Modes of antibody immobilization

Within ELIS As, both direct and indirect modes of performance are possible. The former are based on competition between the analyte and an enzyme-labeled analyte for a place on an immobilized antibody. The latter is based on competition between the analyte and an immobilized analyte for a place on an enzyme-labeled... 

This mode of separation, as the name suggests, uses stationary phases with a special affinity for a specific analyte. The affinity ligand immobilized on the stationary phase varies dramatically from peptide, to protein, to oligonucleotide, to monoclonal antibody. In some cases the target molecule is labelled with an affinity tag to simplify the separation. This approach is common in the synthesis of recombinant proteins where the system can be engineered so that the target biomolecule expresses a tag such as polyhistidine. A stationary phase functionalized with aminodiacetic acid and nickel chelate is then used to fish out the required molecule by chelating with the polyhistidine tag. 

For immobilization studies to date, two distinct modes of immobilization have been used. The first utilizes nonspecific covalent bonding to CNBr-activated Sepharose via primary amino groups on the antibody molecule. Since there are many of these available on the antibody, this is expected to result in random orientation of antibody molecules on the support. The other method involves linkage through immobilized protein A, a protein which binds immunoglobulins in the structural F portion of the molecule. 

Figure 2. Resonant frequency shift of the second flexural mode ofPEMC sensor upon binding of Bacillus anthracis spores at various sample concentrations to antibody functionalized cantilever. The results showed that the binding rate strongly depends on concentration. The control was an antibody-immobilized cantilever immersed in PBS. Adapted from reference(24).

Chromatographic (or flow-injection) immimoassays represent another important mode of lAC. This technique utilizes immobilized antibodies, or antigens, in a column to perform various competitive or noncompetitive immunoassays. Detection in these methods involves the use of a labeled antibody or labeled analyte analog (known as the label) to indirectly measure the amount of a target in a sample, as demonstrated in Figure 1.2. The detection format may be based on absorbance. 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

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... 

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