Enzyme-linked immunosorbent assay quantitation

EngvaU, E.,Jonsson, K., Perlmann, P., 1971. Enzyme-linked immunosorbent assay, quantitative assay ofprotein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Bio-chem. Biophys. Acta 251, 427—434. 

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. 

The use of independent methods, other than IFIC, for quantitative demonstration of proteins is particularly important. Both enzyme-linked immunosorbent assay (ELISA) and Western blot may be employed to confirm the amount of protein in a cell/tissue model, and in the protein-embedding bar code model under both comparable fresh and FFPE samples for accurate... 

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. 

Engvall, E. and Perlman, P. (1971) Enzyme-linked immunosorbant assay (ELISA) quantitative assay of Immunoglobulin G. Immunocytochemistry 8, 871-879. 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Schnitzius, J. M., Hill, N. S., Thompson, C. S. and Craig, A. M. 2001. Semi-quantitative determination of ergot alkaloids in seed, straw, and digested samples using a competitive enzyme-linked immunosorbent assay. Journal of Veterinary Diagnostic Investigation, 13 230-237. 

Enzyme-linked immunosorbent assay (ELISA) Immnne-based quantitation of expressed protein Rapid, specifle, quantitative, and efficient Requires one or more high-afflnity antisera or monoclonal antibodies... 

Antibodies can be combined with enzymes and color reagents or radioactive antigens to produce quantitative testing for drugs. The ELISA or Enzyme-Linked ImmunoSorbant Assay uses antibodies generated against the Ag to be tested for covalently linked to an enzyme which can catalyze a color change reaction such as the NADH to NAD conversion (Xmax at 340 nm). When the Ag-Ab complex is formed the enzyme is activated and the color can be detected. 

During in vivo studies under biologically relevant conditions, the cis-Pt loading of the DNA is much lower than for the above-mentioned in vitro studies. It has been calculated that mortality of HeLa cells occurs at an value of 10 5 (i.e., one bound cis-Pt molecule per 105 nucleotides) (64a). This excludes atomic absorption spectroscopy for identification of the in vivo adducts. Immunochemical techniques, however, have shown to be very promising, and high sensitivity and selectivity levels have been reached. At the moment, only a few studies in which antibodies are raised against cis-Pt-treated DNA (64) or against synthetic cis-Pt adducts with mono- or dinucleotides are available (64a). With the latter method, quantitation of the different platinum-DNA adducts formed under in vivo conditions is possible. At the moment, femtomole (10-15 mol) amounts of the adducts can be detected with competitive enzyme-linked immunosorbent assay (ELISA) techniques. It has been demonstrated in this manner that the GG-Pt adduct is also the predominant adduct under in vivo conditions. 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

Butler, J. E., McGivern, P. L., Conterero, L. A. and Peterson, L. 1980. Application of the amplified enzyme-linked immunosorbent assay Comparative quantitation of bovine serum IgGi, IgG2, IgA, and IgM antibodies. Am. J. Vet. Res. 41, 1479-1491. 

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable SPEs have been developed for quantitative determination of OTA. 

Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-... 

Rubio, F.M., J.A. Itak, A.M. Scutellaro, M.Y. Selisker, and D.P. Herzog (1991). Performance characteristics of a novel magnetic-parti-cle-based enzyme linked immunosorbent assay for the quantitative analysis of atrazine and related triazines in water samples. Food Agric. Immunol., 3 113-125. 

Yl. Yamagata, H., Harley, J. B., and Reichlin, M., Molecular properties of the Ro/SS-A antigen and enzyme-linked immunosorbent assay for quantitation of antibody. J. Clin. Invest. 74, 625—633... 

Enzyme-linked Immunosorbent Assay. A promising alternative to the RIA procedure is an enzyme-linked immunosorbent assay (ELISA) which depends upon the conjugation of a functional enzyme to either an antigen or antibody. The amount of enzyme present in a competitive binding assay is quantitated instead of the amount of radiolabeled compound. The concentration of the enzyme can be determined through its subsequent reaction with a substrate which results in a measurable spectroscopic change. 

Hefle, S.L., Bush, R.K., Yunginger, J.W., and Chu, F.S. 1994. A sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of selected peanut proteins in foods. J Food Protect 57 419 -23. 

Ronne E, Behrendt N, Ploug M, Nielsen HJ, Wollisch E, Weidle U, et al. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay. J Immunol Methods 1994 167(1-2) 91-101. 

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