Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies Homogeneous enzyme immunoassay for thryroxine

Ullman, Edwin F.; Yoshida, Robert A.; Blakemore, Judith I.; Maggio, E; Leute, Richard K.

doi:10.1016/0005-2744(79)90173-6

Cited by 30 publications

(7 citation statements)

References 18 publications

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“…This could be achieved by a conformational change in the ␤-gal loop that partially recovered a distortion induced by the antigen upon the active site, or also because antibody-binding would promote direct conformational alteration of amino acids involved in the catalysis. Conformational changes have been proposed as the mechanism of reactivation of malate dehydrogenase-thyroxine conjugates upon binding with anti-thyroxine antibodies (31). Protection performed by anti-␤-gal antibodies against heat denaturation of the enzyme has been previously described (32).…”

Section: Discussionmentioning

confidence: 99%

“…Modification of enzyme activity by anti-haptens antibodies can be blocked by prior incubation of the antibodies with free hapten. This provides the basis for the enzyme-multiplied immunoassay technique (31,36) and cloned enzyme donor immunoassay (37). In our system, the presence of antibodies against the main antigenic site of FMDV can be simply detected by mixing the sample with M278VP1 enzyme and measuring the ␤-gal activity after a short incubation time.…”

Section: Discussionmentioning

confidence: 99%

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β-Galactosidase Enzymatic Activity as a Molecular Probe to Detect Specific Antibodies

Benito

Feliu

Villaverde

1996

Journal of Biological Chemistry

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The main antigenic region of foot-and-mouth disease virus serotype C1, also called site A, has been inserted in zones of the beta-galactosidase important for the stabilization of the active site, causing important changes in the Km and the specific activity of the resulting enzymes. The peptide is displayed at the surface of the recombinant proteins and, in all the cases, presents a good antigenicity. Among the recombinant proteins constructed, in proteins M278VP1 and M275SVP1 the peptide is inserted in a large loop of the beta-galactosidase (amino acids 272-288) involved in the formation of the activating interface. In these constructs, the binding of the specific antibodies directed to the foreign peptide causes an increase of the beta-galactosidase activity up to about 200%. This phenomenon has been proved using monoclonal antibodies and also using polyclonal sera generated against the peptide. Different hypothesis of the mechanism of modulation upon antibody binding are discussed. This insertion site seems to be sensitive enough to enzymatic modulation mediated by antibody binding. We propose further exploring this insertion site as a tool for a rapid detection of specific antibodies in a quick and simple homogeneous assay based on the colorimetric determination of beta-galactosidase activity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

β-Galactosidase Enzymatic Activity as a Molecular Probe to Detect Specific Antibodies

Benito

Feliu

Villaverde

1996

Journal of Biological Chemistry

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“…Non isotopic methods adopting enzymes, [1][2][3][4][5][6][7][8][9][10][11] fluorescent, [12][13][14][15][16][17][18] and chemiluminiscent [19][20][21] labels, using competitive format for measuring T 4 and T 3 , were reported. Due to the presence of three available functional groups in T 4 and T 3 , the necessity to select the group that needed to be attached to carrier protein for raising antibodies, as well as the functional group that had to be labeled with the enzyme, needed specific consideration.…”

Section: Introductionmentioning

confidence: 99%

Influence of Iodothyronine Conjugates of Bovine Serum Albumin and Horseradish Peroxidase on Enzyme Immunosorbent Assay of Thyroid Hormones

Kumari¹,

Kumar²,

Gupta³

et al. 2013

Journal of Immunoassay and Immunochemistry

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Enzyme-linked immunosorbent assays (ELISA's) reported for thyroxine (T₄) and 3,5,3'-triiodothyronine (T₃), involved coupling of the haptens through (i) carboxylic group to carrier protein for producing antibodies and (ii) amino group to detection labels. To improve the titer and specificity of antibodies, immunogens were prepared by coupling of carboxyl group to bovine serum albumin (BSA) either directly or through adipic acid dihydrazide (ADH), after protecting amino group through acetylation of T₄ and T₃. Direct coupling resulted in the incorporation of 40-50 moles of T₄ and T₃ per BSA molecule and helped in improving immunogenic response and specificity, especially of T₄. High epitope density of immunogens evoked better antibody response, since attachement of ADH as spacer, introduced 18-27 moles of haptens into carrier protein and had less effect on antibody development, with T₃ being exception. Detection labels were prepared by coupling horseradish peroxidase (HRP) to amino group of thyroid hormones directly and after preparing their methyl esters, which provided sensitive displacement curves in combination with the antibodies developed against N-acetylated-T₄ and T₃. Unlike methyl esters, T₄-HRP and T₃-HRP showed higher sensitivity and seemed to be related to the affinity of the labels for binding the antibody.

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“…Further, a system based on the β‐gal‐estradiol conjugate, but with estrogen receptor as binding protein (rather than antibody), could be useful in screening for the whole class of anthropomorphic compounds that mimic estradiol, an endocrine hormone, in binding receptor and that therefore pose potential health hazards. Such endocrine hormone mimics constitute an important subclass of toxins that are referred to as endocrine‐disrupting chemicals (EDCs) (23 − 25).…”

Section: Introductionmentioning

confidence: 99%

Photometric and Electrochemical Enzyme-Multiplied Assay Techniques Using β-Galactosidase as Reporter Enzyme

Monbouquette

2006

Biotechnol. Prog.

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Beta-galactosidase (beta-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known beta-gal substrate analog, o-nitrophenyl beta-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl beta-D-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These beta-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for beta-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, beta-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the beta-gal-estradiol conjugate up to approximately 75%. Reporter enzyme inhibition is relieved significantly by addition of < or =2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (beta-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p-aminophenol, is responsive to added estradiol within minutes. These results show that beta-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.

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Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies Homogeneous enzyme immunoassay for thryroxine

Cited by 30 publications

References 18 publications

β-Galactosidase Enzymatic Activity as a Molecular Probe to Detect Specific Antibodies

β-Galactosidase Enzymatic Activity as a Molecular Probe to Detect Specific Antibodies

Influence of Iodothyronine Conjugates of Bovine Serum Albumin and Horseradish Peroxidase on Enzyme Immunosorbent Assay of Thyroid Hormones

Photometric and Electrochemical Enzyme-Multiplied Assay Techniques Using β-Galactosidase as Reporter Enzyme

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