LDLRAD3 Antibody

What is LDLRAD3 and why is it significant in viral research?

LDLRAD3 (Low Density Lipoprotein Receptor Class A Domain Containing 3) is a highly conserved plasma membrane protein of the LDL scavenger receptor family. Its significance dramatically increased after it was identified as a primary receptor for Venezuelan equine encephalitis virus (VEEV), an encephalitic alphavirus responsible for epidemics across the Americas .

LDLRAD3 has three LDL-receptor class A extracellular domains, with Domain 1 (D1) being critical for VEEV binding. Research has shown that LDLRAD3 binds directly to VEEV particles and facilitates virus attachment and internalization into host cells . Gene editing studies demonstrate that mice with deletions in Ldlrad3 are resistant to VEEV challenge, highlighting the protein's essential role in viral pathogenesis .

Methodologically, when studying VEEV infection pathways, LDLRAD3 should be considered a primary target for both diagnostic and therapeutic research applications.

What types of LDLRAD3 antibodies are available and how do they differ?

Several types of LDLRAD3 antibodies are available for research, targeting different domains of the protein:

Most available antibodies are rabbit polyclonal preparations, which recognize different epitopes within LDLRAD3 . For domain-specific applications, researchers should select antibodies targeting the relevant region - particularly Domain 1 (D1) for VEEV interaction studies.

What experimental applications are LDLRAD3 antibodies validated for?

LDLRAD3 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Most common application for detecting LDLRAD3 expression levels in cell or tissue lysates

• ELISA: For quantitative detection of LDLRAD3 in solution

• Immunocytochemistry (ICC): For cellular localization studies

• Immunofluorescence (IF): Both for cultured cells and paraffin-embedded sections

• Immunohistochemistry (IHC): For both frozen and paraffin-embedded sections

Depending on the specific antibody, recommended dilutions vary. For example, HPA038251 is recommended at 0.04-0.4 μg/mL for Western blot, 0.25-2 μg/mL for immunofluorescence, and 1:20-1:50 for immunohistochemistry .

How can LDLRAD3 antibodies be used to study VEEV infection mechanisms?

LDLRAD3 antibodies serve as valuable tools for investigating VEEV infection mechanisms through several methodological approaches:

• Blocking studies: Anti-LDLRAD3 antibodies can block VEEV infection in cell culture, making them useful for studying virus-receptor interactions. Research has shown that pre-treatment of cells with anti-Ldlrad3 immune serum reduced SINV-VEEV-GFP TrD infection .

• Receptor localization: Immunohistochemistry with LDLRAD3 antibodies can map tissue and cellular expression patterns, correlating with VEEV tropism. In situ hybridization studies have shown that Ldlrad3 mRNA is expressed in neurons of the brain, consistent with VEEV neurotropism .

• Binding assays: LDLRAD3 antibodies can be used in competitive binding assays to assess virus-receptor interactions at the molecular level. Studies demonstrate that LDLRAD3 domain 1 (D1) directly engages domains A and B of the VEEV E2 protein and the fusion loop in E1 .

• Therapeutic development: LDLRAD3-D1-Fc fusion proteins, developed using insights from antibody studies, have shown therapeutic potential by abolishing disease caused by multiple VEEV subtypes .

What considerations should be made when selecting domain-specific LDLRAD3 antibodies?

When selecting domain-specific LDLRAD3 antibodies, researchers should consider:

• Target domain relevance: Domain 1 (D1) of LDLRAD3 is necessary and sufficient to support VEEV infection, making antibodies targeting this region particularly valuable for studying VEEV-receptor interactions . For example, anti-LDLRAD3 antibodies targeting D1 can block VEEV infection in cell culture.

• Receptor isoforms: LDLRAD3 exists in multiple isoforms, including a shorter isoform with a 32-amino acid deletion. Experiments show that the full-length, but not truncated isoform of Ldlrad3, can restore VEEV infection in LDLRAD3-deficient cells . Antibodies should be selected based on which isoform is being studied.

• Cross-species reactivity: For comparative studies across species, select antibodies with validated cross-reactivity. Some antibodies react with human, mouse, rat, and other species LDLRAD3 .

• Functional validation: Prioritize antibodies validated in functional assays (e.g., virus blocking) rather than just expression detection, particularly for mechanistic studies of viral entry.

