scholarly journals Differentiation of Two Bovine Lentiviruses by a Monoclonal Antibody on the Basis of Epitope Specificity

2001 ◽  
Vol 8 (2) ◽  
pp. 283-287 ◽  
Author(s):  
Ling Zheng ◽  
Shucheng Zhang ◽  
Charles Wood ◽  
Sanjay Kapil ◽  
Graham E. Wilcox ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Capsid Protein ◽  
Recombinant Fusion Protein ◽  
Bovine Immunodeficiency Virus ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Assays ◽  
Immunodeficiency Virus ◽  
Competitive Binding Assays

ABSTRACT Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are bovine lentiviruses that are closely related genetically. A recombinant fusion protein containing the capsid protein of BIV expressed in Escherichia coli was used to immunize mice and produce monoclonal antibodies. Six hybridomas specific for BIV capsid protein were identified, and one antibody, designated 10H1, was characterized further. Competitive binding assays were performed to analyze the topography of antigenic determinants by enzyme-linked immunosorbent assay and demonstrated the existence of at least three distinct antigenic determinants on capsid protein. The monoclonal antibody reacted specifically with both BIV capsid and the recombinant fusion protein in Western immunoblot analyses. However, it did not react with the recombinant capsid fusion protein of JDV, indicating that BIV contains at least one unique epitope in the capsid protein that is absent in JDV. Further mapping of the epitope by chemical cleavage analysis identified that the epitope is located at the 6.4-kDa N terminus of the 29-kDa capsid protein. This monoclonal antibody assay will be valuable for distinguishing the two closely related lentiviruses by Western blotting.

Reactions of human antibodies with CR1 immobilised by mouse monoclonal antibody E11 Red cell phenotype Murine MAb Human anti- Absorbance Ratio Kn(a+) ] Kn(a-) E11 Kna 0.755 0.195 4:1 McC(a+) 0.538 McC(a-) E11 McC 0.136 4:1 Yk(a+) 0.315 Yk(a-) E11 Yka 0.120 26:1 Sl(a+) 0.342 Sl(a-) E11 Sla 0.074 4.6:1 Cs(a+) 0.139 Cs(a-) E11 Cs 0.108 Mapping relative positions of antigens on a specific protein When several murine monoclonal antibodies to different epitopes on the same protein are available, MAIEA can be used to study the relative position of antigens on that protein. This application of MAIEA depends on mutual inhibition of murine monoclonal antibodies and human antibodies. A negative result is obtained when human and monoclonal antibodies compete for the same epitope, or bind to very closely located epitopes, so no tri-molecular complex is produced. Several monoclonal antibodies to the Kell protein have been used in MAIEA to study the relationships of the Kell system antigens [10]. The decay accelerating factor DAF, CD55, is detected by several monoclonal antibodies. Three antibodies BRIC 230, BRIC 110 and BRIC 216 were known from competitive binding assays to bind to different short consensus repeats (SCR) [11]. So three of the four SCRs of the DAF molecule were positively identified (Table II). Strong positive reactions were observed with all three BRIC antibodies and anti-Cr3, anti-WES8, and anti-WESb showing that MAIEA is a useful techique for studying this system [12]. The results showed that Cr8, WESa, and WESb are not on the first three SCRs and must

1995 ◽  
pp. 190-190
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Molecular Complex ◽  
Specific Protein ◽  
Cell Phenotype ◽  
Binding Assays ◽  
Human Antibodies ◽  
Short Consensus Repeats ◽  
Murine Monoclonal Antibodies ◽  
Competitive Binding Assays

scholarly journals Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody That Recognizes a Novel Conformational Epitope on gp41 and Lacks Reactivity against Self-Antigens

2008 ◽  
Vol 82 (14) ◽  
pp. 6869-6879 ◽  
Author(s):  
Mei-Yun Zhang ◽  
Bang K. Vu ◽  
Anil Choudhary ◽  
Hong Lu ◽  
Michael Humbert ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Immunodeficiency Virus ◽  
Conformational Epitope ◽  
Heptad Repeat ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Antibody ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Self Antigens

ABSTRACT Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.

