Effects of deglycosylation of human thyroperoxidase on its enzymatic activity and immunoreactivity

1992 ◽  
Vol 132 (2) ◽  
pp. 317-323 ◽  
Author(s):  
A. Giraud ◽  
J.-L. Franc ◽  
Y. Long ◽  
J. Ruf
Keyword(s):  
Monoclonal Antibodies ◽  
Enzymatic Activity ◽  
Tertiary Structure ◽  
Polyclonal Antibodies ◽  
Thyroid Peroxidase ◽  
Immunosorbant Assay ◽  
Autoimmune Thyroid ◽  
Pngase F ◽  
Enzyme Linked Immunosorbant Assay ◽  
Microsomal Antigen

ABSTRACT Thyroid peroxidase (TPO) is a glycoprotein enzyme which catalyses the iodination of thyroglobulin and the coupling of iodinated tyrosines. Human TPO (hTPO) is the microsomal antigen recognized by the autoantibodies in the serum of patients with autoimmune thyroid disease. An active detergent-solubilized immunoaffinitypurified hTPO was deglycosylated, either by peptide N-glycosidase F (PNGase F) or by endo-β-N-acetylglucosaminidase H (endo H), and the enzymatic activity and immunoreactivity of the native and degylcosylated forms were compared. Electrophoretic controls and affinoblotting with concanavalin A showed that deglycosylation was not total and that it was more pronounced with endo H than with PNGase F. The enzymatic activity of hTPO was inhibited by endo H deglycosylation, but not by PNGase F deglycosylation; this inhibition was not due to aggregation and/or insolubilization of the molecule subsequent to deglycosylation. Immunoreactivity was monitored by enzyme-linked immunosorbant assay (ELISA) with 13 mouse monoclonal antibodies, rabbit polyclonal antibodies and antibodies from serum of patients with Hashimoto's thyroiditis. In contrast with enzymatic activity, immunoreactivity was not modified or was slightly enhanced (with four monoclonal antibodies) by deglycosylation. The results indicate that strong, if not total, deglycosylation induces a modification of the tertiary structure of hTPO, which affects the enzymatic site but does not modify markedly the epitopes implicated in the recognition of the molecule by the antibodies tested. Journal of Endocrinology (1992) 132, 317-323

Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies

1983 ◽  
Vol 3 (4) ◽  
pp. 381-388 ◽  
Author(s):  
P. Hérion ◽  
D. Portetelle ◽  
J. -D. Franssen ◽  
J. Urbain ◽  
A. Bollen
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Biological Fluids ◽  
Immunosorbant Assay ◽  
Rapid Procedure ◽  
Preliminary Purification ◽  
Enzyme Linked Immunosorbant Assay

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng/ml) and evaluation of the enzyme in biological fluids such as urine, pleural elfusions, and ascitic fluids without preliminary purification.

scholarly journals Immunological Reactivity Using Monoclonal and Polyclonal Antibodies of Autoimmune Thyroid Target Sites with Dietary Proteins

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Datis Kharrazian ◽  
Martha Herbert ◽  
Aristo Vojdani
Keyword(s):  
Thyroid Hormones ◽  
Dietary Protein ◽  
Polyclonal Antibodies ◽  
Thyroid Peroxidase ◽  
Immune Reactivity ◽  
Dietary Proteins ◽  
Thyroxine Binding Globulin ◽  
Autoimmune Thyroid ◽  
Target Sites ◽  
Thyroid Axis

Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods. Additionally, food interactions may play a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage. Our study was designed to investigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sites. We identified immune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones, thyroid receptors, enzymes, and transport proteins. We also measured immune reactivity of either target specific monoclonal or polyclonal antibodies for thyroid-stimulating hormone (TSH) receptor, 5′deiodinase, thyroid peroxidase, thyroglobulin, thyroxine-binding globulin, thyroxine, and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms. Dietary protein determinants included unmodified (raw) and modified (cooked and roasted) foods, herbs, spices, food gums, brewed beverages, and additives. There were no dietary protein immune reactions with TSH receptor, thyroid peroxidase, and thyroxine-binding globulin. However, specific antigen-antibody immune reactivity was identified with several purified food proteins with triiodothyronine, thyroxine, thyroglobulin, and 5′deiodinase. Laboratory analysis of immunological cross-reactivity between thyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potential immunoreactive role in autoimmune thyroid disease.

scholarly journals Microimmunoassay permits determination of concentrations in immunohistochemistry controls.

1986 ◽  
Vol 34 (4) ◽  
pp. 543-545
Author(s):  
D J Goldstein ◽  
M M Davis ◽  
R K Naviaux ◽  
T L Dailey ◽  
T M Ulbright
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbant Assay ◽  
Enzyme Linked Immunosorbant Assay

A competition enzyme-linked immunosorbant assay (CELIA) can be used to determine the amount of antigen needed to immunoabsorb an antiserum. This is demonstrated using both polyclonal and monoclonal antibodies against human amylase. This method is of especial value when the tissue is particularly difficult to obtain, and especially valuable when CELIA has already been completed.

