Indirect competitive enzyme-linked

Indirect competitive enzyme-linked immunosorbent assay... 

Pinacho, D.G., F. Sanchez-Baeza, and M.P. Marco. 2009. Development of a class selective indirect competitive enzyme-linked immunosorbent assay (ELISA) for detection of fluoroquinolone antibiotics. J. Agric. Food Chem. submitted. 

Yu, W., X. Zhen-Lin, X. Yan-Yun et al. 2011. Development of polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for sodium saccharin residue in food samples. Food Chem. 126 815-820. 

Competitive enzyme-linked immunosorbent assays (ELISA) Two types of ELISA have been used for the analysis of mycotoxins and both types are heterogenous competitive assays. One type, i.e. direct ELISA, involves the use of a mycotoxin-enzyme conjugate and the other system, i.e. indirect ELISA, involves the use of a protein-mycotoxin conjugate and a secondary antibody to which an enzyme has been conjugated. Although horseradish peroxidase (HRP) is most commonly used as the enzyme for conjugation, other enzymes such as alkaline phosphatase and beta-galactosidase, also have been used (5, 9, 13). 

An indirect competitive ELISA has been also developed for the determination of streptomycin and dihydrosticptomyciri in milk (24). Prior to the analysis, the milk sample was skimmed and treated with oxalic acid. The antiserum was raised in rabbits using streptomycin linked to a bacterial protein as the antigen. To perform the test, microtiter plates were coated with streptomycin, and antiserum and milk samples were mixed to be added in the wells where they were incubated for 1 h. Depending on the amount of residues in the sample, more or less antibody remained available for binding to the streptomycin coat. A pig antirabbit antibody-enzyme conjugate was subsequently added and incubated for 90 min. Using a suitable substrate, streptomycin and dihydrostreptomycin could be detected down to 1.6 ppb, whereas quantification could be made possible up to 100 ppb when samples were used undiluted. 

Immunoassays vary by the different labels they use. The most common labels include chromophores, fluorophores, radioisotopes and enzymes. Of those labels, enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA) is the most popular technique. ELISA has as an advantage the amplification of the analytical signal and/or increase of the sensitivity of the immunoassay. There are four types of ELISA direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA. 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

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