Enzyme flow immunoassay

The ICMs used for pesticide analysis include immimoassays (lAs) and the use of antibodies for sample preparation (e.g., for SPE and the cleanup of samples) [153], detection in flow-injection analysis, and biosensors. The earliest ICMs to be developed for pesticides analysis were lAs. There are various t) es of lAs, but the most frequently used in this context is the enzyme-linked immunosorbent assay (ELISA) [185]. ELISA is a heterogeneous assay because the antibodies or antigens are immobilized on a solid phase. Table 18.3 lists selected ELISA methods for the determination of pesticides in water samples [186-190]. Bjamason et al. have proposed an enzyme flow immunoassay (EFIA) using a protein G column for the determination of triazine herbicides in surface and wastewaters with a linear range between 0.1 and 10 pg/L [191]. 

Bjarnason, B. et al.. Enzyme flow immunoassay using a Protein G column for the screening of triazine herbicides in surface and waste water. Anal. Chim. Acta, 426,197,2001. 

Table 9.3 shows the effect of increase in molar ratio of an atrazine hapten coupled to the enzjune p-galactosidase (p-GAL) in a flow immunoassay for the determination of atrazine. A high hapten density had a negative impact on the assay sensitivity, i.e., the lowest MDC and IC50 values were obtained for the lowest molar ratio of hapten to enzyme. 

Mecklenburg M, Lindbladh C, Li H, Mosbach K and Danielsson B 1993 Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin Anal. Biochem. 212 388-93 Danielsson B, Flygare L and Velev T 1989 Biothermal analysis performed in organic solvents Anal. Lett. 22 1417-28... 

T. A. Kelly, Separation-Free Enzyme Fluorescence Immunoassay by Continuous Flow Injection Analysis. Proc. Enzyme-Mediated Immunoassay, Plenum, New York (1985) 191. 

The tests used most frequently for speciation of animal products are the enzyme-hnked immunosorbent assay (ELISA), including immunosticks and, more recently, lateral flow immunoassays (LFIAs), the latter of which now use latex particles or colloidal gold for detection rather than the older enzyme immunoassay (EIA) technology (seeTable 13.1). 

See also Enzymes Immobilized Enzymes. Flow Injection Analysis Detection Techniques. Forensic Sciences DNA Profiling. Immunoassays, Techniques Luminescence Immunoassays. Liquid Chromatography Column Technology Instrumentation. 

See alsa Bioassays Overview. Chemiluminescence Overview. Derivatization of Analytes. DNA Sequencing. Enzymes immobilized Enzymes. Flow Injection Analysis Detection Techniques. Fluorescence Overview. Immunoassays Oven/iew. Luminescence Overview. Molecularly Imprinted Polymers. Optical Spectroscopy Detection Devices. Phosphorescence Principles and Instrumentation. Sensors Photometric. 

Enzyme immunosensors are used in flow injection systems and Hquid chromatography to provide automated on-line analyses (71—73). These systems are capable of continuously executing the steps involved in the immunoassays, including the binding reactions, washing, and the enzyme reaction, in about 10 minutes. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

Electroosmotic flow, 195 End column detection, 89 Energy barrier, 16 Enzyme electrodes, 172, 174 Enzyme immunoassays, 185 Enzyme inhibition, 181 Enzyme reconstitution, 178 Enzyme wiring, 178 Equilibrium potential, 15 Ethanol electrodes, 87, 178 Exchange current, 14... 

Figure 18 Flow chart of the automated on-line flow injection immunoassay (FllA). Six steps are involved in each cycle (1) addition of antibody and incubation (2) addition of analyte (or standard) and incubation (3) addition of enzyme-tracer and incubation (4) addition of substrate and incubation (5) downstream measurement of fluorescence (6) regeneration of affinity column...

Most of the reported methods of analysis of valproic acid and its sodium salt in biological fluids rely on the use of chromatography, especially gas chromatography, although high performance liquid chromatography (HPLC) is also reported. Other methods, such as flow injection analysis, enzyme-immunoassay, fluorescence-polarization capillary electrophoresis, and potentiometry are sometimes used. The reported methods can be classified as follows. 

J. Gascon, A. Oubina, B. Ballesteros, D. Barcelo, F. Camps, M.P. Marco, M.A. Gonz lez-Martmez, S. Morais, R. Puchades, and A. Maquieira, Development of a highly sensitive enzyme-linked immunosorbent assay for atrazine. Performance evaluation by flow injection immunoassay. Anal Chim. Acta 347, 149-162 (1997). 

In the past, general chapters and reviews have been published, related to the characteristics of CL as analytical technique [7-9], mainly in the liquid phase [10-14], and its use as detection mode in flowing streams and immunoassay [15-17]. Two extensive reviews reported on the specific application of CL reactions according to the nature of the analyte (inorganic species, enzymes and nucleotides, acids and amines, carbohydrates, steroids, polycyclic aromatic compounds, and drugs) and covering the literature from 1983 to 1991 [18] and from 1991 to mid-1995 [19]. 

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). 

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