False negative results observed in anti-thyroid peroxidase autoantibody determination by competitive radioimmunoassays using monoclonal antibodies

1994 ◽  
Vol 130 (6) ◽  
pp. 552-558 ◽  
Author(s):  
Stefano Mariotti ◽  
Giuseppe Barbesino ◽  
Patrizio Caturegli ◽  
Francesca Atzeni ◽  
Luca Manetti ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Thyroid Disease ◽  
Autoimmune Thyroid Disease ◽  
False Negative ◽  
Thyroid Peroxidase ◽  
Passive Hemagglutination ◽  
Autoimmune Thyroid ◽  
Negative Results ◽  
False Negative Results

Mariotti S, Barbesino G, Caturegli P, Atzeni F, Manetti L, Marinò M, Grasso L, Velluzzi F, Loviselli A, Pinchera A, Martino E. False negative results observed in anti-thyroid peroxidase autoantibody determination by competitive radioimmunoassays using monoclonal antibodies. Eur J Endocrinol 1994;130:552–8. ISSN 0804–4643 Objective: Anti-thyroid peroxidase autoantibody (anti-TPO) and anti-thyroid microsomal antibody (anti-M) are strictly related, but discrepancies are sometimes observed. The aim of this study was to assess the incidence and to identify the causes of these discrepancies. Design and antibody measurements: Anti-M by passive hemagglutination and anti-TPO by two competitive monoclonal antibody-assisted radioimmunoassays (RIA-1 and RIA-2) were measured in 10 103 sera from 4232 subjects (663 male, 3569 female) screened for thyroid disease. Results: Anti-TPO and anti-M correlated quite well (r = 0.7 and p < 0.0001 by RIA-1; r = 0.74 and p < 0.0001 by RIA-2), with discrepancies mostly limited to sera with low antibody titers. After exclusion of the latter samples, anti-TPO were detected in only 79 (1.4%) out of 5317 anti-M negative sera, but were undetectable in a more consistent proportion (130/2880 = 4.5%) of sera from patients with autoimmune thyroid disease and positive anti-M. In 61 sera of the latter group, anti-TPO was measured by a non-competitive RIA (RIA-3). Forty-one (67.7%) were positive by RIA-3, suggesting the presence of anti-TPO not competing with the monoclonal antibodies of RIA-1 and RIA-2. The remaining 20 sera had undetectable anti-TPO also by RIA-3. Nineteen (95%) of these sera had positive anti-thyroglobulin (anti-Tg) autoantibody and preincubation with thyroglobulin inhibited the agglutination reaction of anti-M tests. Conclusion: Anti-TPO by competitive monoclonal antibody-assisted RIA is negative in a minority of sera of patients with autoimmune thyroid disease and positive anti-M. This could be accounted for by anti-Tg producing false positives in the anti-M assay and by a subset of anti-TPO not competing with the monoclonal antibodies in the RIA. When autoimmune thyroid disease is suspected on clinical grounds, a negative anti-TPO test with a competitive RIA should be confirmed always by a non-competitive assay. Stefano Mariotti. Institute of Endocrinology. University of Pisa, Viale del Tirreno 64,1-56018 Tirrenia-Pisa, Italy

An enzyme-linked immunosorbant assay using polyclonal antibodies against bacopaside I

2007 ◽  
Vol 584 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Watoo Phrompittayarat ◽  
Waraporn Putalun ◽  
Hiroyuki Tanaka ◽  
Sakchai Wittaya-Areekul ◽  
Kanchalee Jetiyanon ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Immunosorbant Assay ◽  
Enzyme Linked Immunosorbant Assay

Microparticle-enhanced nephelometric immunoassay of anti-thyroid peroxidase autoantibodies in thyroid disorders

1994 ◽  
Vol 40 (3) ◽  
pp. 442-447 ◽  
Author(s):  
A A Harchali ◽  
P Montagne ◽  
J Ruf ◽  
M L Cuillière ◽  
M C Bene ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hashimoto Thyroiditis ◽  
Human Thyroid ◽  
Thyroid Peroxidase ◽  
Graves Disease ◽  
Thyroid Disorders ◽  
Autoimmune Thyroid Diseases ◽  
Autoimmune Thyroid ◽  
Human Thyroid Peroxidase ◽  
Antigenic Domains

Abstract Crude thyroid peroxidase extracted from human thyroid microsomes was covalently bound onto polyacrylic and polyfunctional copolymerized microparticles. We observed agglutination of the thyroid peroxidase-microparticle conjugate with 13 monoclonal antibodies (mAbs) specific for epitopes on four different antigenic domains of human thyroid peroxidase (TPO; EC 1.11.1.7), after addition of anti-mouse immunoglobulins. We quantified agglutination by measuring with a specially designed nephelometer the light scattered by the conjugates. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for human anti-TPO autoantibodies (aAbs) with defined epitopic specificity, based on the ability of aAbs to inhibit mAb-induced agglutination. Applied to patients with autoimmune thyroid diseases, this assay confirmed the polyclonality of anti-TPO aAbs and their preferential reactivity toward epitopes located on the A and B antigenic domains of the TPO molecule. The same specificities seem to be present in patients with Hashimoto thyroiditis or Graves disease.

scholarly journals Characterization of the thyroid microsomal antigen, and its relationship to thyroid peroxidase, using monoclonal antibodies.

1988 ◽  
Vol 81 (4) ◽  
pp. 1217-1224 ◽  
Author(s):  
L Portmann ◽  
F W Fitch ◽  
W Havran ◽  
N Hamada ◽  
W A Franklin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Thyroid Peroxidase ◽  
Thyroid Microsomal Antigen ◽  
Microsomal Antigen
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