Immunoassay sampling/sample preparation

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. 

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. 

Consistent with other analytical methods, immunoassays must be validated to ensure that assay results are accurate. Initial validation involves an evaluation of the sensitivity and specificity of the immunoassay, while later validation includes comparison with a reference method. Because a goal of immunoassays is to minimize sample preparation, validation also includes testing the effects of sample matrices and(or) sample cleanup methods on results. The final steps in validation involve testing a limited number of samples containing incurred residues to determine if the method provides reliable data. 

An immunoassay was developed to determine the penicillinase stable isoxazolyl penicillins cloxacillin and dicloxacillin in milk by Usleber et alJ The assay detected lOpgkg" of cloxacillin and 30pgkg of dicloxacillin with recoveries of 102% and 84%, respectively. The calibration curve was prepared by fortifying skimmed milk powder (lOOgL ) with standards. Fortified samples were prepared in pasteurized milk and analyzed directly after decreaming by centrifugation. This immunoassay was performed with minimal sample preparation, probably because the extensive water solubility of the penicillins prevents problems associated with more lipid-soluble analytes. 

Kennedy et al. developed a lasalocid immunoassay for application to residues in chicken meat and liver samples. The antibody was specific and did not cross-react with salinomycin, maduramicin, or monensin. Sample preparation consisted of homogenization in aqueous acetonitrile, removal of fat from an aliquot of the aqueous acetonitrile by hexane extraction, and evaporation of acetonitrile. The sample was then reconstituted with assay buffer. Liver required an additional solid phase extraction step. The LOQ was 0.02 xgkg for muscle and 0.15 agkg for liver. These workers were able to use the system to determine the half-life of lasalocid in the tissues. 

Ivermectin, a macrocyclic lactone, is also utilized to control parasites. An immunoassay was developed to determine ivermectin residues in bovine liver by Crooks etal. The sample preparation procedure was complex, involving tissue homogenization in acetonitrile, centrifugation, extraction with hexane (to remove lipids), evaporation and reconstitution in ethyl acetate, and passage through an SPE column followed... 

Third, the bulk of the items in Table 1 address method performance. These requirements must be satisfied on a substrate-by-substrate basis to address substrate-specific interferences. As discussed above, interferences are best dealt with by application of conventional sample preparation techniques use of blank substrate to account for background interferences is not permitted. The analyst must establish a limit of detection (LOD), the lowest standard concentration that yields a signal that can be differentiated from background, and an LOQ (the reader is referred to Brady for a discussion of different techniques used to determine the LOD for immunoassays). For example, analysis of a variety of corn fractions requires the generation of LOD and LOQ data for each fraction. Procedural recoveries must accompany each analytical set and be based on fresh fortification of substrate prior to extraction. Recovery samples serve to confirm that the extraction and cleanup procedures were conducted correctly for all samples in each set of analyses. Carrying control substrate through the analytical procedure is good practice if practicable. 

These methods have been used since the 1970s they usually require little or no sample preparation and are rapid and easy to use. However, immunoassay has two limitations. First, it does not differentiate between active drugs and similar molecules such as metabolites or co-administered drugs.9,11 Thus, cross-reactivity is a common problem. Second, its use is limited to only those drugs for which antibodies are available. 

The separate items of equipment necessary for the preliminary (i.e. sample preparation) stages of partially automated (i.e. prior to the final measurement instrument) immunoassay available from Denby Instruments Ltd are listed in Appendix 1. 

Beckman Robotic Biomek 1000 automated laboratory The Biomek 1000 integrates the work formerly done by four instruments sample preparation system, diluter/dispenser, plate washer and a spectrometer finish. In can handle assays such as radio-immunoassays (RIA), fluorescence immunoassays (FIA), enzyme immunoassays EIA and enzyme-linked immunoassays (ELISA). 

A number of different testing kits based on immunoassay technology are available for rapid field determination of certain groups of compounds, such as benzene-toluene-ethylbenzene-xylene (EPA 4030) or polynuclear aromatic hydrocarbons (EPA 4035, Polycyclic Aromatic Hydrocarbons by Immunoassay). The immunoassay screening kits are self-contained portable field kits that include components for sample preparation, instrumentation to read assay results, and immunoassay reagents. 

Matrix Sample preparation Type of immunoassay Antibody Sensitivity Ref. 

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