Recombinant Human TL1-A

Recombinant human TL1A (TNF-ligand-related molecule 1A) is a valuable research tool for investigating immune regulation and inflammatory pathways due to its multifaceted biological activities and therapeutic relevance.

Immunological Research Applications

TL1A functions as a T cell costimulator with significant effects on adaptive immunity. On primary T cells, it induces NF-κB activation and enhances IL-2 responsiveness while promoting secretion of proinflammatory cytokines. This makes it particularly useful for studying T cell activation mechanisms and costimulatory signaling pathways.

The protein demonstrates dichotomous effects on helper T cell differentiation. TL1A synergizes with IL-12 and IL-18 to boost Th1 immune responses through increased IFN-γ and TNF-α secretion. Additionally, it enhances Th17 cell differentiation through upregulation of RORc expression, a Th17 lineage-specific transcription factor. These properties enable researchers to investigate T helper cell polarization and the molecular mechanisms governing immune subset development.

Disease Modeling and Therapeutic Development

Recombinant TL1A is instrumental for studying inflammatory and autoimmune diseases. The TL1A/DR3 axis has been implicated in rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, and other autoimmune conditions. Using recombinant TL1A allows researchers to model disease pathology in vitro and validate therapeutic interventions targeting this pathway.

The protein is particularly valuable for antibody drug screening and functional validation. Researchers can use recombinant TL1A to evaluate neutralizing antibodies, assess binding affinity, and measure inhibition of DR3 signaling. This application supports the development of therapeutic antibodies designed to block pathological TL1A activity in inflammatory diseases.

Functional Assay Development

Recombinant human TL1A enables multiple assay formats including bioassays, binding assays, and apoptosis assays. The protein can be used in direct ELISA formats with anti-TL1A antibodies for protein-protein interaction studies. Additionally, it serves as a positive control for cell-based functional assays examining cytokine-induced signaling and cellular responses.

Mechanistic Studies

The protein facilitates investigation of cell-type-specific responses. While TL1A promotes proliferation in T cells through DR3 costimulation, it reduces proliferation in B cells activated by BCR stimulation. This differential activity makes recombinant TL1A valuable for comparative studies examining how the same ligand produces opposing effects across immune cell populations.

Recombinant TL1A also enables research into innate immune modulation, as the protein enhances natural killer cell infiltration and cytotoxicity, providing opportunities to study innate-adaptive immune crosstalk.

Yes, recombinant human TL1A can be used as a standard for quantification or calibration in ELISA assays, provided it is of sufficient purity and its concentration is accurately determined. This approach is widely accepted for cytokine quantification in ELISA, especially when natural protein standards are unavailable.

Supporting details:

• ELISA kits and protocols for TL1A routinely use recombinant human TL1A as the standard for generating calibration curves. These standards are typically serially diluted to create a range of known concentrations, which are then used to interpolate the concentration of TL1A in unknown samples based on their optical density readings.

• Specificity and compatibility: Monoclonal antibodies in validated TL1A ELISA kits are designed to detect both natural and recombinant human TL1A, ensuring accurate quantification regardless of the protein source.

• Standard preparation: It is critical that the recombinant TL1A used as a standard is highly purified and its concentration is precisely measured, ideally by methods such as HPLC or spectrophotometry. Carrier proteins (e.g., BSA) may be included to stabilize the recombinant protein, but carrier-free formulations are preferred if BSA could interfere with your assay.

• Validation: Published protocols and commercial ELISA kits demonstrate that recombinant TL1A yields reliable standard curves, with typical sensitivity in the low pg/ml to ng/ml range. The standard curve should be validated in your assay system to ensure linearity and accuracy.

• Best practices:

  • Use the same buffer for diluting standards and samples to minimize matrix effects.
  • Confirm that your recombinant TL1A matches the epitope recognized by the ELISA antibodies (most kits target the extracellular domain, e.g., aa 72–251).
  • Prepare standards fresh or aliquot and store appropriately to avoid degradation.

In summary: Recombinant human TL1A is suitable and commonly used as a standard for ELISA quantification, provided it is properly validated and prepared according to best practices.

