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

Abstract. Evidence has been accumulated that human thyroid microsomal/microvillar autoantigen (M) is expressed both in the cytoplasm and on the surface of thyroid follicular cells. The availability of this autoantigen to the immune system, possibly associated with abnormally expressed HLA-DR antigens may be relevant both to the triggering and to maintenance of thyroid autoimmune reactions. Preliminary biochemical characterization of M suggested that it was a glycoprotein with a mol. wt. of about 100–110 kD. recent studies carried out in our laboratories taking advantage of monoclonal antibodies provided evidence that the structure presently referred as M-Ag is represented by thyroid peroxidase (TPO). The identity between TPO and M is further supported by four-layer immunofluorescence analysis showing a complete overlap of the two antigens both in the surface and in the cytoplasm of thyroid cells and by the observation that the expression of M and TPO is similarly modulated by TSH, possibly through a cAMP-dependent mechanism.

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

Journal of Endocrinology ◽

10.1677/joe.0.1320317 ◽

1992 ◽

Vol 132 (2) ◽

pp. 317-323 ◽

Cited By ~ 23

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

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Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

Journal of Clinical Investigation ◽

10.1172/jci113181 ◽

1987 ◽

Vol 80 (4) ◽

pp. 1205-1208 ◽

Cited By ~ 48

Author(s):

P Seto ◽

H Hirayu ◽

R P Magnusson ◽

J Gestautas ◽

L Portmann ◽

...

Keyword(s):

Thyroid Peroxidase ◽

Complementary Dna ◽

Thyroid Microsomal Antigen ◽

Microsomal Antigen

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The Effect of Dithiotreitol on thyroid Peroxidase and Microsomal Antigen Epitopes Recognized by Auto and Monoclonal Antibodies

Autoimmunity ◽

10.3109/08916939008993387 ◽

1990 ◽

Vol 7 (2-3) ◽

pp. 149-156 ◽

Cited By ~ 14

Author(s):

Andrzej Gardas ◽

Hanna Domek ◽

Arbara Czarnocka

Keyword(s):

Monoclonal Antibodies ◽

Thyroid Peroxidase ◽

Microsomal Antigen

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Characterization of the human thyroid microsomal antigen involved in thyroid autoimmunity

Biochemical Society Transactions ◽

10.1042/bst0121118 ◽

1984 ◽

Vol 12 (6) ◽

pp. 1118-1119 ◽

Cited By ~ 5

Author(s):

J. PAUL BANGA ◽

GARETH PRYCE ◽

LINDA HAMMOND ◽

IVAN M. ROITT

Keyword(s):

Thyroid Autoimmunity ◽

Human Thyroid ◽

Thyroid Microsomal Antigen ◽

Microsomal Antigen

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Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614229 ◽

1998 ◽

Vol 79 (01) ◽

pp. 104-109 ◽

Cited By ~ 6

Author(s):

Osamu Takamiya

Keyword(s):

Monoclonal Antibodies ◽

Tissue Factor ◽

Human Factor ◽

Solid Phase ◽

Three Dimensional ◽

Coagulation Factors ◽

Factor Vii ◽

Dimensional Structure ◽

Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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