Epitope mapping of human thyrotropin

1989 ◽  
Vol 120 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Yuichi Endo ◽  
Kiyoshi Miyai ◽  
Yasushi Iijima ◽  
Toshihiro Nakajima ◽  
Yasuyuki Eda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Epitope Mapping ◽  
Antigenic Determinants ◽  
Conformational Structure ◽  
Glycoprotein Hormones ◽  
Two Dimensional ◽  
Β Subunit ◽  
Binding Studies ◽  
Α Subunit ◽  
Affinity Constants

Abstract. Epitope mapping of hTSH was carried out using 19 monoclonal antibodies prepared with hTSH or its β-subunit as antigen. The affinity constants of the monoclonal antibodies ranged from 9.6 × 107 to 5.7 × 109 mol/l for hTSH. The binding activities of monoclonal antibodies were maintained or in some cases rather enhanced after removal of the sugar moeity of the subunits of hTSH, and completely diminished after reduction of intramolecular S-S bonds in the subunits of hTSH. Ten monoclonal antibodies recognized the epitopes on hTSH (α:β subunit combined form) and on free α-subunit form. Eight other antibodies recognized the epitopes on free/or combined form of β-subunit, all of which did not recognize any other human glycoprotein hormones. The monoclonal antibodies directed against the α-subunit could bind also other human glycoprotein hormones to a varying extent. On the basis of results from competitive binding studies, the antibodies directed against α-subunit and those against β-subunit were each classified into five subgroups recognizing different antigenic determinants. The remaining one antibody recognized an epitope expressed only by hTSH and not by the free subunits. In addition, a positive cooperativity on the binding of hTSH was observed between monoclonal antibodies directed towards a particular epitope on the α-subunit and those towards an epitope on the β-subunit. From these data, two-dimensional map of epitopes on hTSH was constructed. The epitopes on each subunit were found to form a cluster with complicated overlapping, suggesting a highly conformational structure.

PLATELETS OF A PATIENT LACKING GLYCOPROTEINS lib AND Ilia AGGREGATE TO HIGH CONCENTRATIONS OF THROMBIN OR COLLAGEN

1987 ◽  
Author(s):  
J L McGregor ◽  
L McGregor ◽  
M Hans ◽  
A Sayegh ◽  
M C Trzeeiak ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Two Dimensional ◽  
Binding Studies ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Sds Page ◽  
Bleeding Episodes ◽  
High Concentrations ◽  
Induced Aggregation

The aim of this study was to investigate the platelets of a patient having bleeding episodes that began in infancy. The patient’s platelets in citrated-PRP did not aggregate when stimulated with ADP (5 and 10 uM), collagen (2.5 ug/ml), or sodium arachidonate (1 uM). However, washed patient platelets, in the presence of 2mM calcium, aggregated and secreted when stimulated with high concentrations of thrombin (0.36, 0.72 and lU/ml) or collagen (2, 4, 10 ug/ml). Monoclonal antibodies (Mab) LYP18 (directed against the IIb-IIIa glycoprotein complex) and LYP8 (anti-thrombospondin) inhibited thrombin and collagen induced aggregation of control but not the patient platelets. Patient thrombin -stimulated platelets did not bind 125I-labelled fibrinogen (40 to 320 ug/ml). Moreover, stimulating the washed patient's platelets with ADP (10-100 uM), in the presence of fibrinogen (2mg/ml), did not result in aggregation. Binding studies using Mab 125I-LYP2 (directed against the IIb-IIIa glycoprotein complex) showed the absence of the complex on the patient's platelets. The absence of the IIb-IIIa complex on the patient's platelets was also observed using crossed immunoelectro -phoresis and Mab 125I-LYP2 or 125I-LYP18. Individual glycoproteins (lib or Ilia) were not detected on silver stained two-dimensional (non-reduced/reduced) SDS-PAGE. Moreover, Western blots of |he patients platelets used in combination with anti-PLA or anti-LEK polyclonal antibodies failed to detect the presence of these two glycoproteins. These results indicate that this patient has Glanzmann's thrombasthenia or a variant of this disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium.

scholarly journals Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.

