Recombinant Human TLR3

Using Recombinant Human TLR3 in research applications enables precise investigation of TLR3-mediated immune responses, facilitates drug screening, and supports the development of immunotherapies and vaccine adjuvants. Recombinant TLR3 provides a controlled, reproducible tool to study receptor-ligand interactions, signaling pathways, and functional outcomes in both basic and translational research.

Key scientific reasons to use recombinant human TLR3 include:

• Mechanistic Studies of Innate Immunity: TLR3 is a pattern recognition receptor that detects viral double-stranded RNA, triggering innate immune responses such as type I interferon and pro-inflammatory cytokine production. Recombinant TLR3 allows for in vitro reconstitution of these pathways, enabling detailed analysis of downstream signaling events and immune modulation.

• Cancer Immunotherapy Research: TLR3 activation can induce immunogenic apoptosis in tumor cells, enhance antigen presentation, and promote the activation of cytotoxic T cells and natural killer (NK) cells. Recombinant TLR3 is used to screen and characterize agonists or antagonists, optimize combination therapies, and evaluate the immunostimulatory potential of candidate drugs or adjuvants.

• Vaccine Adjuvant Development: TLR3 agonists are being explored as adjuvants to boost vaccine efficacy by enhancing dendritic cell maturation, cytokine secretion, and adaptive immune responses. Recombinant TLR3 enables high-throughput screening of novel adjuvant candidates and mechanistic studies of adjuvant action.

• Drug Discovery and Screening: Recombinant TLR3 is essential for in vitro assays to identify and characterize small molecules, peptides, or biologics that modulate TLR3 activity, supporting the development of new therapeutics for infectious diseases, cancer, and inflammatory conditions.

• Reproducibility and Standardization: Using recombinant protein ensures batch-to-batch consistency, defined activity, and the absence of confounding cellular components, which is critical for generating reliable, interpretable data in both basic and applied research.

• Functional Assays: Recombinant TLR3 can be used in binding studies, cell-free signaling assays, and as a standard in ELISA or other immunoassays to quantify TLR3 or its ligands.

In summary, recombinant human TLR3 is a versatile and essential reagent for dissecting TLR3 biology, evaluating therapeutic strategies, and developing immunomodulatory agents in a controlled and reproducible manner.

You can use recombinant human TLR3 as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is compatible with the capture and detection antibodies used in your specific ELISA kit.

Essential context and supporting details:

• ELISA Standard Requirements: For quantitative ELISA, the standard curve is typically generated using a purified or recombinant protein that matches the analyte of interest—in this case, human TLR3. This allows for accurate determination of TLR3 concentrations in unknown samples by comparison to the standard curve.

• Antibody Compatibility: Not all recombinant proteins are recognized equally by all ELISA kits. The recombinant TLR3 must contain the relevant epitopes recognized by both the capture and detection antibodies in your kit. Some ELISA kits specifically caution that recombinant proteins may not always match the antibody specificity, potentially resulting in undetectable or inaccurate assays. It is recommended to verify that your recombinant TLR3 is recognized by the antibodies used in your assay, either by consulting the kit documentation or performing a pilot experiment.

• Standard Preparation: When preparing standards, ensure the recombinant TLR3 is properly diluted in the same buffer as your samples to minimize matrix effects and maintain consistency. Follow the kit’s recommended concentration range for standards, which is typically provided in the manual (e.g., 0.16–10 ng/mL for some TLR3 ELISA kits).

• Validation: If you are using a recombinant TLR3 not supplied with the kit, it is good practice to validate its performance by running a standard curve and checking for linearity, recovery, and parallelism with endogenous TLR3 in your sample matrix.

Additional relevant information:

• Carrier Proteins: Some recombinant proteins are supplied with carrier proteins (e.g., BSA) to enhance stability. If your recombinant TLR3 contains a carrier, ensure it does not interfere with the assay or cause non-specific binding.

• Documentation: Always consult the ELISA kit’s technical manual for specific recommendations regarding standards, as some kits may require or recommend using the supplied standard for optimal results.

In summary, recombinant human TLR3 can be used as a standard for ELISA quantification if it is recognized by the kit antibodies and prepared according to best practices for ELISA standard curves. Validation is essential to ensure accurate quantification.

