Recombinant Invertebrate iridescent virus 3 Transmembrane protein 066L (IIV3-066L)

What is Invertebrate Iridescent Virus 3 and how is it classified taxonomically?

Invertebrate Iridescent Virus 3 (IIV-3), also known as mosquito iridescent virus (MIV), belongs to the genus Chloriridovirus within the Iridoviridae family. It is characterized by its restricted host range, primarily infecting mosquitoes (order Diptera), and its relatively large particle size of approximately 180 nm in diameter . This distinguishes it from members of the Iridovirus genus, which typically have a broader host range including organisms from the orders Diptera, Lepidoptera, Hemiptera, and Coleoptera, and are smaller at approximately 120 nm in diameter . Phylogenetic analyses indicate that IIV-3 is distantly related to other iridovirus genera, showing low levels of amino acid identity in predicted proteins compared to other iridovirus homologues .

What expression systems are suitable for producing recombinant IIV3-066L protein?

Based on available research, Escherichia coli has been successfully employed as an expression system for producing recombinant IIV3-066L protein . The protein can be expressed as a full-length construct (amino acids 1-193) with an N-terminal histidine tag to facilitate purification . This bacterial expression system provides several advantages:

• High protein yield

• Established protocols for induction and harvesting

• Cost-effectiveness for research-scale production

• Compatibility with common purification techniques such as immobilized metal affinity chromatography (IMAC)

The resulting recombinant protein is typically harvested in the form of a lyophilized powder, which can be reconstituted in an appropriate buffer system for experimental use .

How should recombinant IIV3-066L protein be stored for optimal stability?

To maintain the structural integrity and functional properties of recombinant IIV3-066L protein, the following storage recommendations should be implemented:

It is crucial to avoid repeated freeze-thaw cycles as they can lead to protein denaturation and loss of activity . After reconstitution, the protein should be aliquoted before storage at -20°C/-80°C to minimize the need for multiple thawing events.

What methodologies are most effective for investigating the role of IIV3-066L in virus-host interactions?

Investigating the role of IIV3-066L in virus-host interactions requires a multi-faceted approach combining molecular, cellular, and systems biology techniques. The following methodological framework is recommended:

• Protein-Protein Interaction Studies

  • Yeast two-hybrid screening with mosquito cell proteins

  • Co-immunoprecipitation followed by mass spectrometry

  • Bimolecular fluorescence complementation in insect cell lines

  • Surface plasmon resonance to determine binding kinetics

• Functional Genomics Approaches

  • CRISPR-Cas9-mediated gene editing of IIV3-066L to create mutant viruses

  • RNA interference to knockdown IIV3-066L expression during infection

  • Transcriptome analysis of host cells in response to wild-type vs. mutant IIV3-066L

• Structural Biology Methods

  • Cryo-electron microscopy of virus particles with wild-type or modified IIV3-066L

  • X-ray crystallography of purified IIV3-066L alone or in complex with host factors

  • Molecular dynamics simulations to predict conformational changes

• In Vivo Studies

  • Transgenic mosquito models expressing IIV3-066L

  • Comparative infection studies in different mosquito species

  • Tissue-specific localization using immunohistochemistry

These methodologies should be applied in a systematic manner, with initial in vitro studies informing subsequent in vivo experiments, to build a comprehensive understanding of IIV3-066L's role in the virus life cycle and host interactions.

How can researchers address challenges in expressing and purifying functional IIV3-066L for structural studies?

Membrane proteins like IIV3-066L present significant challenges for expression and purification, particularly when aimed at structural studies that require high purity and native conformation. A systematic approach to overcome these challenges includes:

Expression System Selection:
While E. coli is commonly used for initial expression attempts , membrane proteins often require eukaryotic expression systems for proper folding and post-translational modifications. Consider:

Solubilization Strategies:

• Screen multiple detergents (DDM, LDAO, OG, etc.) at various concentrations

• Employ novel solubilization agents such as SMALPs (styrene-maleic acid lipid particles)

• Consider nanodiscs or amphipols for maintaining native-like environment

Purification Protocol Optimization:

• Implement two-step affinity chromatography (His-tag IMAC followed by size exclusion)

• Incorporate on-column detergent exchange during purification

• Minimize exposure to room temperature during all procedures

• Add stabilizing agents such as glycerol or specific lipids

Quality Control Assessments:

• Circular dichroism to verify secondary structure

• Thermostability assays to identify optimal buffer conditions

• Dynamic light scattering to assess monodispersity

• Limited proteolysis to identify stable domains

By systematically addressing these challenges, researchers can improve the likelihood of obtaining functionally active IIV3-066L suitable for downstream structural and functional studies.

