Immunoassay direct

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Toxicity associated with chronic use is not as well described as acute toxicity, but it appears to include cerebral atrophy, cardiomyopathy, and chronic pulmonary disease. Cocaine and its metabolites are most commonly identified in patient urine. An immunoassay directed toward identification of benzoyl-ecgonine will frequently indicate the presence of cocaine and its metabolites for many days after use. The duration of qualitatively detected cocaine and metabolites in urine is probably dose dependent and may be up to 3 weeks in length. Chronic use of cocaine may lead to dependence. 

These screenings are basically performed by immunoassays directly on the urine or blood, or after specific manipulation of the samples (e.g., ultrafiltration and concentration, blotting transfei and electrophoretic separation). In principle, formal confirmation shall be obtained by using other immunological tests acting on a different epitope of the targeted molecule. 

There are three main formats of immunoassays direct noncompetitive assays, competitive (direct or indirect) assays. 

The ability to combine small volumes with excellent sensitivity has made amperometric detection with RDE ideal for bead-based immunoassays. Direct comparison of the results obtained in the above Bugbead assay with those obtained with FIAEC detection showed that the detection limit of RDE was 2 orders of magnitude better than for EIAEC [53]. The other important feature of RDE is the extremely short detection time that could be reached by combining enzyme turnover with electrochemical detection. The RDE detection time of 2 min in the above Bugbead assay is over 10 times faster than the typical detection time for HAEC that requires a 20 min enzyme substrate incubation. It is also clear... 

Immunodiffusion and immunoprecipitation, developed ia the 1940s as a means to identify and semiquantitate specific proteias, were the direct precursors to the development ia 1953 of Immunoelectrophoresis, a method used ia many clinical laboratories (5). Single- and double-gel immunodiffusion and immunoelectrophoresis were, ia effect, the first standardized and routinely used immunoassay methods (see Electroseparations, electrophoresis). 

Fluorescence Immunoassay. Basic FIA follows the same formats and approaches as EIA. The difference Hes in the indicator a fluotophote is used instead of an enzyme. This allows direct quantification of the indicatot—antibody—antigen complex, or free indicator-reagent, without the need for a substrate. 

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Sulfonylureas are not directly amenable to gas chromatography (GC) because of their extremely low volatility and thermal instability. GC has been used in conjunction with diazomethane derivatization, pentafluorobenzyl bromide derivatization, and hydrolysis followed by analysis of the aryl sulfonamides. These approaches have not become widely accepted, owing to poor performance for the entire family of sulfonylureas. Capillary electrophoresis (CE) has been evaluated for water analysis and soil analysis. The low injection volumes required in CE may not yield the required sensitivity for certain applications. Enzyme immunoassay has been reported for chlorsulfuron and triasulfuron, with a limit of detection (LOD) ranging from 20 to 100 ng kg (ppt) in soil and water. 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

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. 

The development of sensitive and inexpensive immunoassays for low molecular weight pesticides has been an important trend in environmental and analytical sciences during the past two decades. 0.27-29 jq design an immunoassay for a pesticide, one can rely on the immunoassay literature for agrochemicals, " but many of the innovations in clinical immunoanalysis are also directly applicable to environmental analysis. - Conversely, the exquisite sensitivity required and difficult matrices present for many environmental immunoassay applications have forced the development of technologies that are also useful in clinical immunoassay applications. In the following discussion we will describe widely accepted procedures for the development of pesticide immunoassays. 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. 

Beasley et al. developed a panel of immunoassays to monitor DDT, its metabolites, and structurally related compounds, but they found that milk has a severe effect on the assay performance. They found that when directly utilizing whole milk, color development was completely inhibited. Even when using 1 100 dilutions of whole milk, the assay sensitivity was reduced by 90% (based on the IC50 shift, not simply the dilution factor). A number of procedures were evaluated to eliminate the interferences from the fat-soluble analytes. However, many of the procedures that removed interferences also removed the analytes. Extraction with a mixture of solvents and the use of similarly processed blank milk to prepare the standards ultimately yielded more accurate results. This article demonstrates the difficulties encountered in analyzing lipid-soluble analytes. 

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. 

Test strip and immunoflltration devices were developed by Ostermaier et al to detect sulfadiazine, sulfamethazine, and sulfamethoxypyridazine in milk. Direct competitive immunoassay was utilized with sulfonamide-horseradish peroxidase as the detector. The LOD for sulfamethazine for both the dipstick and immunoflltration was 10 pg kg for sulfadiazine, the LOD was 12 pg kg for the dipstick and 30 pg kg for immunoflltration. For sulfamethoxypyridazine, the LOD was 10 pgkg for the dipstick and 20 pgkg for immunoflltration. The devices were found to be suitable for on-site use with undiluted milk. 

Silverlight and Jackman developed an immunoassay for levamisole in meat and milk. The LOD in both milk and meat samples was 1 xg kg The assay was applied to milk directly, and muscle samples required only homogenization in the presence of 10-fold of buffer prior to analysis. The linear range of the assay was between 5 and 50 pg kg for meat and between 0.2 and 25 qg kg for milk. The linear range of the assay was below the MRL for milk (10 qg kg ) and meat (50 qg kg ). 

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