How can researchers validate the specificity of LDLRAD3 antibodies in their experimental systems?

Rigorous validation of LDLRAD3 antibodies should include:

• Genetic controls: Compare antibody staining/detection in wild-type versus Ldlrad3-deficient cells or tissues. Studies have used gene-edited Ldlrad3-deficient mice (e.g., Δ14/Δ14 mice) and cells (ΔLdlrad3 cells) as negative controls .

• Peptide competition: Pre-incubate antibodies with immunizing peptides to confirm signal specificity. For example, antibodies like ABIN953156 are peptide-affinity purified and could be validated using their immunizing peptide (aa 314-345) .

• Multiple antibody validation: Use antibodies targeting different LDLRAD3 epitopes and confirm concordant results.

• Recombinant expression: Overexpress tagged LDLRAD3 and confirm co-localization or detection with anti-LDLRAD3 antibodies. Research has used complementation of ΔLdlrad3 cells with Ldlrad3 to validate antibody specificity .

• TaqMan verification: Develop TaqMan primer sets spanning deletion regions to verify the absence of Ldlrad3 in gene-edited models as a complementary approach to antibody validation .

What are the methodological approaches for studying LDLRAD3-independent mechanisms of VEEV infection?

Despite LDLRAD3's critical role, evidence suggests alternative entry pathways exist for VEEV. To study these LDLRAD3-independent mechanisms:

• Residual infection analysis: Carefully quantify low-level VEEV infection in Ldlrad3-deficient models. Studies show that Ldlrad3-deficient mice still exhibit some VEEV RNA in tissues, albeit at significantly reduced levels (100-1000 fold lower than wild-type) .

• Tissue-specific analysis: Examine differential dependencies on LDLRAD3 across tissues. Research indicates lymphoid tissues from Ldlrad3-deficient mice maintain measurable viral RNA levels through 14 days post-infection, suggesting potential LDLRAD3-independent mechanisms in these tissues .

• Alternative receptor screening: Use receptor competition assays with anti-LDLRAD3 antibodies to identify cells/tissues where blocking LDLRAD3 does not completely abrogate infection.

• Temporal analysis: Recent research shows LDLRAD3-independent infection appears in Ldlrad3-deficient mice over time, suggesting potential adaptation or alternative entry mechanisms that could be characterized using time-course studies .

What are the optimal storage and handling conditions for LDLRAD3 antibodies?

For optimal performance of LDLRAD3 antibodies:

• Storage temperature: Store at -20°C according to manufacturer specifications. For example, HPA038251 antibody should be stored at -20°C .

• Formulation: Most LDLRAD3 antibodies are supplied in buffered aqueous glycerol solutions, which helps maintain stability during freeze-thaw cycles .

• Shipping conditions: Antibodies are typically shipped on wet ice and should be transferred to -20°C immediately upon receipt .

• Aliquoting: For antibodies used frequently, prepare small working aliquots to minimize freeze-thaw cycles.

• Dilution buffer: Use manufacturer-recommended buffers for dilution. For immunohistochemistry applications, proper antigen retrieval methods should be optimized for each specific antibody.

How can researchers troubleshoot non-specific binding or weak signals when using LDLRAD3 antibodies?

When encountering technical issues with LDLRAD3 antibodies:

• Non-specific binding:

  • Increase blocking time/concentration (5% BSA or normal serum from the same species as the secondary antibody)

  • Optimize antibody concentration - titrate from manufacturer's recommended range

  • Include additional washing steps with higher detergent concentration

  • Pre-adsorb antibody with tissue/cell lysates from Ldlrad3-deficient samples

• Weak signals:

  • Optimize antigen retrieval methods for fixed tissues

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Confirm LDLRAD3 expression levels in your sample using qRT-PCR as a complementary approach

• Signal verification:

  • Compare results with multiple antibodies targeting different epitopes of LDLRAD3

  • Include known positive controls (tissues with confirmed LDLRAD3 expression, such as neurons )

  • Use recombinant LDLRAD3-expressing cells as positive controls

What considerations should be made when using LDLRAD3 antibodies for co-localization studies with viral proteins?

For LDLRAD3-virus co-localization studies:

• Antibody compatibility: Ensure primary antibodies against LDLRAD3 and viral proteins are raised in different host species to avoid cross-reactivity of secondary antibodies.

