Heterogeneous Immunoassays

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]. 

Fluorescent immunoassay was also conducted. For instance, physisorption of goat anti-mouse IgG (biotin-conjugated) was first made to the PDMS surface for the immunoassay of IgG. Then neutravidin was introduced to bind to the biotin on the surface. Immobilization of biotinylated goat anti-human IgG on the neu-travidin-coated channel was first achieved by confining the reagent flow at a T-intersection on the PDMS chip. The antigen (Cy5-human IgG) was applied via a perpendicular channel. Fluorescence measurement allowed the 

In another report, an assay of a cardiac marker (human C-reactive protein, CRP) was achieved on a Si-PDMS chip based on a solid-phase sandwich immunoassay [459]. 

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The main advantage of a homogeneous immunoassay, compared to a heterogeneous immunoassay, is the absence of a separation step. This translates into a simpler procedure and easier automation. However, homogeneous assays are typically less sensitive and more susceptible to sample interferences which are removed in a separation step. 

M.J. Doyle, H.B. Halsall, and W.R. Heineman, Heterogeneous immunoassay for serum proteins by differential pulse anodic stripping voltammetry. Anal. Chem. 54, 2318-2322 (1982). 

Heterogenous immunoassays, 14 151-152 Heteroglycans, 4 697, 702 23 64 Hetero-interface, 24 71 Heterojunction, 23 34 Heterojunction bipolar transistors (HBTs), 22 166-169... 

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

In the past ten years, numerous applications of fluorescence methods for monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted the development of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation for lifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician s office testing, future innovations in fluorescence immunoassay development will be expected to center on the simplification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing. 

Many immunoassays are performed at 37 C or above (7). An advantage of the novel immunoassay methodology disclosed in the previous paper is that it avoids a significant problem encountered with many heterogeneous immunoassays, that of slow kinetics when binding of a molecule in solution to a solid surface is involved. 

Enzyme-linked immunosorbent assay is a heterogenous immunoassay. Reactions involve a solid phase to which components are sequentially presented and successively bound. This method is very effective in the determination of the total alkaloid content. The positive characteristics of this method are the use of non-toxic reagents and basic equipment with low costs, a small sample volume and the ability to measure alkaloids in crude sample extracts. According to the literature, compared with results obtained from GLC, the precision of ELISA for quinolizidine alkaloids is not as high as that of the gas chromatography procedure, but is adequate for plant breeding purposes. The use of enzymes in developing the methods of quinolizidine alkaloids analysis looks likely to increase in the future. 

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.

Immunoassays can be classified according to different criteria. The particular type selected has a strong influence on the assay performance with regard to precision and sensitivity. The main criteria include [3, 22, 23] (1) labeled or unlabeled assay formats, with different type of labels (2) competitive or non-competitive immunoassays, and (3) homogeneous or heterogeneous immunoassays. These classifications can also be extended to MIP-ILAs (Fig. 2) ... 

In heterogeneous assays the bound and free label forms must be separated by different means, such as precipitation of antibody or coupling of the antibody to a solid support. Heterogeneous immunoassays are more versatile as inclusion of a separation step eliminates most of the interferences before quantification. However, these assays are also more labor-intensive and time-consuming. Any of these methods can be performed in either competitive or non-competitive format. 

Heineman s group [13,14] proposed the method of heterogeneous immunoassay to detect human serum albumin (HSA) labeled with In... 

An electrochemical immunoassay has been developed by Chu et al. [75], based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by ASV at a glassy carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an IgG... 

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 another report, pFNs were also formed from PDMS chips to create patterns of immobilized chicken IgG. Heterogeneous immunoassay with antispecies IgG (fluorescently labeled) was then conducted [247]. 

After patterning antibodies as an array using the PDMS microchannel, TNT (2,4,6-trinitrotoluene) was detected using the heterogeneous immunoassay format... 

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)... 

Dodge, A., Fluri, K., Verpoorte, E., de Rooij, N.F., Electrokinetically driven microfluidic chips with surface-modified chambers for heterogeneous immunoassays. Anal. Chem. 2001, 73(14), 3400-3409. 

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