What bioinformatic approaches can reveal the evolutionary history of IIV3-066L in relation to other viral transmembrane proteins?

Understanding the evolutionary history of IIV3-066L requires sophisticated bioinformatic analyses that integrate sequence, structure, and phylogenetic information. The following comprehensive approach is recommended:

• Sequence-Based Analyses

  • Position-Specific Iterative BLAST (PSI-BLAST) to identify distant homologs

  • Multiple Sequence Alignment (MSA) using MUSCLE or MAFFT algorithms, with manual refinement of transmembrane regions

  • Calculation of conservation scores across iridovirus transmembrane proteins

  • Identification of sequence motifs unique to IIV3-066L versus shared with other viral proteins

• Phylogenetic Reconstruction

  • Maximum Likelihood methods using RAxML or IQ-TREE

  • Bayesian inference approaches using MrBayes or BEAST

  • Reconciliation of gene trees with species trees to identify potential horizontal gene transfer events

  • Molecular clock analyses to estimate divergence times

• Structural Prediction and Comparison

  • Ab initio and template-based structural modeling

  • Structural alignment with homologous viral transmembrane proteins

  • Comparison of predicted transmembrane topology across evolutionary lineages

  • Analysis of coevolving residues to identify functional constraints

• Selection Pressure Analysis

  • Calculation of dN/dS ratios to identify sites under positive or purifying selection

  • Sliding window analysis to identify domains with distinct evolutionary patterns

  • Branch-site models to detect episodic selection in specific lineages

Existing phylogenetic analyses already indicate that IIV-3 is distantly related to other iridovirus genera, with low amino acid identity in predicted proteins compared to other iridovirus homologues . The absence of obvious colinearity with any sequenced iridovirus further suggests a distinct evolutionary trajectory for IIV-3 proteins, including IIV3-066L . These findings should guide more targeted analyses of this specific transmembrane protein.

How can researchers design effective experiments to elucidate the immunomodulatory properties of IIV3-066L?

Investigating potential immunomodulatory properties of IIV3-066L requires a systematic experimental approach that addresses both innate and adaptive immune responses. The following experimental design framework is recommended:

Phase 1: In vitro Screening for Immunomodulatory Effects

• Cell Culture Models:

  • Mosquito cell lines (e.g., C6/36, Aag2)

  • Human immune cell lines (for zoonotic potential assessment)

  • Primary mosquito hemocytes

• Recombinant Protein Application Methods:

  • Direct addition of purified IIV3-066L to culture medium

  • Transfection with IIV3-066L expression constructs

  • Transduction using viral vectors expressing IIV3-066L

• Readout Measurements:

  • Transcriptome analysis focusing on immune-related genes

  • Quantification of antimicrobial peptide production

  • Measurement of ROS/NOS production

  • Assessment of cell viability and apoptosis markers

Phase 2: Signaling Pathway Analysis

• Target Pathway Investigation:

  • NF-κB pathway activity using reporter assays

  • JAK-STAT pathway activation assessment

  • Toll and Imd pathway component phosphorylation

  • RNAi machinery function evaluation

• Protein Interaction Studies:

  • Co-immunoprecipitation with known immune regulators

  • Yeast two-hybrid screening against immune protein libraries

  • Proximity ligation assays in intact cells

  • FRET/BRET studies to detect real-time interactions

Phase 3: Functional Validation

• Gene Silencing Approaches:

  • RNAi-mediated knockdown of identified interaction partners

  • CRISPR-Cas9 knockout of target genes in amenable systems

  • Complementation studies with mutant proteins

• Domain Mapping:

  • Generation of truncated IIV3-066L constructs

  • Site-directed mutagenesis of key residues

  • Chimeric proteins with homologs from other iridoviruses

• Infection Models:

  • Comparison of wild-type virus versus IIV3-066L mutants

  • Competitive infection assays

  • Time-course studies of immune response dynamics

This experimental framework allows for systematic identification and characterization of any immunomodulatory functions of IIV3-066L, providing insights into how this viral protein may contribute to immune evasion or manipulation during infection.

What are the optimal conditions for expressing recombinant IIV3-066L in E. coli systems?