1985 ◽  
Vol 101 (6) ◽  
pp. 2047-2054 ◽  
Author(s):  
F M Brodsky
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Heavy Chain ◽  
The Other ◽  
Light Chains ◽  
Antigenic Determinants ◽  
Binding Studies ◽  
Myeloma Cells ◽  
Antigenic Sites ◽  
Immunogenic Proteins

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.

scholarly journals Alternatively Folded Choriogonadotropin Analogs

2001 ◽  
Vol 276 (50) ◽  
pp. 46953-46960 ◽  
Author(s):  
Yongna Xing ◽  
Win Lin ◽  
Mei Jiang ◽  
Rebecca V. Myers ◽  
Donghui Cao ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Carboxyl Terminus ◽  
Glycoprotein Hormones ◽  
Β Subunit ◽  
Hormone Activity ◽  
Α Subunit ◽  
Receptor Interactions ◽  
Lutropin Receptor ◽  
Carboxyl Terminal ◽  
Loop 2

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their β-subunits that is wrapped around α-subunit loop 2 “like a seatbelt.” During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally “latches” it to the β-subunit, its carboxyl-terminal end can “scan” the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the α-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41–43, and 56 of α-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10–20% the activity of hCG. Attachment of the seatbelt to α-subunit residues 45–51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the β-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of α-subunit loop 2, and the end of the α-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.

Monoclonal antibodies against human, bovine and rat prolactin: epitope mapping of human prolactin and development of a two-site immunoradiometric assay

1987 ◽  
Vol 114 (2) ◽  
pp. 311-318 ◽  
Author(s):  
B. Staindl ◽  
P. Berger ◽  
R. Kofler ◽  
G. Wick
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Epitope Mapping ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Placental Lactogen ◽  
Antigenic Determinants ◽  
Cross Reactions ◽  
Human Prolactin ◽  
Hybridoma Cell Lines

ABSTRACT Nine mouse hybridoma cell lines producing monoclonal antibodies (MCA) against human prolactin (hPRL), 19 cell lines against bovine prolactin (bPRL) and one MCA against rat prolactin (rPRL) were established. The MCA were characterized by one- and two-site radioimmunoassays (RIA) as well as indirect immunofluorescence (IIF) and used for epitope mapping of hPRL and immunoradiometric assays (IRMA). Interspecies cross-reactivity studies by RIA revealed two groups of anti-hPRL MCA: seven which reacted only with hPRL and two additionally recognizing bPRL and ovine prolactin (oPRL). The anti-bPRL MCA, which were tested on pituitary sections by IIF could be divided into 17 MCA cross-reacting with the closely related oPRL, and two MCA which showed additional cross-reactions with equine prolactin. The anti-rPRL antibody reacted exclusively with rPRL in direct binding RIA studies. No intraspecies cross-reactions with the closely related protein hormones placental lactogen and GH were detected. To elucidate the antigenic surface of hPRL all MCA directed against hPRL were then used for two-site epitope mapping studies in which pairs of MCA were assessed for simultaneous reaction with the same antigen. The native hormone was incubated with the first, solid-phase bound, so called 'capture MCA', and this complex treated with a second, 125I-labelled 'detection MCA'. Based on the results of these combinations, at least three sterically non-overlapping and (taking RIA cross-reaction studies into consideration) two additional epitopes could be defined. Two antibodies (code numbers INN-hPRL-1 and INN-hPRL-9) recognizing different antigenic determinants were selected and used to elaborate a two-site IRMA with an operating range wider and a reaction time shorter than those obtained with a conventional one-site RIA. J. Endocr. (1987) 114,311–318

The production of monoclonal antibodies to human chorionic gonadotrophin and its subunits

