Enzyme-linked immunosorbent assays formats

Kornegay JR, Shepard AP, Hankins C, Franco E, Lapointe N, Richardson H, et al. Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format. J Clin Microbiol 2001 39 3530-6. 

EIAs are more desirable for the measurement of agrochemicals than enzyme-linked immunosorbent assays (ELISAs) for several reasons. EIAs are easier to run, require minimal liquid transfers, and are completed in brief time frames, approximately 40 min for tube assays to 2.5 h for microtiter plate assays. In contrast, ELISAs are more complex, have many steps involving transfer of reagents, and require 6-8 h to complete. Most commercially available immunoassays utilize the EIA format. 

A very versatile piece of equipment that is affordable for individual laboratories is the microplate reader. This allows multiple samples to be analyzed at once, commonly in a 96-well format, although 384- and 1536-well formats are available. Typical measurements that can be performed include UV-Vis absorbance, fluorescence, or luminescence, allowing a range of assays to be performed, such as cell growth, enzyme kinetics, enzyme stability, or enzyme-linked immunosorbent assay [60-62]. Functionality can be increased by the use of liquid dispensing systems or automatic plate handling. 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Immune detection is a key utility of antibodies in biotechnology [3, 5]. Antiden-drimer sera efficiently detect dendrimers in multiple assay formats, including enzyme-linked immunosorbent assays (ELISA), and in Western and dot blots [3, 5], ELISA assays are commonly used to quantitate proteins, and a quantitative ELISA could be developed for dendrimers using our sera, though doing so would require development of dendrimer standards of known concentration that could be used for calibration. 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

The CML is the most characterized AGE and is referred to as a glycoxidation product. The inhibitory effects of C-glycosylflavones on the CML formation were tested by enzyme-linked immunosorbent assay in kidney diabetic subjects. The results showed that the percent inhibition was about 53% for chrysoeriol 6-C-boivinosyl 7-0-glucoside, 64% for chrysoeriol 6-C-boivinosyl, 80%i chrysoeriol 6-C-fucosyl, and only 2% for 4"-OH-3 -methox-ymaysin versus 60%i for the standard glycation inhibitor, aminoguanidine. 

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 5-28b). Proteins in a sample are adsorbed to an inert surface, usually a 96-well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody-linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. 

This chapter presents an approach to perform enzyme linked immunosorbent assays (ELISA) in a microfluidic format with electrochemical detection. This field of analytical chemistry has shown a strong activity in recent years, and many reports have presented the use of capillary-sized reactors for running immunoassays either in homogeneous format (where the antigen-antibody complex and the labelled revelation reagents are separated prior to detection, as for instance by capillary electrophoresis [1-3]) or in heterogeneous format (where the antibody is immobilised on the inner surface of the microsensor device [4] or on microbeads [5,6]). 

Test wells exhibiting growth for the presence of monoclonal antibodies when the hybridoma growth covers approx 25% of the base of the cell well. The assay system used should mimic the final test format required. Commonly, Triple Antibody Sandwich (TAS) (5) enzyme-linked immunosorbent assay (ELISA) is use for screening hybridomas for specific antibody. It is important when testing hybri-... 

Specific antibodies can also be used to quantify the amount of the corresponding antigen in a biological sample. Several types of immunological assays exist. An increasingly popular version is enzyme-linked immunosorbent assay (ELISA) (see Fig. 1) which can readily detect and quantify less than a nanogram of a specific antigenic protein. In ELISA, the specific antibody is coupled to a solid support. A convenient format for ELISA is to use a plastic tray that has molded... 

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). 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

The cultured root segments were divided into proximal, distal and newly developed lateral root parts and their endogenous lAA levels were periodically analyzed using enzyme-linked immunosorbent assay (ELISA) kits for lAA (PHYTODETEK iAA, Idetek Inc., USA) according to the procedure of Weiler et al. [39]. The lAA levels in the root segments are indicated in Fig. (26). The lAA levels in the roots cultured on HF B5 medium that initiated shoot formation increased once at day 9 and consequently decreased in both proximal and distal parts. On the other hand, lAA levels in proximal part of the roots cultured in the presence of 0.5 mg/1 TIBA increased along with the culture period. The roots cultured on the MS medium containing 0.5 mg/1 NAA (source for the experiments) accumulated low amounts of lAA and the lateral roots did not accumulate detectable amounts of lAA in all the cases. 

In most cases, competitive immunoassays for the analysis of small molecules are carried out in microtitre plates. The majority of these assays use an enzyme as label, thus leading to the term enzyme immunoassay, and the most commonly used is the ELISA (enzyme-linked immunosorbent assay) that has a heterogeneous format (separation of bound and unbound). This format can be set up either in the enzyme-tracer format (Figure 3.3.1 A) or in the coating antigen format (Figure 3.3. IB). The result shows a... 

The myelin formation is most often studied using microscopic analysis (including electron microscopy) of the immunocytochem-istry for specific proteins, such as myelin basic protein (MBP), protein zero, myelin associated glycoprotein (MAG), and peripheral myelin protein 22 [107, 108]. The same markers can also be evaluated by other protein assays (e.g., enzyme-linked immunosorbent assay (ELISA) and western blot) or at mRNA level using for example PCR or microarray assays. 

To use immunoassays one normally employs a reporter substance or label. This label can be one of many materials including a radiochemical, a heavy metal, or commonly, an enzyme. A widely used format is the enzyme linked immunosorbent assay or ELISA. 

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