Immunoassay sampling/sample handling

Oubina A, Ballesteros B, Bou Carrasco P, Galve R, Gascdn J, Iglesias F, Sanvicens N, Marco MP (1999) Immunoassays for environmental analysis. In Barcelo D (ed) Sample handling and trace analysis of pollutants techniques, applications and quality assurance. Elsevier, Amsterdam, p 287... 

Oubina, A., B. Ballesteros, P. Bou, et al. 2000. Immunoassays for environmental analysis. In D. Barcelo (ed.), Sample Handling and Trace Analysis of Pollutants Techniques, Applications and Quality Assurance, pp. 289-340. Amsterdam Elsevier. 

See also-. Blood and Plasma. Clinical Analysis Sample Handling. Electrophoresis Clinical Applications. Enzymes Overview Industrial Products and Processes Enzyme-Based Assays. Immunoassays, Techniques ... 

See also Clinical Analysis Sample Handling. Derivatization of Analytes. Forensic Sciences Systematic Drug Identification Thin-Layer Chromatography. Fourier Transform Techniques. Gas Chromatography Mass Spectrometry Forensic Applications. Immunoassays, Applications Clinical Forensic. Liquid Chromatography Normal Phase Reversed Phase. Spectrophotometry Pharmaceutical Applications. 

See also Blood and Plasma. Capillary Electrophoresis Overview. Cerebrospinal Fluid. Clinical Analysis Sample Handling. Electrophoresis Principles Isoelectric Focusing Polyacrylamide Gels Clinical Applications. Immunoassays Overview. Immunoassays,... 

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). 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

In hair analysis, three methodically different lA with different kinds of labeling are used radioimmunoassay (RIA), which is the most common lA in hair analysis, enzymeimmunoassay (EIA), and fluorescence polarisation immunoassay (FPIA). In hair analysis, lA have the same advantages and disadvantages as compared to their use in urinalysis. They are fast, easy to handle, and can be automated. Their use can save time and expenses when a great number of negative samples has to be expected. 

Figure 7 Fluorescence image of a sandwich immunoassay that was generated with the microchannel assay system. Capture antibodies were patterned as 11 vertical lines. Subsequently, 17 different biological samples were patterned as horizontal lines. At the intersections, an interaction pattern of 11 X17 = 187 features is thus generated with 11 + 17 = 28 liquid-handling steps...

For immunoassay techniques, cellulose acetate filters offer no advantage over glass fiber filter paper, and the slow flow rate and the handling problems of cellulose acetate when compared with glass fiber make it less suitable for use when large numbers of samples are involved. The retention characteristics of GF/B filter paper are adequate for most immunoassay techniques. However, in receptor assays, the particle sizes are close to the minimum retention size of the GF/B filter paper, and cellulose acetate filters are being used for filtration of these samples. 

The effects of the appropriate environmental matrices (soil, water, air, biological - for biomarker or exposure assessment studies) on assay performance must be well characterized and documented. The SOP must also include the degree of quality control necessary to ensure the satisfactory performance of the method. Quality control procedures must address the required sample preparation steps, reagent stability, instrumentation, data handling and analysis. In many immunoassay SOPs that the EPA has reviewed, quality control is totally lacking or minimally addressed particularly for the sample preparations. The Agency can provide direction on what is an appropriate degree of quality control based on the objective of the method. 

Figure 7. Standard curve of gel purified 60 kd protein endotoxin of Btk was generated using a double antibody sandwich ELISA. The arrows indicate dilutions of two Btk formulations absorbance values were used to determine the endotoxin concentrations of the formulations, based on the standard curve. The formulation dilutions gave curves that were virtually superimposable on the standard curve. Such similarity in shape and slope indicate that the antibody is likely binding to a specific determinant common to the purified Btk and the Btk in the formulation. In general immunoassays for biopolymers are much easier to develop than assays for small molecules. However, only recently has an interest in trace analysis of such materials begun to develop in the environmental field. Thus, sample cleanup and handling is not as sophisticated as with small molecules.

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... 

These results illustrate two important factors in working with biological and biochemical pesticides. First, sample clean-up is probably needed before environmental samples can be applied to an immunoassay procedure. Second, to correlate field and laboratory data, samples should be handled in such a way as to minimize biodegradation. 

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