ELISA formats

Sandwich assay — This format works well for ELISA. The success and potential shortcomings for microarrays are discussed in the next section. 

Mass spectrometry — It has excellent sensitivity but low throughput and is semiquantitative. The ICAT (isotope-coded affinity tag) labeling method for proteins enables differential display analyses using mass spectrometry (Griffin and Abersold, 2001). This may be 

Protein expression profiling (protein differential display) using microarrays is considered an important new tool for proteomic discovery. It is similar in concept and approach to the gene expression microarray for mRNA profiling. Sreekumar and Chinnaiyan (2002) describe a general approach for using the microarray to monitor protein expression in cancer and normal tissues. Here are the steps  

Extract total protein from cancer and normal cells using a detergent, e.g., 1% NP40. 

Remove excess detergent from the lysate by adsorption onto a solid phase (e.g., beads). 

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The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

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. 

Immunoassays designed for environmental applications are mostly sold as some variation of the ELISA format. ELISA-like formats dominate the field because they are inexpensive and because they provide high sensitivity and precision without requiring complex instrumentation. The basic ELISA format supports both field and laboratory-based applications but is limited by multiple steps and inadequate sensitivity for some applications, excessive variability and sometimes long analysis times. Some of the other formats discussed in this article may replace the ELISA for selected applications however, because many laboratories are familiar with the ELISA technology, there will be a significant delay before alternative formats are widely accepted. 

The involvement of several tyrosine kinases in various cancers requires efficient screening methodologies for the inhibitory compounds. Screening is divided into three steps (1) primary screening against the pure isolated PTK in a cell-free system. The objective is always an ELISA format. The compounds are screened against a battery of PTKs and Ser/Ther kinases in order that the pattern of selectivity can be established quickly [2]. 

Figure 6.17 Concordance of standard ELISA and microarray ELISA formats in PSA determinations. (From Wiese, R. et al., Clin. Chem., 47,1451-1457, 2001. With permission.)...

FIGURE 8.4 Scheme of the ELISA format most commonly used for the analysis of heavy metals, where antibodies recognize chelated forms of metal ions. 

ELISAs for detecting cadmium(II), nickel(II), lead(II), and mercury(II) in water samples have also been reported.134139 The most common ELISA format used for the analysis of these compounds can be seen in Figure 8.4. 

Schneider, P. and B.D. Hammock. 1992. Influence of the ELISA format and the hapten enzyme conjugate on the sensitivity of an immunoassay for S-triazine herbicides using monoclonal-antibodies. J. Agric. Food Chem. 40 525-530. 

A competitive ELISA was developed to detect stem peptide generated from the coupled enzyme reaction as well as to measure the cross-reactivity of the antibody with small peptides. The ELISA format is pictured in Figure 4. BSA-pentapeptide (BSA-PP) was immobilized onto the wall of a microtiter plate. Samples or standards containing pentapeptide were then added, followed by rabbit anti-pentapeptide antibody. The amount of anti-pentapeptide antibody bound to immobilized BSA-PP was visualized by adding a second anti-rabbit IgG antibody... 

De Luis R, Lavilla M, Sanchez L, Calvo M, Perez MD (2009) Development and evaluation of two ELISA formats for the detection of b-lactoglobulin in model processed and commercial foods. Food Control 20 643-647... 

In addition to using radiolabeled antibodies in the ELISA format, you can also use them in the... 

Another interesting and novel set of GEM models has linked regulatory elements of relevance to toxicology to genes encoding secreted reporter proteins. For example, the antioxidant response element (ARE) has been linked to the human chorionic gonadotropin (hCG) beta chain gene to produce a urinary marker that is detectible with an antibody in an ELISA format.64 Mice with this construct are presently under development. 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

The competition assay was designed which followed the standard indirect ELISA format (17-18 . The methoprene conjugate was bound to a solid support in the form of a microtiter plate. Free methoprene in methanol (5 fib) was added to the pre-coated wells followed by methoprene-specific antiserum. The antibodies were allowed to compete for both immunogen-bound and free methoprene. Enzyme-conjugated, goat-antirabbit antibody was added, followed by substrate, and the color was allowed to develop. The absorbance of substrate over a range of methoprene concentrations can be drawn as a standard curve, which is presented as percent inhibition of the assay (Figure 6). The 50% inhibition (I5g) of methoprene was at a concentration of approximately 50 ng/mL. 

BTA stat. test strip. A niulticenter trial compared the BTA stat. test with voided urine cytology studies in 499 patients undergoing surveillance cystoscopy for recurrent bladder cancer. The BTA stat. test identified 40% of patients with positive cystoscopy results, and cytology detected 17%. A positive test may provide a higher degree of suspicion for recurrence. A quantitative test in ELISA format, BTA TRAK, is also availabie. 

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