1983 ◽  
Vol 98 (3) ◽  
pp. 323-330 ◽  
Author(s):  
M. C. Stuart ◽  
P. A. Underwood ◽  
D. F. Harman ◽  
K. L. Payne ◽  
D. A. Rathjen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Free Form ◽  
Binding Affinities ◽  
Β Subunit ◽  
Α Subunit ◽  
Specific Antisera ◽  
Hybridoma Technology ◽  
Circulating Levels

The difficulties encountered in producing highly specific antisera to human chorionic gonadotrophin (hCG) were overcome by the use of hybridoma technology. A panel of monoclonal antibodies directed toward hCG and its subunits was produced. Of the four antibodies which were fully characterized, one recognized the intact hCG molecule only, a second recognized only the free β-subunit, a third recognized only the free α-subunit and the fourth bound to the β-subunit of hCG both when it was in the free form and when it was associated with the α-subunit forming the intact hCG molecule. There was no significant cross-reaction of any of these antibodies with the pituitary glycoprotein hormones. The four antibodies had high binding affinities which should permit their use in immunoassays for measurement of circulating levels of hCG and its subunits.

Conformation of the α-subunit of glycoprotein hormones: a study using polyclonal and monoclonal antibodies

1990 ◽  
Vol 72 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Rajan R. Dighe ◽  
G.Satyanarayana Murthy ◽  
Basangowda S. Kurkalli ◽  
N.Raghuveer Moudgal
Keyword(s):  
Monoclonal Antibodies ◽  
Glycoprotein Hormones ◽  
Α Subunit

scholarly journals Monoclonal antibodies against the chronic lymphatic leukemia antigen cCLLa: characterization and reactivity

Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 437-443 ◽  
Author(s):  
GB Faguet ◽  
JF Agee
Keyword(s):  
Monoclonal Antibodies ◽  
Target Cell ◽  
Competitive Inhibition ◽  
Cell Subset ◽  
Cytolytic Activity ◽  
Antigenic Determinants ◽  
Chronic Lymphatic Leukemia ◽  
Binding Studies ◽  
Cell Reactivity ◽  
Lymphatic Leukemia

Abstract Monoclonal antibodies (MoAbs) were developed against the cCLLa, a 69- kilodalton leukemia-associated antigen expressed on malignant cells of B-type chronic lymphatic leukemia (B-CLL) and its variants: prolymphocytic (PLL) and hairy cell leukemias (HCL). Two hybridomas yielded approximately 2 and approximately 7.5 mg/mL of IgG2a kappa and IgM kappa, respectively. Monoclonal surface immunoglobulin-bearing cells of all B-CLL patients studied (n = 30) reacted with the MoAbs (r greater than .99) regardless of stage or lymphocyte count. This suggests that the malignant clone in CLL can be identified and its size monitored by using our MoAbs. In contrast, normal B lymphocytes, a large panel of normal, reactive and neoplastic cells, and malignant cell lines failed to react with either MoAb as judged by indirect immunofluorescence and by flow cytometry. Only two patients (one with non-Hodgkin's lymphoma, the other with acute myeloblastic leukemia) exhibited a small cell subset reactive with the MoAbs. cCLLa specificity was suggested by selective target cell reactivity and competitive inhibition-absorption and confirmed by immunoprecipitation. MoAbs IgG2a kappa and IgM kappa appeared to share antigenic determinants and were moderate and avid complement binders inducing 100% and 40% target cell lysis, respectively. cCLLa density on malignant CLL and HCL cells was estimated by equilibrium binding studies using the IgG2a kappa MoAb at 1.7 and 9 X 10(6)/cell, respectively. The restricted expression of the cCLLa and the specificity and cytolytic activity of the anti-cCLLa MoAbs support these antibodies as probes for the classification of lymphoproliferative diseases and for the specific diagnosis and treatment of B-CLL and its variants.

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