Disparities in estimates of IgG bound to normal platelets

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 200-202 ◽  
Author(s):  
N Blumberg ◽  
D Masel ◽  
M Stoler
Keyword(s):  
Direct Measurement ◽  
Immunosorbent Assay ◽  
Competitive Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Binding Assays ◽  
Competitive Assay ◽  
Standard Curve ◽  
Normal Platelet ◽  
Direct Binding ◽  
Competitive Binding Assays

Abstract Estimates of the number of IgG molecules bound to normal platelets have ranged from several hundred to several tens of thousands. The lower estimates were generated from direct binding assays and stoichiometric assumptions. The higher values derive from competitive binding assays, in which platelet-associated IgG (PAIgG) is calculated from a standard curve using soluble IgG standards. Using a kinetic-ELISA (enzyme-linked immunosorbent assay) antiglobulin assay, we measured normal platelet IgG to be 21,200 +/- 9,400 molecules per platelet when a competitive assay and soluble IgG standards were used. Direct measurement of bound antiglobulin by kinetic-ELISA and stoichiometric assumptions yielded a measurement of 259 +/- 117 IgG molecules per platelet. Soluble IgG and PAIgG are not comparable in their ability to bind anti-IgG. Disparities in estimates of normal PAIgG are probably due to methodological differences. The estimate most likely to be correct is several hundred IgG or less per normal platelet.

scholarly journals Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5

2001 ◽  
Vol 75 (22) ◽  
pp. 10906-10911 ◽  
Author(s):  
Carol E. Parker ◽  
Leesa J. Deterding ◽  
Christine Hager-Braun ◽  
James M. Binley ◽  
Norbert Schülke ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Immunodeficiency Virus ◽  
Human Monoclonal Antibody ◽  
Maldi Ms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ionization Mass ◽  
Protection Assay ◽  
Virus Envelope ◽  
C Terminus ◽  
Immunodeficiency Virus

ABSTRACT Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

scholarly journals Identification of Genogroup I and Genogroup II Broadly Reactive Epitopes on the Norovirus Capsid

2005 ◽  
Vol 79 (12) ◽  
pp. 7402-7409 ◽  
Author(s):  
Tracy Dewese Parker ◽  
Noritoshi Kitamoto ◽  
Tomoyuki Tanaka ◽  
Anne M. Hutson ◽  
Mary K. Estes
Keyword(s):  
Monoclonal Antibody ◽  
Immune Response ◽  
Monoclonal Antibodies ◽  
Capsid Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Linear Epitope ◽  
Norwalk Virus ◽  
Diagnostic Assays ◽  
Separate Genus ◽  
The Family

ABSTRACT Norwalk virus, a member of the family Caliciviridae, is an important cause of acute epidemic nonbacterial gastroenteritis. Norwalk and related viruses are classified in a separate genus of Caliciviridae called Norovirus, which is comprised of at least three genogroups based on sequence differences. Many of the currently available immunologic reagents used to study these viruses are type specific, which limits the identification of antigenically distinct viruses in detection assays. Identification of type-specific and cross-reactive epitopes is essential for designing broadly cross-reactive diagnostic assays and dissecting the immune response to calicivirus infection. To address this, we have mapped the epitopes on the norovirus capsid protein for both a genogroup I-cross-reactive monoclonal antibody and a genogroup II-cross-reactive monoclonal antibody by use of norovirus deletion and point mutants. The epitopes for both monoclonal antibodies mapped to the C-terminal P1 subdomain of the capsid protein. Although the genogroup I-cross-reactive monoclonal antibody was previously believed to recognize a linear epitope, our results indicate that a conformational component of the epitope explains the monoclonal antibody's genogroup specificity. Identification of the epitopes for these monoclonal antibodies is of significance, as they are components in a commercially available norovirus-diagnostic enzyme-linked immunosorbent assay.

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