Antibodies solid-phase immobilized

Fig. 9. Schematic representation of the noncompetitive immunoassay of triiodothyronine using a solid-phase immobilized hapten for masking an unoccupied antibody.

Enzyme-Labeled Antigen. In this method, the sample is first incubated with a moderate excess of solid-phase immobilized antibody (Ab ). After washing, excess enzyme-labeled antigen (Ag ) is allowed to bind to unreacted Ab . 

Assays for insulin antibodies fall into three categories (1) quantitative radioimmunoelectrophoresis, which measures the binding of IgG antibody to radiolabeled insulin by rocket Immunoelectrophoresis into anti-IgG-containing agarose (2) RIAs with separation of bound and free insulin by precipitation with PEG or a second antibody and (3) solid phase immobilization of insulin to test tubes or Sepharose. These are discussed in more detail in Reeves. ... 

In the schemes in this chapter, x and y denote different animal species, H hapten, Ab antibody in general and AB antibody to Ig (anti-Ig), Ag antigen, E enzyme, ] = is the symbol for solid-phase immobilization, covalent links are indicated by a stop (Ab E, H E), immunological or other non-covalent links (biotin-avidin, Ab-H, Ab-Ag, Ab-AB, SpA-Ab) are indicated by long hyphens and links in immune complexes, prepared prior to addition, by a colon (Ab E). 

Non-competitive assays with antibodies immobilized on the solid phase Immobilization of antibodies or their F(ab )2 on... 

Using alkaline phosphatase-labeled antibodies to protein antigens and the meta-phenyl phosphate dioxetane shown in Fig. 41 (referred to as AMPPD or Lumigen PPD), one can conduct sandwich-type immunoassays with a solid phase immobilized capture antibody and an enzyme-labeled signal antibody (B19). After the... 

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. 

H. Zhang and M.E. Meyerhoff, Gold-coated magnetic particles for solid-phase immunoassays enhancing immobilized antibody binding efficiency and analytical performance. Anal. Chem. 78, 609-616 (2006). 

In order to develop a process that is more robust and free from some of the limitations of the double-antibody concept, various methods have been explored to immobilize the primary antibody onto the solid phase. Covalent immobilization of the immunoreactive species directly on a solid support would have the distinct advantage of overcoming the capacity limitations and the possible molecular changes that can take place before or after immobilization. However, this mode has not been widely utilized due to lack of successful and reproducible methods of covalent attachment. A further complication is the uncontrollable heterogeneity of the solid supports used currently for immobilization. Realizing these limitations, the approach used for Stratus has been to choose a design... 

In nearly all cases studied, the amount of primary antibody required in the E5-Ab complex to perform an assay has been found to be substantially less than that required for the double antibody immune complex format. This was found to be the case (Table 19.1) when the E5-Ab complex was either directly immobilized on the solid phase, to imitate the double-antibody immune complex format, or utilized in a solution phase format [12],... 

Fig. 31 (A) Principle of a sandwich immunoassay using FDA particulate labels. The analyte is first immobilized by the capture antibody preadsorbed on the solid phase (a) and then exposed to antibody-coated microparticle labels (b). Every microparticle contains 108 FDA molecules. High signal amplification is achieved after solubilisation, release, and conversion of the precursor FDA into fluorescein molecules by the addition of DMSO and NaOH (c). (B) Calibration curves of IgG-FDA microcrystal labels with increasing surface coverage of detector antibody (a-d) compared with direct FITC-labeled detector antibody (e). The fluorescence signals increase with increasing IgG concentration. FDA microcrystals with a high IgG surface coverage (c,d) perform better than those with lower surface coverage (a,b). (Reprinted with permission from [189]. Copyright 2002 American Chemical Society)...

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