• Fixation optimization: Different fixation methods may affect epitope accessibility. Paraformaldehyde fixation is commonly used, but optimization may be required for dual-detection of LDLRAD3 and viral antigens.

• Temporal dynamics: VEEV-LDLRAD3 interactions occur during early entry steps. Design time-course experiments (4°C binding, followed by 37°C internalization) to capture different stages of the virus-receptor interaction .

• Subcellular compartments: LDLRAD3 engagement with VEEV occurs at the cell surface initially, but internalization follows. Use markers for different endocytic compartments to track the virus-receptor complex.

• Controls for specificity: Include controls where one primary antibody is omitted to ensure signal specificity in each channel.

How might LDLRAD3 antibodies contribute to developing therapeutics against VEEV infection?

LDLRAD3 antibodies hold significant potential for therapeutic development:

• Therapeutic antibody development: Antibodies targeting the VEEV-binding domain of LDLRAD3 could serve as templates for developing therapeutic antibodies. Research has shown that both anti-Ldlrad3 antibodies and Ldlrad3-D1-Fc fusion protein block VEEV infection .

• Decoy receptor strategies: Studies show that administration of Ldlrad3-D1-Fc fusion protein abolishes disease caused by multiple VEEV subtypes, including highly virulent strains . This approach, informed by antibody binding studies, represents a promising therapeutic strategy.

• Epitope mapping: LDLRAD3 antibodies can help map critical interaction interfaces between VEEV and LDLRAD3. Structural studies show that domain 1 of LDLRAD3 wedges into a cleft created by two adjacent E2-E1 heterodimers in the VEEV trimeric spike .

• Comparative studies: Since LDLRAD3 is highly conserved across species, antibodies can help evaluate therapeutic approaches across different animal models before human clinical testing.

• Alternative receptor identification: LDLRAD3 antibodies can help identify alternate VEEV entry pathways that might need to be targeted simultaneously for complete therapeutic efficacy .

What are the current limitations of available LDLRAD3 antibodies for research applications?

Despite their utility, current LDLRAD3 antibodies have several limitations:

How can researchers design experiments to differentiate between LDLRAD3-dependent and independent mechanisms of VEEV infection?

To distinguish between these mechanisms, researchers should:

• Gene editing approaches: Generate complete LDLRAD3 knockout cell lines or animal models. Compare infection kinetics in wild-type versus knockout systems to quantify residual infection .

• Blocking antibody studies: Use dose-response curves with anti-LDLRAD3 blocking antibodies to identify the threshold beyond which increased antibody concentration provides no additional reduction in infection.

• Competitive inhibition: Use soluble LDLRAD3-D1 domain to competitively inhibit VEEV binding to cellular LDLRAD3. Assess whether complete inhibition of infection can be achieved .

• Cell type comparative analysis: Different cell types may exhibit varying dependence on LDLRAD3. Compare infection rates across cell types with different LDLRAD3 expression levels after normalization .

• Time-course and deep sequencing: Analyze VEEV RNA from Ldlrad3-deficient animals at different infection time points to identify potential viral adaptations enabling LDLRAD3-independent entry .

What new approaches might improve the specificity and utility of LDLRAD3 antibodies for viral research?

Emerging approaches that could enhance LDLRAD3 antibody research include:

• Single-domain antibodies: Developing nanobodies or single-domain antibodies against specific LDLRAD3 domains might enable better access to sterically hindered epitopes.

• Recombinant antibody technology: Precisely engineered recombinant antibodies could target specific functional domains with higher specificity than conventional polyclonal antibodies.

• Bispecific antibodies: Creating bispecific antibodies targeting both LDLRAD3 and VEEV envelope proteins could enhance detection of receptor-virus complexes.

• Proximity labeling approaches: Combining LDLRAD3 antibodies with proximity labeling techniques (BioID, APEX) could identify additional proteins in the VEEV entry complex.

• CRISPR epitope tagging: CRISPR-mediated endogenous tagging of LDLRAD3 would allow the use of highly validated tag-specific antibodies for detection, potentially reducing background and specificity issues.

• Structure-guided antibody design: Utilizing the cryo-EM structures of VEEV-LDLRAD3 complexes to design antibodies targeting specific interface residues could yield more functionally relevant reagents.