whole immunoassay range. 

In order to decide the lowest possible working concentrations of competitor and antibody, the development of a competitive immunoassay usually starts by performing a two-dimensional dilution experiment, i.e., the antibody concentration, [Ab], is varied in one dimension and the competitor concentration, [Ag ], is varied in the other. In this way the optimal [Ab] and [Ag ] can be determined experimentally and the lowest possible [Ag ] and [Ab] are usually selected, when 30-70% of the tracer is bound to the antibody ([Ab-Ag ]). However, the contradiction in defining immunoassay sensitivity, as mentioned above, has led to different values of the theoretically predicted... 

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. 

TM Jackson, RP Ekins. Theoretical limitations on immunoassay sensitivity. J Immunol Meth 87 13, 1986. 

In the mid 1970s, immunoassay sensitivity was greatly increased with the arrival of the enzymo immunoassay (EIA) [21-24]. In this type of assay, an enzyme attached to the detection antibody (cf Fig. 8.2B) or to the analyte allows amplification of the signal. These types of assay are currently the most widespread. Derivatives of ferrocene have been used in this type of assay, several of which are discussed below. This is in fact an extension of the term MIA. In fact, the organo-metaUic complex as used here is not the tracer but the substrate or co-substrate (redox mediator) of an enzyme. 

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