To achieve optimal expression of recombinant IIV3-066L in E. coli systems, researchers should consider the following comprehensive protocol:

Strain Selection:
E. coli BL21(DE3) or derivatives are recommended due to their reduced protease activity and tight regulation of T7 RNA polymerase . For membrane proteins like IIV3-066L, specialized strains such as C41(DE3) or C43(DE3) may provide improved expression by accommodating the potential toxicity of membrane protein overexpression.

Vector Design:

• Include an N-terminal His-tag for purification as demonstrated in existing protocols

• Consider fusion partners such as MBP or SUMO to enhance solubility

• Incorporate a precision protease cleavage site for tag removal

• Use T7 or tac promoters for controlled induction

Expression Conditions:
The following parameters should be optimized through factorial design experiments:

Harvest and Lysis:

• Centrifugation at 5,000 × g for 15 minutes at 4°C

• Resuspension in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl

• Addition of protease inhibitors (PMSF, EDTA-free cocktail)

• Cell disruption via sonication or high-pressure homogenization

• Separation of membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

Solubilization and Purification:

• Solubilize membrane fraction with detergents such as DDM (1%), LDAO (1%), or OG (2%)

• Purify using Ni-NTA affinity chromatography with imidazole gradient elution

• Further purify by size exclusion chromatography

• Verify purity by SDS-PAGE (>90% as reported)

By systematically optimizing these conditions, researchers can maximize the yield and quality of recombinant IIV3-066L for subsequent structural and functional studies.

What research design is most appropriate for investigating the potential role of IIV3-066L in host range determination?

Investigating the role of IIV3-066L in host range determination requires a multi-layered research design that integrates comparative genomics, functional assays, and in vivo studies. The following comprehensive research design is recommended:

Phase 1: Comparative Analysis

• Sequence Comparison Across Iridoviruses:

  • Compare IIV3-066L sequences from IIV-3 with homologs from other iridoviruses with different host ranges

  • Identify conserved and variable regions that correlate with host specificity

  • Construct phylogenetic trees to visualize evolutionary relationships

• Host Factor Prediction:

  • Perform in silico protein-protein interaction predictions

  • Identify potential host receptors or interaction partners

  • Compare predicted interactions across susceptible and non-susceptible host species

Phase 2: In Vitro Functional Studies

• Cell Line Susceptibility Testing:

  • Transfect IIV3-066L into cell lines from diverse insect orders

  • Measure binding affinity to cell surface components

  • Assess ability to mediate membrane fusion or entry events

• Domain Swapping Experiments:

  • Generate chimeric constructs with domains from IIV3-066L and homologs from viruses with different host ranges

  • Test these chimeras for altered cell tropism

  • Map specific regions responsible for host-specific interactions

• CRISPR-Mediated Gene Editing:

  • Generate IIV-3 variants with modified IIV3-066L sequences

  • Test infection capacity in different host cells

  • Quantify replication efficiency using qPCR or plaque assays

Phase 3: In Vivo Validation

• Cross-Species Infection Assays:

  • Challenge multiple mosquito species with wild-type and mutant viruses

  • Quantify viral loads in different tissues

  • Monitor pathogenesis and disease progression

• Transgenic Approaches:

  • Express IIV3-066L in normally non-susceptible hosts

  • Assess whether expression confers susceptibility

  • Examine tissue-specific effects of expression

• Competition Assays:

  • Co-infect hosts with wild-type and mutant viruses

  • Measure relative fitness in different host backgrounds

  • Identify selective pressures on IIV3-066L variants

This research design provides a comprehensive framework for determining whether and how IIV3-066L contributes to the restricted host range of IIV-3, which is primarily limited to mosquitoes (Diptera) , contrasting with the broader host range of other iridoviruses.

How can researchers develop reliable assays to measure the interaction between IIV3-066L and host cell membranes?

Developing reliable assays to measure interactions between IIV3-066L and host cell membranes requires both biophysical and cell-based approaches. The following methodological framework provides a comprehensive strategy:

Biophysical Interaction Assays:

• Liposome Binding Assays

  • Preparation of liposomes with compositions mimicking mosquito cell membranes

  • Fluorescently labeled IIV3-066L incubated with liposomes

  • Measurement of binding by:

    • Co-sedimentation followed by SDS-PAGE analysis

    • Fluorescence microscopy to visualize binding

    • FRET assays with dual-labeled membranes and protein

• Surface Plasmon Resonance (SPR)

  • Immobilization of purified IIV3-066L on sensor chip

  • Flow of lipid vesicles or purified membrane components over the sensor

  • Determination of kinetic parameters (kon, koff, KD)

  • Comparative analysis with different lipid compositions

• Atomic Force Microscopy (AFM)

