Immunoassay alkaline phosphatase

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

A competitive electrochemical enzyme immunoassay has been demonstrated for digoxin Alkaline phosphatase, which catalyzes the hydrolysis of phenyl phosphate... 

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

Jeanson, A., Cloes, J.M., Bouchet, M., and Rentier, B. (1988) Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent. Anal. Biochem. 172, 392. 

Porstmann, B., Porstmann, T., Nugel, E., and Evers, U. (1985) Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays Horseradish peroxidase, alkaline phosphatase or b-galactosidase. J. Immunol. Meth. 79, 27-37. 

M.S. Wilson and R.D. Rauh, Hydroquinone diphosphate an alkaline phosphatase substrate that does not produce electrode fouling in electrochemical immunoassays. Biosens. Bioelectron. 20, 276-283 (2004). 

Schaap, A. P., Akhavan, H., and Romano, L. J., Chemiluminescent substrates for alkaline phosphatase Application to ultrasensitive enzyme-linked immunoassays and DNA probes. Clin. Chem. (Winston-Salem, N.C.) 35, 1863-1864 (1989). 

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D-... 

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

As indicator enzymes horseradish peroxidase (HRP or HRPO), alkaline phosphatase (AP), or /i-galactosidase, are favored, since they are relatively robust, have a high product-forming rate, are easy to purify, and are cheap. The most used colloids are from gold, silver, and iron, and iodine isotopes are mostly taken as radioactive labels in immunoassays. 

Methods based on chemiluminescent and bioluminescent labels are another area of nonisotopic immunoassays that continue to undergo active research. Most common approaches in this category are the competitive binding chemiluminescence immunoassays and the immunochemiluminometric assays. Chemiluminescence and heterogenous chemiluminescence immunoassays have been the subject of excellent reviews (91, 92). Detection in chemiluminescence immunoassays is based on either the direct monitoring of conjugated labels, such as luminol or acridinium ester, or the enzyme-mediated formation of luminescent products. Preparation of various derivatives of acridinium esters has been reported (93, 94), whereas a variety of enzyme labels including firefly or bacterial luciferase (70), horseradish peroxidase (86, 98), and alkaline phosphatase are commercially available. 

Instead of using human serum albumin as a carrier protein, other workers (135) utilized ovalbumin for preparing the diazotized clenbuterol antigen in an enzyme immunoassay developed for screening of clenbuterol residues in bovine urine, liver, and eye. Alkaline phosphatase rather than -galactosidase was also used as an enzyme label in the preparation of the enzyme-clenbuterol conjugate. 

Bronstein, I., Voyta, J. C., Thorpe, G. H. G., Kricka, L. J., and Armstrong, G (1989) Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin. Clin Chem 35, 1441—1446. 

C. Fernandez-Sanchez and A. Costa-Garcia, 3-Indoxyl phosphate an alkaline phosphatase substrate for enzyme immunoassays with voltam-metric detection, Electroanalysis, 10 (1998) 249-255. 

The specific electrochemical behaviour of IDAs is result of its design [97], i.e. two arrays intercalated and individually addressed in a bipotentiostatic system where reversible redox species can be cycled between one array (generator) and the other array (collector) (Fig. 32.3). The feedback obtained, greatly enhances the current and high sensitive detection can be achieved. An important application of IDAs is the electrochemical detection of p-aminophenol when it is generated from p-aminophenyl phosphate, by enzymatic reaction with alkaline phosphatase (like enzymatic label), in geno- [98-100] and immunoassays [101-103]. Another interesting feature of IDAs is the possibility of... 

Different immunoassays have been developed in CE microchips for detecting immunoglobulins, such as mouse IgG. Enzymatic (alkaline phosphatase) [39] or electroactive (ferrocene) [38] labelled anti-mouse... 

PERFORMANCES EXEMPLIFIED WITH THE IMMUNOASSAY OF ALKALINE PHOSPHATASE... 

On the other hand, the main types of immunoassays that can be performed by using labelled antibodies or antigens are direct sandwich, competitive and indirect assays. The labels can be enzymes (alkaline phosphatase, peroxidise or glucose oxidase) metal NPs (gold) fluorescent or electrochemiluminescent probes. 

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