Immunoassay capture

IgG (rabbit) Competitive immunoassay capture of analyte by sol-gel carbon ink incorporating IgG Amperometric detection of ALP-generated a-naphthol +400 mV 50-5000 ngml-1 5 ngmU1 (32 pM) Wang et al. [78]... 

Figure 4.12 describes two popular antibody microarrays formats that are constructed for antigen capture in small sample volumes with detection by either sandwich immunoassay or antigen labeling. In sandwich immunoassay, capture antibodies are arrayed and immobilized to select specific proteins which are then found by a second labeled detection antibody. In protein target labeling, all proteins in the sample are prelabeled (i.e., fluorescent dyes) prior to capture by immobilized antibody arrays. In direct assay systems, sample proteins are directly immobilized onto... 

GC = gas chromatography ECD = electron capture detector EIA = enzyme-immunoassay GPC = gel permeation chromatography HPLC = high-performance liquid chromatography ITMS = ion trap mass spectrometer LSE = liquid solid extraction MS = mass spectrometry RSD = relative standard deviation SPE = solid phase extraction... 

Sandwich ELISAs (Eigure 4) are the most common type of immunoassay used for the detection of proteins. A capture antibody is immobilized on the wells of a microplate. The solution containing the analyte is introduced and antibody-analyte... 

Figure 4 Sandwich immunoassay. A capture antibody (Y) is passively adsorbed on a solid phase. The target protein contained in the sample and the enzyme-labeled reporter antibody (Y-E) are added. Both the capture antibody and enzyme-labeled reporter antibody bind to the target protein at different sites, sandwiching it between the antibodies. Following a wash step, the substrate (<>) is added and colored product ( ) formed. The amount of colored product is directly proportional to the amount of target protein captured...

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

The application of conducting polymers such as polyaniline, polypyrrole, and polythiophene for immobilizing capture antibodies in immunoassay systems is widespread. 

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],... 

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