Recombinant Human TL1A has been validated in published research for several key applications, primarily in immunology and cell biology. The most frequently reported applications include:

• Bioassays/Functional Assays: Used to study TL1A’s effects on immune cell activation, cytokine secretion, and apoptosis in various cell types, including T cells, monocytes, and tumor cell lines.
• Apoptosis Assays: Employed to induce and measure caspase-dependent apoptosis, particularly in DR3-expressing tumor cell lines.
• Binding Assays/Protein-Protein Interaction: Utilized in ELISA and other binding assays to characterize interactions with receptors (e.g., DR3) and blocking antibodies.
• Cytokine Induction and Measurement: Used to stimulate immune cells (e.g., PBMCs, MAIT cells) and measure downstream cytokine production (e.g., IL-6, TNF-α, IL-2) via multiplex bead-based ELISA or similar platforms.
• Blocking/Neutralization Assays: Applied in studies evaluating the efficacy of anti-TL1A antibodies or soluble receptors in blocking TL1A-mediated signaling.
• Standard for Quantification: Used as a standard in ELISA and other quantitative assays for TL1A detection.
• Western Blot: Validated for use as a positive control or standard in immunoblotting applications.

Supporting details and published research examples:

• Bioassays/Functional Assays: TL1A has been used to stimulate primary human T cells, leading to NF-κB activation, increased IL-2 responsiveness, and secretion of proinflammatory cytokines. In MAIT cells, TL1A-DR3 signaling drives effector functions and tissue repair responses.
• Apoptosis Assays: In tumor cell lines such as TF-1, recombinant TL1A induces caspase activity and apoptosis, which can be blocked by decoy receptors.
• Cytokine Induction: TL1A, especially in combination with other cytokines (IL-12, IL-15, IL-18), induces robust production of IL-6 and TNF-α from leukocytes, independent of TCR stimulation.
• Binding/Protein-Protein Interaction: Direct ELISA and other binding assays have been used to confirm TL1A’s interaction with DR3 and to screen for blocking antibodies.
• Blocking/Neutralization: Recombinant TL1A is used in screening assays to evaluate the neutralizing capacity of therapeutic antibodies or soluble receptors targeting TL1A.
• ELISA Standard/Western Blot: TL1A is used as a standard for quantification in ELISA and as a control in Western blotting.

Summary Table of Validated Applications

These applications are supported by numerous peer-reviewed studies and product validation data, particularly in the context of autoimmune disease, inflammation, and T cell biology.

To properly reconstitute and prepare Recombinant Human TL1A (TNFSF15) protein for cell culture experiments, follow these best practices based on manufacturer guidelines and general recombinant protein handling protocols:

1. Reconstitution Protocol

• Centrifuge the vial briefly before opening to ensure all lyophilized powder is at the bottom.
• Reconstitute the protein with sterile PBS (or as specified by the Certificate of Analysis/COA). For most products, reconstitute at 100 µg/mL (e.g., add 100 µL of sterile PBS to 10 µg of protein).
• Gently mix by swirling or tapping the vial; do not vortex or shake vigorously to avoid foaming and protein denaturation.
• Allow the vial to sit at room temperature for 15–30 minutes with gentle agitation to fully dissolve the protein.

2. Carrier Protein Addition (Recommended for Stability)

• For optimal stability and activity, especially if storing the reconstituted protein for more than a few days, add a carrier protein such as 0.1% BSA (bovine serum albumin) or 10% FBS to the reconstituted solution.
• If performing serum-free or in vivo experiments, use trehalose (5–50% final concentration) as a stabilizer instead of BSA or FBS.

3. Storage and Handling

• Short-term storage (≤1 month): Store the reconstituted protein at 2–8°C under sterile conditions.
• Long-term storage: Aliquot the protein, add carrier protein or trehalose, and store at –20°C to –80°C in a manual defrost freezer. Avoid repeated freeze-thaw cycles to maintain activity.

4. Dilution for Cell Culture

• Dilute the reconstituted TL1A protein in your cell culture medium to the desired working concentration (typically 10–100 ng/mL, depending on your experimental needs).
• If using serum-free medium, ensure the dilution buffer contains a stabilizer (e.g., trehalose or BSA) to prevent protein loss.

5. General Tips

• Always refer to the Certificate of Analysis (COA) or product manual for specific instructions, as formulations may vary between suppliers.
• For best results, use freshly reconstituted protein for critical experiments.

By following these steps, you can ensure the stability, activity, and reproducibility of Recombinant Human TL1A protein in your cell culture experiments.