  • Visualization of IIV3-066L insertion into supported lipid bilayers

  • Force measurements of membrane-protein interactions

  • Topographical analysis of membrane alterations

Cell-Based Interaction Assays:

• Fluorescence Microscopy

  • Expression of fluorescently tagged IIV3-066L in mosquito cells

  • Co-localization studies with membrane markers

  • Live-cell imaging to track dynamic interactions

  • FRAP (Fluorescence Recovery After Photobleaching) to measure membrane mobility

• Flow Cytometry-Based Binding Assays

  • Incubation of cells with labeled recombinant IIV3-066L

  • Quantification of binding under various conditions

  • Competition assays with potential inhibitors

  • Comparison across cell types from different insect orders

• Membrane Fractionation Studies

  • Isolation of membrane microdomains (lipid rafts, etc.)

  • Western blot analysis of IIV3-066L distribution

  • Mass spectrometry to identify co-purifying host factors

Functional Membrane Interaction Assays:

• Electrophysiology

  • Patch-clamp recordings of cells expressing IIV3-066L

  • Lipid bilayer conductance measurements with purified protein

  • Assessment of membrane permeabilization or ion channel formation

• Membrane Fusion Assays

  • Lipid mixing assays using fluorescently labeled liposomes

  • Content mixing assays to track aqueous compartment merging

  • pH-dependent fusion studies to mimic endosomal environments

• Electron Microscopy

  • Immunogold labeling of IIV3-066L in infected cells

  • Visualization of membrane alterations using TEM

  • Cryo-EM to capture intermediate states of membrane interaction

By implementing this multi-faceted approach, researchers can comprehensively characterize the interaction between IIV3-066L and host cell membranes, providing insights into its role in the viral life cycle.

What statistical approaches are most appropriate for analyzing protein-protein interaction data involving IIV3-066L?

When analyzing protein-protein interaction data involving IIV3-066L, researchers should implement a comprehensive statistical framework that addresses the specific challenges of membrane protein interactions. The following statistical approaches are recommended based on the experimental methods employed:

For High-Throughput Interaction Screening:

• False Discovery Rate (FDR) Control

  • Apply Benjamini-Hochberg procedure to control for multiple testing

  • Implement q-value cutoffs (typically q < 0.05 or 0.01)

  • Consider more stringent thresholds for exploratory studies

• Enrichment Analysis

  • Calculate fold enrichment over background/control conditions

  • Apply hypergeometric tests or Fisher's exact tests for categorical enrichment

  • Utilize permutation-based approaches for empirical p-value estimation

• Network Statistics

  • Calculate betweenness centrality and clustering coefficients

  • Implement Markov clustering algorithms to identify interaction modules

  • Apply random walk with restart (RWR) algorithms to predict additional interactions

For Quantitative Binding Assays:

• Binding Curve Analysis

  • Fit data to appropriate binding models (simple, cooperative, competitive)

  • Calculate confidence intervals for KD values using non-linear regression

  • Compare binding parameters across conditions using extra sum-of-squares F tests

• Association/Dissociation Kinetics

  • Apply global fitting algorithms to extract kon and koff rates

  • Calculate residence time (1/koff) as a measure of interaction stability

  • Use Akaike Information Criterion (AIC) to select between competing kinetic models

• Thermodynamic Analysis

  • Implement van't Hoff analysis for temperature-dependent studies

  • Calculate entropy and enthalpy changes from temperature-dependent KD values

  • Apply isothermal titration calorimetry (ITC) data analysis when available

For Co-localization Studies:

• Correlation Coefficients

  • Calculate Pearson's correlation coefficient for intensity correlation

  • Implement Manders' overlap coefficient for spatial overlap

  • Use Spearman's rank correlation for non-linear relationships

• Object-Based Analysis

  • Calculate Jaccard index for binary object overlap

  • Implement nearest neighbor distance analysis

  • Apply Ripley's K-function for spatial pattern analysis

• FRET Efficiency Analysis

  • Calculate FRET efficiency using appropriate controls

  • Implement pixel-by-pixel FRET analysis for spatial heterogeneity

  • Apply statistical tests for comparing FRET efficiencies across conditions

By selecting the appropriate statistical approaches based on the experimental method and data type, researchers can ensure robust and meaningful interpretation of protein-protein interaction data involving IIV3-066L.

How should researchers integrate structural predictions and experimental data to model the topology of IIV3-066L in viral membranes?

