Principles of ELISA

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

Fig. 2. Principle of ELISA. Antibodies elicited against a hapten-KLH conjugate are tested against a hapten-BSA conjugate. Only hapten-specific antibodies are detected. A blocking agent is used to prevent direct binding of antibodies to the solid support...

ELISAs can be designed for use with either reading by eye or a spectrophotometer although different conditions and controls have to be included. The principles of ELISA must be thoroughly understood before either system is adopted. In particular testing by eye is not necessarily simpler to standardize. However, when correct standardization is used, it offers sensitive assays. When a correct plate template is used, the range of color product will be fix)m full through partial color to no color. 

Direct ELISA was extensively described because it introduces the investigator to the ELISA. Many of the areas covered will need less explanation, so that protocols shown for ELISAs will have less detail. The major use for the direct ELISA is to be able to titrate antispecies conjugates and thus avoid using preparations that are too strong or two weak. Some of the major principles of ELISA were introduced Cplateau height, end point, nonspecific reactions, backgrounds, and titration curvesj and they will be constantly reviewed in all the assays described. 

Principles of ELISA for Detection of Antibodies against Rinderpest... 

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). 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

Figure 19-13 illustrates the principle of an enzyme-linked immunosorbent assay, abbreviated ELISA in biochemical literature. Antibody 1, which is specific for the analyte of interest (the antigen), is bound to a polymeric support. In steps 1 and 2, analyte is incubated with the polymer-bound antibody to form a complex. The fraction of antibody sites that bind analyte is proportional to the concentration of analyte in the unknown. The surface is then... 

Principles of microfluidic ELISAs with electrochemical detection... 

In summary, the simple and flexible Universal-IPCR, with or without nonfunctionalized primary antibody, is well suited for a proof of principle of the general feasibility of the IPCR for a given problem in clinical laboratory applications, as successfully shown in several examples. It allows for an easy access to ultrasensitive research studies—if the necessary time and expertise for some fine-tuning of assay conditions is available. Under these conditions, a typical 1000- to 10,000-fold improvement of ELISA sensitivity is accessible in the research laboratory (see Table 1). 

The working principle of the ELISA described here involves the use of LHRH peptide-coated wells to capture any anti-LHRH antibodies present in the sera of vaccinated mice. Captured antibodies are detected through the use of a horseradish peroxidase-conjugated antibody that is specific for mouse immunoglobulins and a substrate is then added to induce a color change. In this case, the substrate utilized is 2,2 -azino-bis 3-ethylbenzthiazoline-sulfonic acid (ABTS Sigma-Aldrich, USA), which is converted to a green soluble end product by horseradish peroxidase (see Note 7). The level of substrate-induced... 

Particle Concentration Fluorescence Immunoassay. The PCFIA is a solid-phase immunoassay in which proteins are attached to polystyrene particles by adsorption or covalent coupling for the solid phase and fluorescent-labeled reagents are utilized for product detection.22 The general principles of the assay are similar to those of the enzyme-linked immunosorbent assay (ELISA), which has been reviewed extensively elsewhere.23 PCFIAs are performed in specially designed 96-well format Fluoricon assay plates utilizing an automated Screen Machine (Idexx Corporation, Research Product Division, Portland, ME). 

The most popular form of this technique is solid-phase heterogeneous ELISA, and the inherent use of passive adsorption facilitates flexibility in assay design. ELISA is generally classified as direct, indirect, sandwich (double-antibody) or competitive. Principles of each of these formats are briefly discussed below. 

ELISA and SOPHEIA are two names for the satfie technique. The principles are described elsewhere Both types, of ELISA (or SOPHEIA), i.e. 

Figure 2-13. Principle of radioimmuno assay (RIA) and enzyme-linked immunoabsorbent assay (ELISA). RIA and ELISA both depend on the highly specific interaction of an antibody with an antigen to determine, for example, the amount of antigen immobilised on a surface. In the example illustrated above, that is achieved by first binding a specific antibody to the surface-bound antigen, and then adding a second antibody which binds specifically to the first. For RIA, the second antibody is radioactively...

Baggot J D 1992 Bioavailability and bioequivalence of veterinary drug dosage forms, with particular reference to horses an overview. Journal of Veterinary Pharmacology and Therapeutics 15 160-173 Dirikolu L, Lehner A F, Karpiesiuk W et al 2000 Identification of lidocaine and its metabolites in post-administration equine urine by ELISA and MS/MS. Journal of Veterinary Pharmacology and Therapeutics 23 215-222 Evans W E 1992 General principles of applied... 

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

CLIA is similar to EIA and ELISA techniques except that the final receptor enzyme assay is replaced with a chemiluminescent tracer followed by measurement of light released as a result of the chemical reaction. The principles of a chemiluminescence competitive binding assay are shown in Figure 8-8. 

The development of various EIA procedures has led to confusing terminology and classifications, which are often misleading concerning their fundamental features and tend to obscure their relative merits. Not surprisingly, many comparative studies produced inconclusive results. The frequent comparison of the relative merits of radioactive and enzyme labels, as evaluated in RIA by saturation analysis and in ELISA by immunometric analysis, is basically faulty since the underlying principles of these assays are different (Ekins, 1980). Here, EIA will be classified according to their essential differences to expose the inherent limitations and potentials of each assay EIA are based either on Activity Amplification (AA) or Activity Modulation (AM). 

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