Solid phase antibody

An example of an immunoassay format is shown in Figure 3. This immunoassay format relies on partial saturation of the solid-phase antibody (Ab) by the antigen (Ag) and on its competition with the labeled antigen (Ag-L) for the available antibody sites. At low antibody and tracer concentrations the sensitivity of... 

Figure 3 Layout of competitive solid-phase antibody immunoassay format in an incubation step antigen (Ag) and labeled antigen (Ag-L) compete for the solid-phase antibodybinding sites (Ab) after the solid-phase antibodies are washed, the activity of the bound labeled antigen is measured, and a calibration curve constructed. The measured signal is inversely proportional to the antigen concentration.

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... 

Given their high degree of sensitivity, which is 2-5 times more than assays in which the antigen is directly bound to the solid phase, antibody sandwich ELISA is arguably the most useful of the immunosorbent... 

Figure 6.31 Solid-phase antibody density and steric hindrance effects. (From Matson, R.S. and Little, M.C., /. Chromatogr., 458, 67-77, 1988. With permission.)...

Rinse the unbound sample material from the solid phase antibody. 

Add enzyme-labelled antibody, specific for the target antigen, to the solid phase. The "tagged" antibody will attach to any antigen captured by the solid phase antibody. 

Applicability—the ability to apply a single procedure to a wide variety of different assays—is desirable but not essential. Some procedures, such as second antibody and solid-phase antibody, are universal. Precipitation with ammonium sulfate or polyethylene glycol is not, because in some cases the properties of the antigen in the bound and free phases are not sufficiently distinct. 

Solid-phase antibody (particles, disks, tubes, gel entrapment, polymerized antibody)... 

Preparation of Solid Phase Antibody. There are many chemical and physical methods by which antibodies may be linked to different solid phase materials. Unfortunately relatively little work has been carried out to investigate systematically the properties of the different methods and materials. The main desirable characteristics are (a) convenience of handling and standardization both in preparation and in utilization b) adequate amount of antibody bound and stability of the preparation both in terms of the binding to solid phase and on storage (c) a low degree of nonspecific binding on the subsequent addition of antigen and labeled antibody. 

Similar tests to the RAST have employed radiolabeled allergens and solid-phase antibodies, but these are best restricted to certain well char-... 

McConway MG and Chapman RS. Application of solid-phase antibodies to radioimmunoassay evaluation of two pol3mieric microparticles, dynospheres and nylon, activated by carbonyldiimidazole or tresyl chloride. J. Immunol. Methods 1986 95 259-266. 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

BOX 9-2 Immobilization Schemes for Preparation of Solid Phase Antibodies for Use in Immunoassay... 

Enzyme-Linked Immunosorbent Assay. LlSAis a heterogeneous EIA technique that is widely used in clinical analyses. In this type of assay, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as that of a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates separation of bound- and free-labeled reactants. In the most common approach to using the ELISA technique, an aliquot of sample or calibrator containing the antigen to be quantitated is added to and allowed to bind with a solid phase antibody. After washing, enzyme-labeled antibody is... 

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,... 

Labeled-Analogue Methods, In a typical one-step RIA for FT4, endogenous free hormone and a I-labeledT4 analogue compete for solid-phase antibody binding sites in the presence of serum protein the amount of labeled analogue bound by the specific antibody is inversely related to the amount of FT, in the specimen. Test results are expressed relative to secondary serum calibrators that have been independently calibrated using equilibrium dialysis or ultrafiltration techniques. A number of one-step RIAs have been developed and are commercially available. 

Competitive binding chemiluminescence assay. The chemiluminescent competitve binding assay is similar to a RIA. The acridinium ester-labeled substance and the endogenous substance compete with the specific antibody that is available in limited concentration. The concentration is determined by the quantity of tracer that binds to the antibody. The use of solid-phase antibody simplifies separation of free from bound tracer. 

The quantity of antibody which can be attached to the wall of a well of a microtitre plate is limited by the surface of the well and the fraction of antibodies present in the immunoglobulin preparation. Assuming a maximum adsorption of 1.5 ng/mm and an average molecular weight of 150000 (IgG), the maximum attainable concentration of IgG attached to the wall is about 10 M using monoclonal antibodies, but for affmity-purified antibodies, hyperimmune antisera and postinfection sera, typically 3, 10, and 100 times less would be present, respectively. The fraction of antigen bound by the solid-phase antibody can be calculated with the law of mass action (eq. 3) ... 

Completion of the Incubation time is detected by the LAS error-detector assembly, which monitors the analytical stream at the outlet of the Incubator. The microprocessor activates the magnets and, as stated above, the solid-phase antibody Is trapped while the free antigen washes through. The pinch valve is... 

Antibodies against antigen ai e added, these are from a different species to solid phase antibodies. 

Anti-species conjugate is added which binds to species of serum from which the second antibody was prepared. This cannot react with solid phase antibodies. After incubation the unbound conjugate is washed away. 

Essentially, the same parameters have to be standardized as for capture ELISA for antigen detection. However, the test is used to measure antibodies against a fixed amount of antigen captured on the plate. Thus, we must optimize the system to have the correct amount of capture antibody and antigen necessary to bind any test or control antisera. The test offers the ability to specifically capture antigen using a solid-phase antibody. Thus, relatively crude... 

A hapten-protein conjugate is immobilized on the solid phase. Antibody conjugated to enzyme is mixed with analyte. The antibt y-enzyme combination partitions between the bound hapten-protein and the analyte in solution. 

Compound Solid phase antigen Solid phase antibody ... 

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