ORF73 Antibody
Description
Development and Validation of ORF73 Antibodies
ORF73 antibodies are generated through recombinant protein expression or peptide-based approaches. Key methodologies include:
Recombinant Protein-Based Antibodies
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Polclonal Antibodies:
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PA1–73N: Developed using GST-ORF73 fusion proteins immunized in rabbits. This antibody binds specifically to LANA, showing nuclear dot-like staining in KS spindle cells .
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Western Blot Sensitivity: Bacterially expressed full-length ORF73 demonstrated 67% sensitivity in detecting LANA, with variability across patient populations (e.g., 100% in US classic KS vs. 52% in Italian classic KS) .
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Monoclonal Antibodies
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LN53: A rat monoclonal antibody validated for immunohistochemistry (IHC), ELISA, and immunofluorescence. It detects LANA in paraffin-embedded KS tissues and PEL cell lines .
Peptide Mapping
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Epitope Identification: A screen of 171 17-mer peptides identified 13 immunoreactive epitopes, though no single epitope was universally recognized by KS patients .
Diagnostic Utility
Research Insights
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Pathogenesis: LANA interacts with pRb and p53 tumor suppressors, promoting oncogenesis .
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Viral Persistence: Maintains the KSHV episome during cell division, ensuring latent infection .
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Clinical Correlation: ORF73 antibody levels rise post-KS diagnosis, particularly in HIV-infected individuals (median S/C ratios: 3.7 pre-KS vs. 4.9 post-KS) .
Antibody Performance in Patient Cohorts
Functional Role of LANA
Antibody Types
Product Specs
Q&A
How do researchers validate specificity of ORF73 antibodies in detecting latent nuclear antigen (LANA) in HHV-8-infected cells?
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Methodological Answer:
Validation typically involves immunofluorescence (IF) and western blot (WB) assays using HHV-8-positive cell lines (e.g., TY-1, BCBL-1). For IF, colocalization with nuclear punctate patterns confirmed by patient-derived sera serves as a gold standard . In WB, antibodies should detect ~222–234 kDa bands corresponding to ORF73 isoforms . Cross-reactivity is ruled out by testing HHV-8-negative cell lines and pre-adsorption with recombinant ORF73 protein .Key Validation Data:
What experimental models are optimal for studying ORF73’s role in viral episome maintenance?
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Methodological Answer:
Use latently infected B-cell lines (e.g., BCBL-1) or engineered epithelial cell models transfected with HHV-8 bacterial artificial chromosomes (BACs). Chromatin immunoprecipitation (ChIP) assays with ORF73 antibodies (e.g., anti-FLAG M2 affinity gel) confirm tethering to viral terminal repeats . For functional studies, siRNA knockdown of ORF73 disrupts episomal persistence .Recommended Models:
Model System Application Strength BCBL-1 cells Endogenous ORF73 studies Preserves native chromatin interactions HEK293T + HHV-8 BAC Episome replication assays Enables genetic manipulation
How to select ORF73 antibodies for immunohistochemistry (IHC) in Kaposi’s sarcoma (KS) tissues?
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Methodological Answer:
Prioritize antibodies validated for nuclear dot-like staining in KS spindle cells. Use a catalyzed signal amplification system to enhance sensitivity . Co-staining with endothelial markers (CD34) confirms specificity for KS tumor cells . Avoid antibodies with cross-reactivity to HHV-8 lytic antigens (e.g., ORF59) .
Advanced Research Questions
How to resolve contradictions in ORF73’s interaction with tumor suppressors (p53, pRb) across studies?
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Methodological Answer:
Discrepancies arise from strain-specific ORF73 variants (e.g., HVS vs. KSHV) and assay conditions. Use:-
Co-immunoprecipitation (Co-IP) with tagged proteins (e.g., Myc-tagged ORF73) in HEK293T cells .
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Reporter assays (E2F/p53-responsive promoters) to quantify functional antagonism .
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Structural mapping: Truncation mutants localize interaction domains (e.g., C-terminal 240 aa for p53/pRb binding) .
Example Findings:
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What methods identify genome-wide chromatin binding sites of ORF73?
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Methodological Answer:
Combine ChIP-seq with anti-ORF73 antibodies (e.g., mouse monoclonal IgG2a) and bioinformatics tools (e.g., MACS2 for peak calling). Validate using:
How to address epitope variability in ORF73 antibody performance across HHV-8 subtypes?
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Methodological Answer:
Use peptide mapping with overlapping 17-mer peptides to identify immunodominant regions . For example:-
Screen serum samples against a peptide library (offset by 5 residues) via ELISA .
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Focus on conserved regions (e.g., aa 122–329) for broad reactivity .
Epitope Characterization Data:
Peptide Region Reactivity (%) Subtype Specificity aa 150–200 85% Pan-HHV-8 aa 250–300 45% KSHV-specific -
Product Science Overview
Introduction
Mouse antibodies, also known as murine antibodies, are antibodies derived from mice. These antibodies have been widely used in research and therapeutic applications due to their ability to target specific antigens .
Human Anti-Mouse Antibody (HAMA) Response
One significant challenge with using mouse antibodies in humans is the Human Anti-Mouse Antibody (HAMA) response. When mouse antibodies are introduced into the human body, the immune system may recognize them as foreign and produce antibodies against them. This response can range from mild reactions, such as rashes, to severe reactions, such as kidney failure . The HAMA response can also reduce the effectiveness of the treatment and complicate laboratory measurements .
Advances in Antibody Engineering
To overcome the limitations of mouse antibodies, advances in antibody engineering have led to the development of chimeric, humanized, and fully human antibodies. Chimeric antibodies combine mouse and human antibody sequences, while humanized antibodies have a higher proportion of human sequences. Fully human antibodies are generated using techniques such as phage display and transgenic mouse models . These advancements have improved the safety, specificity, and efficacy of antibody-based therapies .
