Enzyme immunoassay models

Li, X.-M., X.-Y. Yang, and S.-S. Zhang. 2008. Electrochemical enzyme immunoassay using model labels. 

An enzyme immunoassay using adenosine deaminase as the enzyme label has been described (318). Potentiometric rate measurements were made with an ammonia gas-sensing electrode. The immunoassay system employed is based on competition between a model haptenic group, dinitrophenyl (DNP) covalently coupled to adenosine deaminase, and free DNP hapten for the available binding sites on the anti-DNP antibody molecules. The detection limit is 50 ng antibody. 

This relationship is established by measurement of samples with known amounts of analyte (calibrators). One may distinguish between solutions of pure chemical standards and samples with known amounts of analyte present in the typical matrix that is to be measured (e.g., human serum). The first situation applies typically to a reference measurement procedure, which is not influenced by matrix effects, and the second case corresponds typically to a field method that often is influenced by matrix components and so preferably is calibrated using the relevant matrix. Calibration functions may be linear or curved, and in the case of immunoassays often of a special form (e.g., modeled by the four-parameter logistic curve) This model (logistic in log x) has been used for both radioimmunoassay and enzyme immunoassay techniques and can be written in several forms as shown (Table 14-1). Nonlinear regression analysis is applied to estimate the relationship, or a logit transforma-... 

A. Dankwardt, B. Hock, R. Simon, D. Ereitag, A. Ket-trup, Determination of Non-extractable Triazine Residues by Enzyme Immunoassay Investigation of Model Compounds and Soil Fulvic and Humic Acids , Environ. Sci. Technol, 30, 3493-3500 (19%). 

S Daunert, BR Payne, L Bachas. Pyruvate carboxylase as a model for oligosubsti-tuted enzyme-ligand conjugates in homogeneous enzyme immunoassays. Anal Chem 61 2160-2164, 1989. 

In another study, a microchip-based electrochemical enzyme immunoassay was developed by Chatrathi and colleagues [65] and its performance is demonstrated for the determination of monoclonal mouse IgG as a model analyte. The direct homogeneous immunoassay requires the integration of electrokinetic mixing of ALP-labeled anti-mouse IgG antibody (Ab-E) with the mouse IgG antigen (Ag) analyte in a precolumn reaction chamber, injection of immunochemical products into the separation channel, followed by rapid electrophoretic separation of enzyme-labeled free antibody and enzyme-labeled antibody-antigen complex. The separation is followed by a postcolumn reaction of enzyme tracer with p-aminophenyl... 

The principle of the first type of assay is similar to that of RIA a bile acid covalently linked with an enzyme (peroxidase, -galactosidase) is used a tracer instead of a radioisotope. The enzymatic activity of the tracer is recorded spectrophotometri-cally (after antigen-antibody complex separa- on) by measuring specific colour-producing substrates. Mat n developed this method for cholic acid conjugates and Maeda for ursodeoxycholic acid. The sensitivity of these methods is quite similar to corresponding RIA as previously reported (see above). This model of enzyme immunoassay could be extended to other bile acids and this may be used on a large scale in medical laboratories. 

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... 

As a first model system goat-IgG (gtIgG) was chosen as an analyte The use of alkaline phosphatase as label required a change the pH between the immunoassay (pH 8) and the electrode reaction (pH 6.5). In order to reduce background signal activity determination was performed with 1 iM PAPP. In 0.1 M MES buffer the electrode sensitivity towards PAPP is approximately 10 % of the respective response in the presence of the same concentration of PAP At the same time, the sensitivity towards PAPP in phosphate buffer is 200 times lower In comparison to PAP sensitivity. This suggests that the enzyme membrane causes the hydrolysis of PAPP to PAP. Obviously, phosphate ions inhibit this process. 

The microplate model has often been used as a starting platform for electrochemical array development. In a report from Tang et al. [14], a multichannel array consisting of eight channels of Pt electrodes, fitted to the dimensions of standard microplate wells, has been developed, with the required multichannel potentiostat for amperometric detection of the product of an enzymatic immunoassay label. Alkaline phosphatase was used as the model immunoassay label in a model rabbit/anti-rabbit IgG study, where the second IgG is labeled with enzyme. The enzyme product provided the amperometric signal with a detection limit in the low pM range. 

An eight-element array consisting of iridium oxide working electrodes, an iridium counter and a Ag/AgCl reference electrode has been used for amperometric multianalyte sandwich immunoassay with an enzyme label on the second antibody [49]. Each planar array element, patterned on a glass substrate, has an area of 0.78 mm. Simultaneous detection of four model analytes (goat, mouse, human, and chicken antibodies) occurred without amperometric crosstalk between adjacent elements, with 3 ng/mL detection limits. 

The microplate model for immunoassays has been successfully adapted for electrochemical measurements, with a 96-weU screen-printed carbon microplate system [50], shown in Fig. 8. The weU bottoms form the sensing surfaces with carbon working electrodes and Ag/AgCl counter electrodes. The system employs intermittent pulse amperometry at fixed pulse potentials for detectiOTi. In this report, aflatoxin Bi was the analyte, and a spiked com matrix was chosen for examination of extraction efficiencies and matrix effects in a competitive enzyme-linked... 

---