Integrating structural predictions with experimental data to model IIV3-066L topology requires a systematic approach that leverages complementary computational and experimental methods. The following integrative modeling framework is recommended:

Phase 1: Initial Prediction and Experimental Validation

• Computational Topology Prediction:

  • Apply multiple transmembrane prediction algorithms (TMHMM, HMMTOP, Phobius)

  • Generate consensus prediction based on majority voting

  • Identify potential membrane-associated domains using hydrophobicity analysis

  • Predict secondary structure elements using PSIPRED or JPRED

• Experimental Topology Mapping:

  • Cysteine accessibility studies using membrane-impermeable reagents

  • Protease protection assays to identify exposed regions

  • Glycosylation mapping using artificial glycosylation sites

  • Epitope insertion and antibody accessibility studies

• Data Integration:

  • Create scoring function that weights predictions based on experimental support

  • Identify regions of agreement and conflict between methods

  • Generate initial topology model with confidence scores for each region

Phase 3: Final Model Construction and Visualization

• Model Integration:

  • Bayesian integration of all data sources with appropriate weighting

  • Generation of ensemble models that reflect uncertainty in specific regions

  • Calculation of model quality metrics and confidence scores

• Context-Dependent Modeling:

  • Model IIV3-066L in different states (pre-fusion, post-fusion)

  • Consider oligomeric states based on experimental evidence

  • Model interactions with other viral and host proteins

• Visualization and Communication:

  • Generate high-quality structural visualizations highlighting key features

  • Create dynamic representations of potential conformational changes

  • Develop interactive models accessible to the research community

This integrative approach ensures that the final topology model of IIV3-066L is consistent with both computational predictions and experimental observations, providing a reliable foundation for understanding its function in the viral life cycle.

What are the most promising research directions for understanding the functional role of IIV3-066L in viral pathogenesis?

Based on current knowledge of IIV3-066L and related viral proteins, several promising research directions emerge for elucidating its functional role in viral pathogenesis. The following research avenues offer significant potential for advancing our understanding:

• Structural Biology Approaches

  • High-resolution structure determination of IIV3-066L using cryo-EM or X-ray crystallography

  • Investigation of conformational changes during different stages of the viral life cycle

  • Structural comparison with homologous proteins from other iridoviruses to identify conserved functional elements

• Host-Pathogen Interaction Studies

  • Identification of specific host receptors or binding partners in mosquito cells

  • Investigation of potential immunomodulatory functions in suppressing host antiviral responses

  • Analysis of tissue tropism determinants that contribute to the restricted host range of IIV-3

• Functional Genomics

  • Development of reverse genetics systems for IIV-3 to create IIV3-066L mutants

  • CRISPR-Cas9 screening in host cells to identify essential factors for IIV3-066L function

  • Transcriptomic and proteomic profiling of host responses to wild-type versus mutant IIV3-066L

• Evolutionary Biology

  • Comparative analysis of IIV3-066L across isolates from different geographical regions

  • Investigation of selective pressures shaping IIV3-066L evolution

  • Reconstruction of ancestral sequences to understand evolutionary trajectories

• Translational Applications

  • Evaluation of IIV3-066L as a target for antiviral strategies in vector control

  • Assessment of IIV3-066L as a potential vaccine antigen for related viruses

  • Development of diagnostic tools based on IIV3-066L-specific antibodies

These research directions should be pursued using integrative approaches that combine computational, in vitro, and in vivo methodologies. The distinct evolutionary history of IIV-3 compared to other iridoviruses suggests that IIV3-066L may possess unique functional properties that contribute to the virus's biology and pathogenesis. Understanding these functions has potential implications not only for basic virology but also for applications in biotechnology and vector control.

How can researchers effectively publish and share IIV3-066L research findings to advance the field collectively?

To maximize the impact of IIV3-066L research and accelerate collective advancement in the field, researchers should implement a comprehensive strategy for publishing and sharing their findings. The following approaches are recommended:

• Strategic Publication Planning

  • Target appropriate journal tiers based on the nature of the findings:

    • High-impact general science journals for breakthrough discoveries

    • Specialized virology journals for detailed mechanistic studies

    • Open access platforms to maximize accessibility

  • Consider preprint servers (bioRxiv, ResearchSquare) for rapid dissemination

  • Develop a publication pipeline that balances comprehensive studies with focused reports

• Enhanced Data Presentation

  • Include interactive visualizations for complex structural data

  • Provide standardized data tables that facilitate meta-analysis

  • Create graphical abstracts that effectively communicate key findings

  • Utilize supplementary materials for detailed protocols and raw data

• Comprehensive Resource Sharing

  • Deposit sequence data in public databases (GenBank, UniProt)

