Antigen enzymes

EIAs can be used per se or with a spectrophotometer. Traditionally, EIAs have been developed in 96-weU microtiter plates which provide the immobilization support for the assay, the reaction vessel, and, when linked to a spectrophotometer-based reader, a rapid means to detect and quantify the color resulting from interaction of a substrate with the antibody—antigen—enzyme complex. Automated immunoassay analyzers targeted primarily for use in the clinical laboratory have taken automation one step further, utilizing robotics to carry out all reagent additions, washings, and final quantification including report preparation. 

Figure 7.15 Enzyme-multiplied immunoassay (EMIT). The three reactants, test (or standard) antigen, enzyme-labelled antigen and a limited amount of antibody are allowed to react and reach an equilibrium position. The unbound labelled antigen which remains is the only source of enzyme activity, the bound enzyme being inactivated. This free enzyme can be quantitated using a direct kinetic assay method and is proportional to the amount of unlabelled antigen originally present.

B.K. Van Weemenand A.H.W.M. Schuurs Immunoassay Using Antigen—Enzyme Conjugates. FEBS Lett. 15, 232 (1971). 

As we will soon discover, microarray-based technologies have found utility in a number of fields. While DNA arrays are the most technically mature and have the broadest application portfolio, we have witnessed the ever-increasing generation of new kinds of probe arrays antibody, antigen, enzyme, aptamer, carbohydrate, tissue, cell, and small molecule microarrays. The list undoubtedly will continue to expand. We can also describe microarrays in terms of prognostic, diagnostic, and predictive roles. A few examples that examine these applications are provided. 

M17. Miedema, K., Boelhouwer, J., and Otten, J. W., Determinations of proteins and hormones in serum by an immunoassay using antigen-enzyme conjugates. Clin. Chim. Ada 40, 187-192 (1972). 

If the iodinated protein will be used in bioassays, check for biologic activity (antigenicity, enzymic activity, ligand binding, etc.) in comparison with the not-labeled starting material. 

The optimum antigen/enzyme conjugate concentration to use is the one giving approx 90% of the maximum value obtained using this curve. 

The additional optimization of other ELISA factors may be necessary after determining the best concentration of antigen/enzyme conjugate to use (see Note 8). Three parameters may be varied to make improvement. Each one should be adjusted independently and an optimum dilution factor or value determined for routine use. Molecules of interest may be masked by components of the test fluid leading to unexpectedly low signals. This may be remedied by mixing the sample with PBS/Tween supplemented with 10% fetal bovine serum (see Note 9). 

The enzyme immunoassay (EIA) applies a single antibody to measure small molecules. The assay works on the principle that two antigens, enzyme-labeled and unlabeled analytes, compete for binding to the limited number of binding sites on the primary antibody, which is subsequently bound to the immobilized anti-IgG. The amount of labeled antigen bound is inversely proportional to the amount of unlabeled antigen (e.g., a cytokine) present in the sample (S7). 

In the future, we will see developments involving surface enhanced Raman scattering technologies in combination with AuNPs and waveguides as well as combinations of immobilized and solution-bom AuNPs and functional bridges in-between them. It can also be expected that applied medical research, namely the detection of antigens, enzymes, and proteins in body fluids, will benefit fi om the sensor developments with its extreme sensitivities. 

Van Weeman, B.K. Schuurs, A.H.W.M. Immunoassay using antigen-enzyme conjugates. FEES Letter 1971, 15, 232-236. 

More recently, automated immunoanalyzer methods for DPD have become commercially available. The most widely used is a solid-phase, competitive EIA with chemiluminescence detection. The solid-phase antibody is incubated with serum or calibrator and ALP conjugated to DPD. After washing, the antibody-antigen-enzyme complexes are determined after the addition of substrate,... 

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

Add 0.1 ml antigen-enzyme conjugate and incubate for another 30 min at room temperature (determine in a preliminary test the amount of conjugate required to saturate 0.1 ml antiserum). 

This chapter summarizes our recent efforts to establish two different protein/ enzyme dosing models in mice, using intratracheal and intranasal dosing. Both models have been used to measure antigen (enzyme) specific antibody responses as a function of the dose of antigen administered, the immunization procedure and regimen, and the isotype of antibody produced. 

Prostate-specific antigen Enzyme that liquefies the seminal coagulum Present in rare basal cells mainly in secretory luminal cells... 

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