Antigens heterogenous immunoassay

Fig. 25. (A) DELFIA (Dissociation Enhanced Lanthanide Fluoro-ImmunoAssay) system. This heterogeneous immunoassay system uses a primary antibody bound to a solid support, to which a variable amount of unlabeled antigen is bound. The secondary antibody is labeled with a non-phospho-rescent lanthanide chelate, which becomes phosphorescent after dissociation from the antibody, due to the addition of an enhancement solution [which typically contains a mixture of sensitizer (typically a (1-diketonate) and micelle inducing surfactant (5). (B) Heterogeneous fluoroimmunoassay using a secondary antibody directly labeled with a phosphorescent lanthanide chelate.

One form of heterogeneous immunoassay is called enzyme-linked immunosorbent immunoassay (ELISA). In one instance, electrochemical immunoassay was performed for anti-ferritin (antibody) in a PDMS/PMMA chip. First, DTSSP was self-assembled on the gold electrode deposited on the PMMA plate. Then horse spleen ferritin (antigen) was attached to the DTSSP layer. A 100-p.g/mL solution of anti-horse ferritin (rabbit serum) was added. Then a secondary anti-rabbit antibody (HRP-linked) was introduced. A substrate (4-CN) was finally added which was converted to a precipitate product. The precipitate caused a reduction... 

Heterogeneous immunoassay has also been conducted with the antibody immobilized on beads. For instance, mouse IgG (50-100 ng/mL) was detected by ELISA in a glass chip. First, mouse IgG (antigen) was captured by magnetic beads coated with sheep anti-mouse antibody (1.02 x 107 beads/mL). Then the secondary antibody, which was rat anti-mouse conjugated with alkaline phosphatase (0.7 pg/mL), was delivered. Thereafter the substrate, PAPP, was added. It was enzymatically converted to p-aminophenol (PAP), which was electrochem-ically detected by the on-chip interdigital microelectrodes [1016]. 

Heterogeneous immunoassay has also been conducted without the use of an enzyme label. For instance, electrochemical immunoassay of mouse IgG (antigen) was carried out in glass chip. The chip contained magnetic beads coated with the sheep anti-mouse antibody. After flowing in the secondary antibody (rat antimouse conjugated with PAPP), electrochemical oxidative detection of PAP was achieved (i.e., PAP was oxidized to p-quinoneimine) [1016]. 

Heterogeneous immunoassay of anti-IgG was achieved in PDMS channels where the antigen IgG was coated on beads that were partially embedded in PDMS (see also Chapter 9, section 9.3.1 for the use of embedded beads for DNA hybridization) [1024]. 

In the on-chip heterogeneous immunoassay for human carcinoembry-onic antigen (CEA) in serum, three antibodies (mouse anti-human CEA, rabbit anti-human CEA, and anti-rabbit IgG-colloidal gold) were used, (a) What is the purpose of colloidal gold (b) Why isn t the colloidal gold directly attached to rabbit anti-human CEA to save a step [1021] (3 marks)... 

The fluorescence of a tagged antigen is often quenched upon immunochemical reaction, and the decrease in fluorescence can be measured without going through a physical separation. This serves as the basis for homogeneous immunoassays, in contrast to heterogeneous immunoassays, which require a separation. 

In the heterogeneous immunoassay, it is crucial to coat the antibody/antigen on a solid surface. In the microfiuidic chip, the antibody/antigen is usually immobilized on the surface of the microchannel wall which is located in the certain zone of the microchip. The immobilization of the... 

Avidin (an egg yolk protein) exhibits an extremely strong afEnity (ca. lO L/mol) for biotin (vitamin H). This irreversible interaction can be utilized in the design of heterogeneous immunoassays with biotinylated. antigen-specific antibodies [68], [69]. Biotinylation can be performed without interfering with antibody function. The antibodies are applied in the usual manner in a competitive immunoassay (Fig. 8). In the last step, enzyme-labeled avidin is added in excess. Since there are four possibilities for the binding of avidin... 

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