Chromogenic assay specificity

S Roychoudhury, RE Kaiser, DN Brems, WK Yeh. Specific interaction between (3-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus development of a chromogenic assay. Antimicrob Agent Chemother 40 2075-2079, 1996. 

Recently, an assay using chromophoric substrate (Chromozym TH) was developed [7], When Chromozym TH is added, the absorbance at 405nm will increase, and the linear section from 15 to 30 sec is used to calculate the activity (international unit, one unit is defined as the specific activity required to convert 1 pM substrate/min/mg of enzyme), Fig. (7). The increasing absorbance is resulted from the p-nitroanilide released off the substrate during hydrolyzation. As compared with the fibrin plate method, this chromogenic assay is more rapid, convenient and reproducible. The specificity of the assay, however, is lower than that of fibrin-plate method because the chromogenic substrate could be hydrolyzed by some other proteases, for instance thrombin. 

Drugs can also Interfere with laboratory results by negating certain nonspecific oxidation and reduction reactions essential for the chemical assay. Penicillin, streptomycin and ascorbic acid are known to react with cupric Ion thus, false positive results for glucose may occur If a copper reduction method Is used. If the specific enzymatic glucose-oxidase method Is employed, ascorbic acid can cause a false negative result by preventing the oxidation of a specific chromogen In the reaction. 

Recently, two fiuorogenic assays were also described that can be used for detecting BVMO activityHowever, these methods are biased toward specific chromogenic and fiuorogenic substrates and will only yield enzymes that are active toward these and similar compounds. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

The appearance of anionic phospholipids, particularly phosphatidylserine, on the cell siuface activates prothrombinase complex culminating in the formation of thrombin (Bevers et al., 1982 Connor et al., 1989). The assay can be performed with pure coagulation proteins and specific chromogenic substtates to produce a very sensitive test to detect the appearance of phosphatidylserine on ceU siufaces. Nevertheless, it has been shown that changes in the disposition of phosphatidylethanolamine and sphingomyelin may interfere with the ability of phosphatidylserine-containing membranes to activate prothrombinase (Smeets et al., 1996). 

The assay involves a chromogenic tag such as pNA linked to a specific peptide corresponding to the cleavage site of the respective caspase. Proteolysis of this peptide releases the chromogenic tag, thereby increasing the chromophore intensity. 

The substance to be assayed—e.g., the hormone thyroxine in a serum sample—is pipetted into a microtiter plate (1), the walls of which are coated with antibodies that specifically bind the hormone. At the same time, a small amount of thyroxine is added to the incubation to which an enzyme known as the "tracer" (1) has been chemically coupled. The tracer and the hormone being assayed compete for the small number of antibody binding sites available. After binding has taken place (2), all of the unbound molecules are rinsed out. The addition of a substrate solution for the enzyme (a chromogenic solution) then triggers an indicator reaction (3), the products of which can be assessed using photometry (4). 

The most specific and frequently used assay to quantify 2-deoxy-2-amino sugars employs the condensation of the carbohydrate with 2,4-pentanedione in basic solution. The product (a pyrrole derivative) is then reacted with p-dimethylaminobenzaldehyde to form a chromogen that has a maximum absorbance at 530 nm (Fig. 11-2). Although both 6-deoxy-6-amino and 3-deoxy-3-amino sugars can also be analyzed using the Elson-Morgan reaction, the chromogens... 

Enzyme-linked immunosorbent assays (ELISA) are by far the most common immunological assay used in biochemical and clinico-chemical laboratories (Crowther 1995). The method is just as specific as RIA and it can achieve comparable sensitivities under optimal conditions. It can be carried out in various ways, but the common feature is that detection relies on turnover of a chromogenic substrate by an... 

It should be noted that the relationship between the final signal output and concentration of the analyte (dose-response) may be one of direct or inverse proportionality, and is dependent on the specific assay format. In addition, a number of different reporter enzymes may be used (e.g., horseradish peroxidase, alkaline phosphatase, p-galactosidase), along with a number of different signaling systems (e.g., substrates that yield chromogenic or fluorescent or chemiluminescent products, activation of signaling enzymes, amplification by biotin-avidin system or polymerase chain reaction). 

The initial assays for detection of CB used chromogenic substrates containing an Arg-Arg sequence and 2-naphthyl-amide and 7-amino-4-methylcoumarin as chromophores. These early assays lacked specificity and were likely to have suffered from interference by endogenous uihibitors (e.g., cystatins and stefins). CB and CL are now measured by ELISA however to date no comparison has been made with the older CB methods. Immunohistochemistry has also been used to detect CB in tissue however no detailed evaluations have been conducted. 

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