scholarly journals Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin

1995 ◽  
Vol 309 (1) ◽  
pp. 355-359 ◽  
Author(s):  
G E Morris ◽  
C Nguyen ◽  
Nguyen thi Man
Keyword(s):  
Monoclonal Antibodies ◽  
Point Mutations ◽  
Hypervariable Region ◽  
Variable Region ◽  
Binding Studies ◽  
Cdna Sequences ◽  
N Terminus ◽  
Random Library ◽  
Blast Cells

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.

scholarly journals cDNA clones encoding rabbit immunoglobulin λ chains. Evidence for length variation of the third hypervariable region and for a novel constant region

1987 ◽  
Vol 245 (3) ◽  
pp. 691-697 ◽  
Author(s):  
D J Hayzer ◽  
R M Duvoisin ◽  
J C Jaton
Keyword(s):  
Germ Line ◽  
Sequence Data ◽  
Hypervariable Region ◽  
Variable Region ◽  
Gene Clusters ◽  
Cdna Clones ◽  
Length Variation ◽  
Nucleotide Sequence Data ◽  
Cdna Sequences ◽  
Rabbit Immunoglobulin

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.

scholarly journals Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

2015 ◽  
Vol 89 (24) ◽  
pp. 12245-12261 ◽  
Author(s):  
Yousef Alhammad ◽  
Jun Gu ◽  
Irene Boo ◽  
David Harrison ◽  
Kathleen McCaffrey ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hypervariable Region ◽  
Variable Region ◽  
Antigenic Structure ◽  
Variable Regions ◽  
Core Domain ◽  
Hypervariable Region 1 ◽  
Glycoprotein E2

ABSTRACTHepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661and E2661lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion.IMPORTANCEThis study reveals conformational and antigenic differences between the Δ123 and intact E2661glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.

Variable region cDNA sequences and characterization of murine anti-human interferon γ receptor monoclonal antibodies that inhibit receptor binding by interferon γ

1995 ◽  
Vol 32 (17-18) ◽  
pp. 1329-1338 ◽  
Author(s):  
Angela Bridges ◽  
Ashley Birch ◽  
Geoffrey Williams ◽  
Michel Aguet ◽  
Daniel Schlatter ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Variable Region ◽  
Interferon Γ ◽  
Human Interferon ◽  
Cdna Sequences

Tissue Factor (TF) and Urokinase Plasminogen Activator Receptor (uPAR) and Bleeding Complications in Leukemic Patients

1997 ◽  
Vol 77 (01) ◽  
pp. 062-070 ◽  
Author(s):  
Chary López-Pedrera ◽  
Merce Jardí ◽  
Maria del Mar Malagón ◽  
Julia Inglés-Esteve ◽  
Gabriel Dorado ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Leukemic Cell ◽  
Bleeding Complications ◽  
Urokinase Plasminogen Activator Receptor ◽  
Leukemic Cell Lines ◽  
Number Of Patients ◽  
Blast Cells ◽  
Plasminogen Activator Receptor ◽  
Hemostatic Disorder

SummaryTissue factor (TF) and urokinase receptor (uPAR) are key cellular receptors triggering, respectively, coagulation and fibrinolysis. Bleeding complications among leukemic patients have been related to an abnormal expression of TF by blast cells and/or to an abnormal fibrinolytic response. In this study the expression of TF and uPAR has been assessed in 18 acute non-lymphoblastic and 8 lymphoblastic leukemic blast cells using several methodological approaches. TF mRNA was evaluated by in situ hybridization and TF and uPAR antigen were evaluated immunologically in cell lysates and on the cell surface by flow cytometry. In addition, TF-procoagulant activity was measured in coagulation-based assays. The reliability of these methods was corroborated in six leukemic cell lines of different lineages and states of maturation. Disseminated intravascular coagulation was detected in two M3 leukemia patients whose blast cells expressed high amounts of TF. Hyperfibrinolysis was detected in one M1 and two M2 patients, whose blast cells displayed a high content of uPAR antigen, but no TF. Furthermore, M5 leukemia blast cells expressed both TF and uPAR, although no hemostatic defects or bleeding complications were detected in these patients. Taken together, although a limited number of patients was included in this study, these data suggest that in leukemia patients exhibiting bleeding, either TF or uPAR are expressed by their blast cells. However, the presence of these receptors does not necessarily imply the existence of a hemostatic disorder.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

scholarly journals Novel idiotypic and antigen-binding characteristics in two anti-dinitrophenyl monoclonal antibodies.

