Enzyme-linked limitations

Biological techniques, e.g., immunoassays, are among the most sensitive analytical methods, but are limited by the availability of the specific antisera and are subject to cross-reactivity. Huang et al. [36] employed an enzyme-linked immunosorbent assay (ELISA) for determination of estradiol, its conjugates, and ethynylestradiol in wastewaster treatment plant effluents (see Table 4). The reported limit of detection (LOD) of 0.1 ng L 1 reflects the sen-... 

The traditional microbiological methods are very time consuming and sometimes limited concerning their interpretation. For that reason fast analysis methods as well as automated methods have been developed the latter are often used in specialised microbiological laboratories. During the last few years more and more modern biotechnological methods have been implemented into quality control, for example the enzyme-linked immunosorbent assay or more recently the polymerase chain reaction, which allows the detection of very specific microorganisms. 

The assessment of DNA adducts may provide a sensitive indicatCH of previous exposure. The enzyme-linked immunosorbent assay (ELISA) has a lower limit of detection of about 0.08 femtomol per microgram of DNA (Perera et oL, 1982). This assay requires (1) the development of an antibody specific for a certain chemical metabolite bound covalently to DNA and (2) the isolation of DNA from some tissue sample, ag., skin biopsy, or lymphocytes of an exposed individual. It is anticipated that further refinement of such immunologic techniques may lower the threshold of sensitivity by one or two orders of magnitude. One such refined test is the ifitrasensitive ymatic radioimmunoassay (USE-RIA), purported to be about five times more sensitive than ELISA (Hsu et oL, 1981 Shamsuddin et oL, 1985 Harris et oL, 1985). Quantification by the development of monoclonal antibodies to aflatoxin Bj metabolites bound to DNA (Groopman et oL, 1982 Sizaret et oL, 1982) has now been reported. 

Use of a surrogate end point that is quick and easy to obtain Permeation experiments using a radiolabeled, fluorescent, HPLC-detectable, or radio immuno assay/enzyme linked immuno sorbent assay-detectable marker necessitate the need of extensive sample handling and sample analysis. This accentuates the cost of sample analysis and overall time spent in characterizing the efficacy of formulations. Furthermore, current state of the art fluidics systems put a fundamental limit on the number of samples handled in a given time. 

Some suppliers provide enzyme-linked primary antibodies that can be used in these protocols, thus avoiding the necessity for a secondary antibody incubation. This does reduce the length of time required for the procedure, but does have some disadvantages with regard to sensitivity The treatment with a secondary antibody does provide a degree of amplification in the system that allows detection of smaller amounts of protein Also, the choice of visualization procedure is limited to the enzyme type conjugated to the primary antibody... 

Three main techniques have been used for detecting HER-2 oncoprotein overexpression Western blot analysis, enzyme-linked immunosorption assay (ELISA), and immunohistochemistry. Western blot analysis is limited to basic research rather than routine clinical... 

Methods for the determination of polychlorobiphenyls seem to be limited mainly to those based on gas chromatography and enzyme-linked immunosor-bant assay (ELISA). 

For all of these targets the versatility of enzyme-linked immunosorbent assays (ELISAs) qualifies this method as an almost universal detection platform, using the highly specific recognition potential of antibodies in conjugation with a detection enzyme and various signal-generating substrates [8, 9]. In contrast to the enormous amplification power of the PCR, these enzymatic methods normally have a detection limit of several millions of molecules. 

In the multiplex-IPCR assays carried out by Joerger and Hendrickson, DNA of different lengths was used for separation of the PCR amplificates (see Fig. 4). Results of their experiments are discussed below in Section 3.5. The ability to discriminate between different DNA markers by length is nevertheless limited by the separation capabilities of the gel. For a large number of different DNA probes, a sequence-specific detection carried out, for example, by PCR-enzyme-linked oligonucleotide sorbent assay (ELOSA Section 2.2.2) should be preferable. 

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

Pharmacokinetic data analysis requires determination of the analyte in various body fluids. In the case of therapeutic antibodies, serum is the most common matrix to be analyzed. For a critical interpretation of pharmacokinetic data the chosen bioanalytical methods must be considered. The most frequently used for mAbs include enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis (CE)/polyacrylamide gel electrophoresis (PAGE), fluorescence-activated cell sorting (FACS), and surface plasmon resonance (SPR). The challenges and limitations of bioanalytical methods used for the analysis of mAb concentrations are discussed in detail in Chapter 6. 

As can be seen in Table 8.2, several immunochemical techniques, principally enzyme-linked immunosorbent assays (ELISAs), have been developed and applied to environmental samples such as water, soil, and sediments with very good limits of detection (LOD).49-51... 

The ELISA procedure for the analysis of parathion as described above requires nearly eight hours, although many samples can be simultaneously assayed. However, incubation times can be shortened to one-half hour, in most cases, resulting in only a 10% reduction in sensitivity. Also the polystyrene microtiter plates containing bound RSA-AP can be mass produced and stored in a freezer. Since the enzyme-linked antibody can be purchased, the limiting factor of the applicability of the ELISA procedure, as well as the RIA procedures, for other pesticides is the development of the antiserum to the pesticide. 

---