  • Submit structural data to the Protein Data Bank (PDB)

  • Share reagents through repositories like Addgene or BEI Resources

  • Develop and share computational tools and scripts via GitHub

• Collaborative Research Frameworks

  • Establish consortium approaches for large-scale projects

  • Implement standardized protocols across laboratories

  • Develop material transfer agreements that facilitate sharing

  • Create collaborative online platforms for real-time data sharing

• Knowledge Dissemination Beyond Publications

  • Present at relevant conferences in virology, structural biology, and vector biology

  • Develop educational resources for researchers entering the field

  • Conduct workshops on specialized techniques relevant to IIV3-066L research

  • Engage with the broader scientific community through science communication

• Translational and Cross-Disciplinary Communication

  • Connect with researchers in applied fields such as vector control

  • Engage with computational biologists for advanced modeling approaches

  • Collaborate with structural biologists for specialized techniques

  • Establish dialogues with evolutionary biologists to contextualize findings

By implementing these strategies, researchers can ensure that findings related to IIV3-066L are effectively shared, validated, and built upon by the scientific community. This approach acknowledges that the restricted host range of IIV-3 to mosquitoes makes this virus and its proteins potentially valuable for understanding host-specific viral mechanisms and possibly for developing targeted vector control strategies.

What is a recommended protocol for generating antibodies against IIV3-066L for research applications?

Generating high-quality antibodies against IIV3-066L requires careful consideration of its transmembrane nature and potential structural complexity. The following comprehensive protocol is recommended for developing research-grade antibodies:

Antigen Design and Preparation:

• Epitope Selection:

  • Perform computational analysis to identify hydrophilic, surface-exposed regions

  • Select multiple epitopes (15-25 amino acids) from different domains

  • Avoid transmembrane regions that might be poorly immunogenic

  • Consider both N-terminal and C-terminal regions for targeting

• Antigen Production Options:

  • Synthetic peptides conjugated to carrier proteins (KLH or BSA)

  • Recombinant protein fragments expressed in E. coli

  • Full-length protein in detergent micelles or nanodiscs

  • DNA immunization encoding IIV3-066L

Immunization Strategies:

• Animal Selection:

  • Rabbits for polyclonal antibodies (larger serum volumes)

  • Mice for monoclonal antibody development

  • Consider chickens for IgY production as an alternative

• Immunization Protocol:

  Time Point	Procedure	Adjuvant
  Day 0	Primary immunization	Complete Freund's Adjuvant
  Day 21	First boost	Incomplete Freund's Adjuvant
  Day 42	Second boost	Incomplete Freund's Adjuvant
  Day 63	Final boost	PBS (for hybridoma development)
  Day 70-75	Serum collection or spleen harvest	-

• Monitoring Immune Response:

  • ELISA testing of serum samples after each boost

  • Western blot validation using recombinant protein

  • Immunofluorescence with IIV-3 infected cells when available

Antibody Production and Purification:

• For Polyclonal Antibodies:

  • Collect serum and heat-inactivate at 56°C for 30 minutes

  • Purify IgG using Protein A/G affinity chromatography

  • Consider epitope-specific affinity purification

  • Validate specificity through Western blot and immunoprecipitation

• For Monoclonal Antibodies:

  • Harvest splenocytes and fuse with myeloma cells

  • Screen hybridoma supernatants by ELISA

  • Select positive clones and subclone by limiting dilution

  • Expand selected clones for antibody production

  • Purify using Protein A/G chromatography

Validation and Characterization:

• Specificity Testing:

  • Western blot against recombinant IIV3-066L

  • Immunoprecipitation from virus-infected cells

  • Immunofluorescence microscopy

  • Pre-absorption controls with immunizing antigen

  • Testing against related viral proteins to evaluate cross-reactivity

• Functional Characterization:

  • Epitope mapping using peptide arrays or truncation mutants

  • Neutralization assays if applicable

  • Determination of binding affinity by ELISA or SPR

  • Assessment of application suitability (WB, IP, IF, IHC)

• Documentation and Storage:

  • Complete documentation of all production and validation steps

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Store at -20°C or -80°C for long-term preservation

  • Include preservatives (0.02% sodium azide) for 4°C working stocks

This comprehensive protocol provides a framework for generating research-quality antibodies against IIV3-066L that can be used for various applications including localization studies, protein-protein interaction analyses, and functional investigations.