Advantages of Immunoassays

Immunoassays. Immunoassays (qv) maybe simply defined as analytical techniques that use antibodies or antibody-related reagents for selective deterrnination of sample components (94). These make up some of the most powerflil and widespread techniques used in clinical chemistry. The main advantages of immunoassays are high selectivity, low limits of detection, and adaptibiUty for use in detecting most compounds of clinical interest. Because of their high selectivity, immunoassays can often be used even for complex samples such as urine or blood, with Httle or no sample preparation. 

The advantages of immunoassay technology relative to other analytical techniques have been discussed in several reviews, and include the following ... 

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. 

We will first review several of the Immunoassays developed in this laboratory as they illustrate some of the advantages of immunoassay in pesticide residue analysis. Then we will move on to a more detailed discussion of our recent analytical work with the herbicide paraquat. 

Conventional analyses for the insecticide, endosulfan, in environmental samples requires time-consuming multistep cleanup procedures prior to the final quantification by gas chromatography (l). For this reason, a sensitive and rapid ELISA could be of great value if large numbers of samples have to be analyzed. The advantages of immunoassays for residue analysis have been described by Hammock and Mumma (2). 

The main disadvantage of immunoassays is that they measure total cytokine, which contains both functional and nonfunctional levels. Cross-reactivity with the precursors or degradation products of the cytokine is frequently observed. The detection limit (about 1 to lOpg/mL) of immunoassays is higher than bioassays. A 1995 study reported a lack of comparability between the different kits for IL-2, IL-6, IL-8, and TNFa. The major advantages of immunoassays are their excellent analytical performance and their ability to be automated. 

This table illustrates one of the major impediments to the rapid assimilation of immunochemical technology into pesticide residue analysis labs. Because of the amount and variety of work involved, new method development costs may be high when compared to routine chromatographic methods. However, the low cost per run allows for rapid recovery of the initial investment with sufficiently high sample loads. For example, the cost of reagents and supplies for an ELISA for diflubenzuron was estimated to be 0.20/sample as compared with 4 for HPLC or 11 for GC (35). In addition to the lower reagent and supply costs, the major economic advantage of immunoassay is the dramatic decrease in labor costs. 

Immunoassays are powerful analytical tools that provide sensitivity and specificity comparable to traditional chemical analyses. The advantages of immunoassays lie in their speed, simplicity, and relatively low cost. These factors have fostered the rapid growth of the medical diagnostic industry and have made possible an agricultural diagnostics industry. 

Both methods have their advantages, disadvantages, and commonalities. The principal advantage of immunoassays over DNA assays is the fact that they detect the actual component of interest (i.e., food allergens, mycotoxins, and species-specific proteins used in speciation, among others), whereas DNA is used only as a marker for the presence of these components in food ingredients or processed foods. Further comparisons between the two are described below. 

In quantitative immunoassays, e.g., enzyme-linked immunosorbent assay and radioimmunoassay, a known amount of labeled vitamin is mixed with sample extract in which the vitamin content should be determined. Methods of labeling include radioisotopes (e.g., cobalamine), fluorescence, or luminescence markers (e.g., folate). The mixture is subjected to binding agent, equally forming complexes with both labeled and unlabeled vitamin. This complex is then isolated, and the amount of labeled vitamin present is measured. Sample vitamin concentration can be deduced from the ratio of labeled vitamin added to labeled vitamin measured after isolation. Advantages of immunoassays are short analysis time, and the possibility of automating them on clinical analyzers. 

The major advantages of immunoassay are its specificity, versatility and sensitivity. Specificity and versatility may be attributed to the selective and specific nature of the antigen-antibody binding reaction and the large number of molecules of interest that themselves, or coupled to a carrier (i.e., haptens),... 

The advantages of homogenous immunoassays are simple formats and rapid data output producing user-friendly and cost-effective products. Technical challenges to consider, however, are the necessity to remove or minimize background interference from the reagents and nonspecific binding reactions. 

Conventional ion-selective electrodes have been used as detectors for immunoassays. Antibody binding measurements can be made with hapten-selective electrodes such as the trimethylphenylammonium ion electrode Enzyme immunoassays in which the enzyme label catalyzes the production of a product that is detected by an ion-selective or gas-sensing electrode take advantage of the amplification effect of enzyme catalysis in order to reach lower detection limits. Systems for hepatitis B surface antigen and estradiol use horseradish peroxidase as the enzyme label and... 

The main advantage of a homogeneous immunoassay, compared to a heterogeneous immunoassay, is the absence of a separation step. This translates into a simpler procedure and easier automation. However, homogeneous assays are typically less sensitive and more susceptible to sample interferences which are removed in a separation step. 

The use of immunoassays for the determination of pesticides and veterinary medicines in food animals has increased since the early 1990s. The advantages of simple analysis, quick results, and high throughput make immunoassays a powerful technique for problematic matrices commonly encountered in animal agriculture. Careful development and validation are required to obtain accurate results, however. This review has demonstrated that most immunochemical techniques have been designed for use with milk samples, but a number of applications have also been developed for liver and muscle samples. The development of immunoassay techniques for residue analysis in eggs has clearly not been pursued to the extent of other edible tissues. 

Lanthanides also have potential as DEFRET energy donors. Selvin et al. have reported the use of carbostyril-124 complexes (53) with europium and terbium as sensitizers for cyanine dyes (e.g., (54)) in a variety of immunoassays and DNA hybridization assays.138-140 The advantage of this is that the long lifetime of the lanthanide excited state means than it can transfer its excitation energy to the acceptor over a long distance (up to 100 A) sensitized emission from the acceptor, which occurs at a wavelength where there is minimal interference from residual lanthanide emission, is then measured. 

As an alternative, extremely sensitive detection can be achieved with reporter antibody probes tagged with intensely SERS-active compounds or with enzymes that react with substrates to yield SERS-active products. These methods often involve sandwich immunoassay techniques, which increase the number of required steps but offer the advantages of excellent sensitivity and the potential for label multiplexing. For example, Nie and coworkers recently reported the simultaneous detection of two types of antigens in a... 

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