ELISA materials

Figure 2 Immobilized antigen ELISA format. Antigen is immobilized to a solid phase by passive adsorption. Following removal of unbound antigen, analyte (free H) and antigen (H-protein) compete for a fixed number of primary antibody (Y) binding sites. Unbound materials are removed (dotted line). Secondary antibody-enzyme conjugate (Y-E) is added to bind to primary antibody followed by another wash step. Substrate (A) for the enzyme is added to detect the bound enzyme. The amount of colored product ( ) detected is inversely proportional to the amount of analyte present...

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. 

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. 

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

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. 

A. Heginbotham, V. Millay, M. Quick, The use of immunofluorescence microscopy (IFM) and enzyme linked immunosorbent assay (ELISA) as complementary techniques for protein identi fication in artists materials, J. Am. Inst. Cons., 45, 89 105 (2006). 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

At an early stage in product development, there must be some material made available for use as ELISA reagents. ELISA requires a standard for quantitation and specific antibodies to the drug protein of interest. If these are not already available, either commercially or internally, then time, effort, and expense must be reserved for reagent development as well as assay development activities. The specific antibody is the key reagent in an ELISA. The antibody defines the specificity and sensitivity of the assay. To date there is no successful substitution for the routine production of specific antibodies by immunization of an animal with the antigen target of interest. 

Of all the possible contaminants and impurities of a biopharmaceutical product, organisms (bacteria, virus, mycoplasma) and their products (DNA, endotoxin, host protein), media components, and raw materials, it is most appropriate to use an ELISA for the HCP impurities and some of the process residuals (media components and raw materials). Impurities from media components are known or expected unlike those from the host cell. 

Because of antibody-based selectivity, ELISAs are capable of handling samples that are impure or only semipurihed. It is possible to perform ELISAs in a variety of matrices. This is in contrast to other methods such as HPLC that require relatively pure material. During the development and validation of the ELISA method, it needs to be demonstrated that the ELISA is not affected by interfering substances that could be in the test sample, such as buffers, salts, contaminating proteins, and excipients. It also needs to be demonstrated that the conjugated antibody does not bind nonspecihcally to the coated solid phase. 

Membranes cast upon glass slides also fall into the 3D surface category. Most notable are the nitrocellulose-coated FAST slides offered by Schleicher Schuell (S S) BioScience. A cytokine micro-ELISA product xmder the trade name Provision has been introduced in single-slide and 96-well spacing (64 usable wells) formats. It is called FAST Quant (Harvey, 2003). Historically, nitrocellulose membrane has been used for the sequestering of both proteins and nucleic acids. The adaptation by S S of this microporous (0.2-p pores) material cast into a microarray format has been relatively straightforward. 

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

In 1993, another immunoassay for the detection of monensin was developed but, unfortunately, was never applied to biological material (91). Quite recently a competitive ELISA and a compatible extraction procedure suitable for screening monensin in poultry liver samples was described (92). In this assay, a polyclonal antiserum raised against a monensin-transferrin conjugate and prepared via an acid anhydride intermediate (93) was used. Significant cross-reactivity with other polyethers commonly used by the broiler industry, such as maduramicin, lasalocid, salinomycin, and narasin, was not found. A detection limit of 3 ppb could be readily attained when liver samples were submitted to extraction with aqueous acetonitrile, partitioning between aqueous sodium hydroxide solution and a hexane-diethyl either mixture, evaporation of the organic phase, and reconstitution in ethanol/sodium acetate solution. 

Enzyme-linked immunosorbent assay (ELISA) is a very useful technique for the specific and sensitive assay of certain compounds, in which suitable antibodies, monoclonal or polyclonal, to the compounds are available. The technique has found particular application m the monitoring of environmental contaminants and toxins, either studying the primarily contaminated materials, e.g., foodstuffs, or body fluids of potentially exposed humans. The technique has been increasingly applied to monitoring the carcinogenic mycotoxins, the aflatoxins. 

Muldoon, M.T. and J.O. Nelson (1994). Evaluation of the effects of selected agricultural materials on an ELISA for s-triazines. Food Agric. Immunol., 6 357-370. 

Another format to test for newly expressed proteins is provided through different ELISA assays. Typically, one antibody is coated on a microtiter plate and serves as a capture antibody while a second antibody (added later in the process) is labeled with a reporter molecule allowing the read-out with optical devices. These ELISAs can be operated in a quantitative manner, but need to be calibrated. The measurement unit can be traced back to the amount of protein present in the calibrator, independent of whether the calibrator consists of purified proteins or other biological materials (e.g., seeds, leaves). The amount of proteins within a plant-derived matrix (leaves, seeds, grains), however, depends on several factors, including environmental conditions and can thus not directly be related to thresholds expressed in weight-%. 

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