Immunoassay robustness

PAbs against 2,4,5-TCP have been prepared after theoretical and molecular modeling chemical studies [226]. Competitive direct and indirect ELIS As have been developed, but as before the latter format was shown to be more robust. The indirect immunoassay has an excellent LOD near 0.05 pg L . The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples, but as with the 2,4,6-TCP assay the brominated analogs may also be recognized. The 2,4,5-TCP immunoassay is stable in media with pH... 

Several successful attempts were done to transfer classical CEIA to a microchip-based format. This kind of miniaturization is a trend that can overcome the limitations of CE in high-throughput systems. On-chip CE offers both parallel analysis of samples and short separation times. Koutny et al. showed the use of an immunoassay on-chip (32). In this competitive approach fluorescein-labeled cortisol was used to detect unlabeled cortisol spiked to serum (Fig. 8). The system showed good reproducibility and robustness even in this problematic kind of sample matrix. Using serum cortisol standards calibration and quantification is possible in a working range of clinical interest. This example demonstrated that microchip electrophoretic systems are analytical devices suitable for immunological assays that can compete with common techniques. 

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

Further progress of ECL probes immobilization methods should result in new robust, stable, reproducible ECL sensors. Especially, the use of electrochemilumi-nescent polymers may prove to be useful in this respect. There are also good prospects for ECL to be used as detection in miniaturized analytical systems particularly with a large increase in the applications of ECL immunoassay because high sensitivity, low detection limit, and good selectivity. One can believe that miniaturized biosensors based on ECL technology will induce a revolution in clinical analysis because of short analysis time, low consumption of reactants, and ease of automation. 

A robust assay for theophylline was established within the clinical range of 10-20 pg/mL [330]. Since the Ab-Ag complex can be mobilized, no additional internal standard was needed [330], as in an immunoassay study for cortisol [1006],... 

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. 

Obviously, the universal goal of any measurement technique is to obtain reproducible results regardless whether the samples come from different sources with different matrix effects, are run by different operators, in different laboratories, on different occasions and using different lots of reagents. This is usually accomplished by characterization of the newly developed assay in terms of sensitivity, selectivity, robustness and correctness (i.e., accuracy and precision). Evaluation of sensitivity and precision does not normally constitute a problem for a newly developed immunoassay, and accuracy can be attained by comparison with the results obtained by a reference ( standard ) method. However, the evolution of assay standardization from this point on is much more difflcult. The following section deals with application-specific problems related to validation and standardization of immimoassay. 

A wide range of chemical compounds have been imprinted successfully, ranging from small molecules,40 2 to large proteins and cells.43 MIPs have been developed for a variety of applications including chromatography,4445 solid-phase extraction (SPE),46 47 enzyme catalysis,48 sensor technology,44>49>50 biomimetic sensors,5153 and immunoassays.54-56 MIPs are robust, inexpensive and, in many cases, possess affinity and specificity that are suitable for industrial applications. The high specificity and... 

Elements in an immunoassay that could impact its robustness include incubation temperatures, light exposure (enzyme-linked immunosorbent assay, ELISA), and different lots of matrix (plasma, serum CSF). The ruggedness of the analytical method can be tested by implementing changes to the analysts, different instruments, batch size, and the day, time, or other environment factors otherwise should not greatly impact the consistency of the assay. 

The optimization and validation of immunoassays for immunogenicity (ADA) testing has been described in detail in several publications [9,14,33,34]. In this section, we will describe the evaluation of relevant performance characteristics (validation parameters) that require the most effort. Some of these are different from the validation of traditional bioanalytical pharmacokinetic (PK) methods for macromolecules [35 37]. Precision, specificity, robustness, and ruggedness are determined similarly between ADA and PK methods. However, recovery/accuracy, sensitivity, stability, linearity, system suitability controls, and selectivity are treated differently between these two types of assays. 

The first step in the validation process involves the establishment of acceptance criteria, which are assay dependent, for the various validation parameters. Assays must meet these criteria in order to receive validation status. The most relevant validation parameters are specificity, accuracy, precision, sensitivity, and robustness. In this section we describe these parameters as they apply to the evaluation of immunoassays. 

Robustness defines the ability of the immunoassay to perform within specifications (remain unaffected) when it is subjected to variations of anal3Tical conditions, such as changes in temperature, incubation times, and changes in test sample volumes. It is a measure of reliability during normal use of the assay. 

More and better QD probes have been designed and constructed during the past several years, and they are getting closer and closer to clinical applications in the near future. The impact of QDs on immunoassay, diagnostics, and molecular pathology can already be seen. We believe that more robust and reproducible conjugation methods for QD probe production are key for their commercialization and widespread use. Additionally, the discovery of non-tra-ditional QDs will pave the way for the in vivo clinical use of these nanomaterials. 

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