Recombinant Human TLR3 protein has been validated for several key applications in published research, primarily centered around its role in innate immunity, antiviral responses, and vaccine development. The main applications include:

1. Ligand Binding Studies:
   Recombinant TLR3 has been used to study its interaction with double-stranded RNA (dsRNA), a natural ligand. For example, recombinant human TLR3 extracellular domain (ECD) has been shown to bind short dsRNA oligonucleotides, enabling structural and functional characterization of the TLR3:dsRNA complex.

2. Cell-Based Signaling Assays:
   Recombinant TLR3 is used in cell-based systems (such as HEK293 cells engineered to express TLR3) to study downstream signaling pathways, including activation of NF-κB and IRF3, and induction of type I interferons. These assays are critical for screening small molecules or proteins that modulate TLR3 signaling.

3. High-Throughput Screening:
   Recombinant TLR3 is employed in high-throughput screens to identify agonists, antagonists, or modulators of the TLR3 pathway. These screens help discover novel compounds for therapeutic intervention in inflammatory diseases and cancer.

4. Vaccine Adjuvant Development:
   TLR3 agonists, including recombinant proteins and synthetic ligands, are used in vaccine formulations to enhance immune responses. Intranasal subunit vaccines incorporating TLR3-stimulating components have been shown to induce mucosal IgA and CD8 T cell responses, offering protection against respiratory viruses.

5. Functional Characterization of TLR3 Agonists and Antagonists:
   Recombinant TLR3 is used to validate the activity of TLR3 agonists (e.g., polyI:C, Ampligen, Hiltonol) and antagonists, as well as newly designed protein minibinders or small molecules, in both in vitro and in vivo models.

6. Structural and Mechanistic Studies:
   Recombinant TLR3 is used for crystallography and other structural biology techniques to elucidate the molecular basis of TLR3 activation and ligand recognition.

7. Therapeutic Applications in Cancer and Infectious Disease:
   TLR3 agonists have been explored in clinical and preclinical studies for cancer immunotherapy and antiviral therapy, leveraging the ability of TLR3 to activate dendritic cells, promote apoptosis, and induce antitumor immune responses.

In summary, recombinant human TLR3 has been validated for ligand binding, signaling pathway analysis, high-throughput screening, vaccine adjuvant development, functional characterization of agonists/antagonists, structural studies, and therapeutic applications in cancer and infectious disease.

To reconstitute and prepare Recombinant Human TLR3 protein for cell culture experiments, centrifuge the vial briefly, then add sterile distilled water to achieve a final concentration of 0.1–0.5 mg/mL. Avoid vortexing or vigorous pipetting to prevent protein denaturation.

Step-by-step protocol:

• Centrifuge the vial at low speed to collect the lyophilized protein at the bottom before opening.
• Add sterile distilled water to the vial to reach the desired concentration (typically 0.1–0.5 mg/mL for cell culture applications).
• Gently pipette to dissolve the protein, washing down the sides of the vial to ensure complete recovery. Do not vortex or shake vigorously.
• Incubate at room temperature for 10–30 minutes with gentle agitation to ensure full dissolution.
• For long-term storage, add a carrier protein or stabilizer (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose), aliquot, and store at ≤–20°C to minimize freeze-thaw cycles.
• For short-term use, reconstitute in sterile water and keep at 2–8°C for up to a week.
• If required for functional assays, dilute the reconstituted protein further in cell culture medium or buffer compatible with your experimental setup, ensuring the final buffer conditions (pH, osmolarity) are suitable for cell viability and protein stability.

Additional notes:

• Always consult the specific product datasheet for recommended reconstitution buffer and concentration, as requirements may vary by protein batch or expression system.
• Avoid repeated freeze-thaw cycles, which can reduce protein activity and stability.
• If using for ELISA or other detection assays, ensure the protein is compatible with the assay buffer and detection antibodies.

Summary of key points:

• Centrifuge vial, add sterile water, avoid vigorous mixing.
• Reconstitute to 0.1–0.5 mg/mL.
• Add stabilizer for long-term storage.
• Aliquot and store at ≤–20°C.

This protocol ensures optimal recovery and stability of recombinant TLR3 for cell culture experiments.