1977 ◽  
Vol 146 (1) ◽  
pp. 282-286 ◽  
Author(s):  
N H Sigal
Keyword(s):  
Monoclonal Antibodies ◽  
Germ Line ◽  
Hypervariable Region ◽  
Weight Basis ◽  
Antigen Binding ◽  
The Novel ◽  
Maximum Frequency ◽  
Somatic Variant ◽  
Binding Characteristics ◽  
Cell Pool

Monoclonal anti-dinitrophenyl antibodies generated in the splenic focus system from B cells of adult BALB/c mice were studied for the presence or absence of murine anti-T15 (M anti-T15) reactivity and for their ability to bind phosphorylcholine (PC). Two foci of the 680 clones analyzed bound PC, and one of these antibodies reacted with M anti-T15 and anti-Fab on a 1:1 weight basis. The discovery of a clonotype reactive with M anti-15 but not with rabbit anti-T15 (R anti-T15) serum, the converse of the R anti-T15+, M anti-T15- clonotype identified in the PC-specific repertoire, points to the novel idiotypic relationships which may be found among homogeneous antibodies binding diverse antigens. The R anti-T15-, M anti-T15+ clonotype may represent a distinct set of hypervariable region sequences inserted into the T15 framework or may be a somatic variant of the T15 germ-line sequence. In addition, the maximum frequency with which this clonotype occurs within the B-cell pool is estimated.

In Situ Growth of a Cationic Polymer from the N-Terminus of Glucose Oxidase To Regulate H2O2 Generation for Cancer Starvation and H2O2 Therapy

2019 ◽  
Vol 11 (10) ◽  
pp. 9756-9762 ◽  
Author(s):  
Hanjun Hao ◽  
Mengmeng Sun ◽  
Pengyong Li ◽  
Jiawei Sun ◽  
Xinyu Liu ◽  
...  
Keyword(s):  
Glucose Oxidase ◽  
Cationic Polymer ◽  
In Situ Growth ◽  
N Terminus ◽  
H2o2 Generation

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. Stedman
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Capsid Protein Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene vp3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

2017 ◽  
Author(s):  
Eric A. Iverson ◽  
David A. Goodman ◽  
Madeline E. Gorchels ◽  
Kenneth M. STEDMAN
Keyword(s):  
Capsid Protein ◽  
Major Capsid Protein ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Vp1 Gene ◽  
Vp1 Protein ◽  
Transposon Insertion ◽  
N Terminus ◽  
Capsid Protein Vp1 ◽  
Virion Formation

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, SSV1, is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein VP3, the vp1 gene does not tolerate deletion or transposon insertion. In order determine the role of the vp1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with vp1 genes from other SSVs. By analyzing these mutants we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions.

scholarly journals Characterization of four new monoclonal antibodies against the distal N-terminal region of PrPc

2014 ◽  
Author(s):  
Alessandro Didonna ◽  
Anja Colja Venturini ◽  
Katrina Hartman ◽  
Tanja Vranac ◽  
Vladka Curin Serbec ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Prion Protein ◽  
Biochemical Characterization ◽  
Prion Diseases ◽  
Physiological Role ◽  
Prion Infection ◽  
Promising Tool ◽  
C Terminus ◽  
N Terminus

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrPC). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrPC using a well-established methodology based on the immunization of Prnp0/0 mice. Additionally, we show their ability to block prion (PrPSc) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrPC.

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