Enzyme-linked immunosorbent assay based methods

Zhang, Y. and Lynd, L.R. (2003) Quantification of cell and cellulase mass concentrations during anaerobic cellulose fermentation development of an enzyme-linked immunosorbent assay-based method with application to Clostridium thermocellum batch cultures. Anal. Chem., 75 (2), 219—227. 

Bioassay-based or enzyme-linked immunosorbent assay (ELISA) methods have been applied in epitope mapping [18, 19]. In this approach, different antigens are coated separately on the surface in different weUs of the analytical plate followed by incubation with a specific antibody that is linked to an enzyme. When the enzyme s substrate is added to the solution, a subsequent reaction can produce a detectable signal. This approach, however, is mostly limited to the characterization of linear epitopes. ELISA can also be applied to multisite binding analysis, where one antibody is attached to the surface, the antigen is bound, and the ability of the second antibody to bind to the attached antibody-antigen... 

Additionally it has been our experience that mass spectrometry as a routine detection/identification technique for bacteria is not well received by microbiologists and clinicians who prefer less expensive, less complicated approaches to bacterial typing and identification, such as methods based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). For that reason we have adapted our MS approach to serve as a means of biomarker discovery that feeds candidate proteins or leads into development as PCR targets or other immunoassay techniques. 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

New detection methods of phenolic compounds are being developed. Based on the principle of the enzyme-linked immunosorbent assay (ELISA), a method has been developed to quantify phenolic compounds such as isoflavones (Vergne and others 2007). 

Most of the analytical methods for the analysis of pesticides in food are based on instrumental approaches based on chromatography coupled to mass spectrometry. However, a great effort of development has been paid to develop rapid screening methods based on biological methods, such as, enzyme linked immunosorbent assays (ELISA). 

Enzyme-linked immunosorbent assay (ELISA) is a new method in alkaloid studies. The application of ELISA to alkaloid study is based on antibody incubations. It differs from classical precipitation-based methods in that specific antigen-antibody interactions are recognized by assaying an enzyme label conjugated to one reactant, usually an antibody. Because of the sensitivity with which enzyme... 

Further improvement of microchemical methods for proteinaceous media was based on immunological techniques. The high specificity of the antigen-antibody reaction enables the discrimination of the same protein coming from different species, or the detection of multiple antigens in the same sample. Application to the analysis of artwork has been reported in two types of immunological techniques immunofluorescence microscopy (IFM), and enzyme-linked immunosorbent assays (ELISA) [31]. 

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-... 

High sensitivity. Detecdon limits are in the range of 0.05-5 pmol/mol of unmodified parent base. This compares well to the sensidvides of commonly used methods, such as high pressure liquid chromatography with fluorescence detection (6,7), radiochromatographic analysis (8), radioimmunoassays, and enzyme-linked immunosorbent assays (9,10). 

The staphylococcal toxin must be separated from food constituents and concentrated to detect trace amounts. The toxin is then identified by specific precipitation with antiserum as follows (1) the selective adsorption of the enterotoxin from an extract of the food onto ion exchange resins and (2) the use of physical and chemical procedures for the selective removal of food constituents leaving the enterotoxin in solution. More recently rapid methods based on monoclonal antibodies (e.g., enzyme-linked immunosorbent assay, reverse passive latex agglutination) have been developed for detecting very low levels of enterotoxin in food. 

Immunoassays offer much potential for rapid screening and quantitative analysis of pesticides in food and environmental samples. However, despite this potential, the field is still dominated by conventional analytical approaches based upon chromatographic and spectrometric methods. We examine some technical barriers to more widespread adoption and utilization of immunoassays, including method development time, amount of information delivered and inexplicable sources of error. Examples are provided for paraquat in relation to exposure assessment in farmworkers and food residue analyses molinate in relation to low-level detection in surface waters and bentazon in relation to specificity and sensitivity requirements built in to the immunizing antigen. A comparison of enzyme-linked immunosorbent assay (ELISA) results with those obtained from conventional methods will illustrate technical implementation barriers and suggest ways to overcome them. 

The IRMM has recently started the validation of presently available methods (mostly based on enzyme-linked immunosorbent assays) in a collaborative trial study with real food samples such as cookies and dark chocolate containing peanuts in minute amounts (1-20 mg/kg). These methods have been already been validated in-house [20]. 

Katsiadaki I, Scott AP, Hurst MR, Matthiessen P, Mayer I. Detection of environmental androgens A novel method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterosteus aculeatus) glue protein. Environ Toxicol Chem 2002 21 1946-54. 

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