Immunoassay problems with

There are proficiency testing programs that are geared toward clinical sensitivity or specificity by seeking to determine whether a disease can be detected versus other types of controls that are use to test sensitivity, selectivity, and most importantly, reproducibility and precision. With mass spectrometry, the controls are and should be no different than those used for other assays, with one interesting exception. Quality assurance materials prepared for MS/MS may not be useful in other assays that are less selective. The example is newborn screening where quality assurance/control QA/QC materials have a mixture of compounds present in the blood specimens. However, in less selective immunoassays, the mixture creates interferences. In addition, material is used to spike a blood sample is key and one should ensure there is no enzyme activity. We have encountered such a problem with a d/1 mixture of metabolites where one form was degraded in the prepared blood. 

In order to become a viable alternative, MIP-based assays need to offer an added value to the conventional antibody-based immunoassays. Some characteristics of the MIP-based assays are summarised in Table 14.1. Superior characteristics of MIPS in comparison to antibodies are observed with respect to chemical, mechanical and thermal stability. The MIPs are compatible with autoclave conditions (120°C, 20 min) and are unaffected by acid and base treatment [7]. In fact, to achieve as complete removal of imprint molecules as possible, in the author s laboratory it is routine to include a wash step with 5 M sodium hydroxide in the MIP synthesis work-up protocol. The possibility of using a wider range of assay solvents, namely both aqueous and organic solvents, enables the solubility of the analyte to be assured and problems with non-specific adsorption minimised. Furthermore, high polymer stability leads to improved shelf life, where the MIP can be stored for several years in the dry state at ambient temperatures. 

The direct detection of electrochemical labels entails problems with sensitivity. For this reason the majority of electrochemical immimoassay development has focused on the measurement of enzyme labels by detection of eiectroactive products arising from enzyme catalyzed reactions. A wide variety of enzyme labels have been used for electrochemical immunoassays. These include glucose oxidase, glucose-6-phosphate dehydrogenase and alkaline phosphatase. ... 

With the development of radioimmunoassays (RIAs) in the 1960s that used radioactive isotopes as labels (see Chapter 9), the measurement of radioactivity became a common and important practice in clinical laboratories. However, concerns about, and problems with, the safe handhng and disposal of radioactive reagents and waste have led to the development of immunoassays that use nonisotopic labels (see Chapter 9). The rapid acceptance and extensive use of nonisotopic immunoassays by the clinical laboratory have resulted in a decreased use of RIA and ultimately a decreased requirement for them to measure radioactivity. Because of this deemphasis on the necessity to measure radioactivity, only a brief discussion of the topic is presented here. Readers requiring more detail on this topic are referred to the chapter entitled Basic Principles of Radioactivity and Its Measurement that is included in a prior edition of this textbook, ... 

The vahdity of assay results is best proved by tests for parallelism of the response curves for both calibrators and randomly selected samples from male and female subjects of various ages. In addition, comparison of extraction/ chromatography immunoassays should be validated by comparison with mass spectrometry methods. Direct immunoassays should not be used for female and prepubertal subjects, especially infants. These groups should be tested using an extraction immunoassay or an LC-MS/MS assay. It is important to note that both types of direct immunoassay demonstrate problems with functional sensitivity for low concentrations. Each lab should determine the low-end detection limit of their assay despite manufacturer claims. 

Assay application. At this point major differences appear between the historical use of clinical immunoassays and the potential applications of environmental and pesticide immunoassays. Most clinical assays have been applied to simple or well defined and consistent matrices such as urine or serum. In contrast, most matrices likely to be analyzed for pesticides are more complex, less well defined, and more variable. The potential for serious problems with matrix effects in the environmental field is far greater than most clinical immunoassays have encountered. The application of immunoassays to environmental analysis requires sampling strategies, cleanup procedures, and data handling fundamentally similar to those presently in use in any good analytical lab. The critical factor in the success of immunochemical technology will likely be competence... 

Bioassays are frequently used as an alternative or in addition to immunoassay techniques. Bioassays, in contrast to immunoassays, quantify not the pharmacologically active substance, but its biological activity, for example, in cell culture models based on cell differentiation, cell proliferation, or cytotoxicity as well as gene expression assays or whole animal models. Frequent major problems with bioassays comprise a high variability in the measured parameters, lack of precision, and their time- and labor-intensive performance. Furthermore, bioassays oftentimes also lack specificity for the measured compound, as they may also detect the response to bioactive metabolites [16,17]. 

Because of some of the problems with bioassays and immunoassays, liquid chromatography (LC)-based techniques are increasingly applied as an alternative. While modern LC-based assays have a comparable sensitivity to immunoassays, they oftentimes are characterized by a higher selectivity [18, 19]. Muller et ah, for example, used LC/mass spectrometry with matrix-assisted laser desorption ionization in ex vivo pharmacokinetic studies in combination with enzyme inhibition experiments to investigate the complex metabolism of dynorphin Al-13, a peptide with opioid activity, up to the fifth metabolite generation [20, 21]. 

The techniques that appear to represent the major advance in identifying and detecting ciguatoxic fish are inununochemical methods radioimmunoassay (RIA), competitive enzyme immunoassay (ElA), and enzyme-linked immunosorbent assay (ELISA). Of these, the enzyme immunoassay stick test is the simplest, fastest, most specific, more sensitive, and does not require complicated instrumentation. The problems with this method are its cross-reactivity with other polyether compounds and the limited antibody supply. Attempts to validate the method have been unsuccessful due to the lack of reference material. 

The commonly used methods for the determination of these drugs are immunoassays and chromatography. Most immunoassays for immunosuppressants are semiautomated since extraction of drugs from the whole blood is needed before analysis. Immunoassays are convenient due to automation, but have problems with cross-reactivity with drug metabolites (3, 6). Both polyclonal and monoclonal antibody-based assays are available. Monoclonal antibody-based immunoassays are more specific. HPLC with ultraviolet detection and tandem mass spectrometry are commonly used chromatographic methods for the assay of immunosuppressants. Due to their specificity and sensitivity, tandem mass spectrometry assays are preferred and are now in wide use (7, 8). The other major advantage of tandem mass spectrometry assays is their ability to simultaneously measure several immunosuppressants (7-10). Pharmacokinetic properties of CSA, sirolimus, and tacrolimus are shown in Table 1 (3, 6, 11). 

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