Immunoassays sample matrix

J.H. Skerritt and B.E. A. Rani, Detection and removal of sample matrix effects in agrochemical immunoassays, in Immunoassays for Residue Analysis, ed. R.C. Beier and L.H. Stanker, American Chemical Society, Washington, DC, pp. 29 3 (1996). 

Several considerations influence the suitability of the immunoassay as a qualitative or quantitative tool for the determination of tissue residues. These include the assay format, the end user (on-farm or laboratory use), effects of sample matrix on the analysis, cross-reactivity considerations, detection levels required of the assay, target tissues to be used in the assay, and the use of incurred or fortified tissues for validation of the immunoassay against accepted instrumental methods. Although these variables are often interrelated, each topic will be discussed in further detail below. 

Since FPIAs are conducted as homogeneous immunoassays, they are susceptible to effects from endogenous fluorophores and from intersample variations. Such problems and others due to the sample matrix are largely avoided by sample dilutions of several hundredfold. Low-affinity, nonspecific binding of tracers to sample proteins, when present in sufficiently high concentrations, can result in a falsely elevated polarization signal. Interference from sample proteins can be eliminated when warranted, by proteolytic hydrolysis with pepsin.(46)... 

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. 

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. 

In an indirect competitive immunoassay, the competitor is commonly immobilized on a solid surface while the antibody and the analyte are added in the adjacent solution. After the competition, the fractions that are unbound to the solid surface are removed and the bound primary antibody is usually measured by the addition of a labelled secondary antibody (see Fig. 9.7). Even though this assay format is not as frequently used as the direct competitive immunoassay, it provides an advantage when analysing complex samples, i.e., the label (usually an enz5rme) does not come into direct contact with the sample matrix, so that interferences with the detection step are minimized. 

Depending on the complexity of the sample matrix, a pre-cleaning step is a possible solution to remove interfering eflFects nevertheless, the choice should be a good compromise in relation to the total time and cost of analysis [33]. Supercritical fluid- [48,49], microwave-assisted- [50] or solid-phase extraction (SPE) [51-53] have been used as pre-cleaning steps prior to immunoassay. However, in certain situations, the extraction step led to a decrease in accuracy and recovery, probably due to analyte loss during clean-up/ evaporation/re-dissolution steps [53]. 

Since the sample matrix generally gives the same eflFect on the analytical response, as if the analyte would be present in the sample, a positive bias when measured by immunoassay vs. a reference method is often observed [54,55]. Simple mathematical models that parallel the strategy of sample addition for correcting these negative eflFects have been proposed [56,57]. 

This chapter covers the theoretical grounds, the possibifities to rationally design the perfect assay in terms of sensitivity for any specific appfication, as well as the limitations of immunoassays, mainly in relation to operation close to the sensitivity limit, susceptibility to saturation kinetics and interferences due to sample matrix components. 

Linearity refers to the relationship between measured and expected values over the analytical measurement range. Linearity may be considered in relation to actual or relative analyte concentrations. In the latter case, a dilution series of a sample may be studied. This is often carried out for immunoassays, in which case it is investigated whether the measured concentration declines as expected according to the dilution factor. Dilution is usually carried out with the appropriate sample matrix (e.g., human serum [individual or pooled serum]). 

Matrix effects can be a problem for immunoassays, especially for a method without any prior sample clean-up. It can be caused by either nonspecific or specific interferences from the sample matrix and reagents. Possible matrix effects can... 

In contrast to the chromatographic techniques where the analyte is extracted from the biological samples and reconstituted into a well-defined matrix prior to assay injection, immunoassay samples are usually analyzed without any... 

The ideal PK immunoassay standard curve, which is nonlinear and heteroscedastic, is derived from solutions of well-characterized macromolecules added to a relatively nonreactive sample matrix. However, rarely does one encounter an ideal situation when describing bioanalytical methodology, thus developing and validating analytical methods for macromolecules, and analyzing samples from preclinical or clinical trials must include an evaluation of these variables and possibly many others. Careful consideration must be given to the topics described in this chapter to achieve the goal of accurate and reproducible quantification of biotherapeutics necessary for pharmacokinetic analysis. 

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... 

To overcome the difficulties encountered with the bioassays and TLC methods, immunoassays using specific polyclonal and monoclonal antibodies have been developed for most of the major trichothecene mycotoxins and their metabolites.73 These antibodies have been used to produce simple, sensitive, and specific radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) for the mycotoxins. In the presence of the sample matrix, the lower detection limits for identification of trichothecene mycotoxins by RIA is about 2 to 5 ppb73 and by ELISA, 1 ppb.74 We conclude that immunoassays are useful tools for screening biomedical samples for evidence of a biological warfare attack with trichothecene mycotoxins. 

In heterogeneous immunoassays in batch format the problem of background fluorescence is reduced by separating the analyte from other fluorescent species present in the sample matrix. The use of enzymes with fluorogenic substrates in fluorescence enzyme-linked immunosorbent assays (F-ELISA) and Eu(in)-chelates in time-resolved fluoroimmunoassay (TRFIA) permits to achieve very low LODs for pesticides (e.g., between 0.023-0.3 and 0.05-0.4pgl for F-ELISA and TRFIA, respectively). 

An immunoassay kit for the measurement of PCP has been developed with a limit of detection around 60 pg/L [109]. The sample matrix had little influence on the immunoassay, but 2,4,5,6-tetrachlorophenol and 2,3,4,6-tetrachlorophenol show some crossreactivity. The methodology can be used as an initial screening of phenols, and it normally does not require any sample preparation. The immimoassay methodology has also been applied for the determination of 4-nitrophenol and substituted 4-nitrophenols [110]. 

Sample preparation techniques vary depending on the analyte and the matrix. An advantage of immunoassays is that less sample preparation is often needed prior to analysis. Because the ELISA is conducted in an aqueous system, aqueous samples such as groundwater may be analyzed directly in the immunoassay or following dilution in a buffer solution. For soil, plant material or complex water samples (e.g., sewage effluent), the analyte must be extracted from the matrix. The extraction method must meet performance criteria such as recovery, reproducibility and ruggedness, and ultimately the analyte must be in a solution that is aqueous or in a water-miscible solvent. For chemical analytes such as pesticides, a simple extraction with methanol may be suitable. At the other extreme, multiple extractions, column cleanup and finally solvent exchange may be necessary to extract the analyte into a solution that is free of matrix interference. 

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. 

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