TGFB3 Antibody

What is TGFB3 and what roles does it play in tissue fibrosis?

TGFB3 (transforming growth factor beta 3) is a protein in humans that belongs to the TGFβ cytokine family. It may also be known as ARVD, ARVD1, LDS5, RNHF, transforming growth factor beta-3 proprotein, or prepro-transforming growth factor beta-3. Structurally, the protein has a molecular weight of approximately 47.3 kilodaltons .

What are the common applications for TGFB3 antibodies in research?

TGFB3 antibodies are utilized in multiple research applications, including:

• Western Blot (WB): For detection and quantification of TGFB3 protein in cell or tissue lysates

• Immunohistochemistry (IHC): For visualization of TGFB3 in tissue sections, such as paraffin-embedded human heart tissue

• Immunofluorescence (IF): For cellular localization studies

• ELISA: For quantitative measurement of TGFB3 in biological samples

• Immunoprecipitation (IP): For isolation and purification of TGFB3 complexes

• Neutralization assays: To block TGFB3 activity in functional studies

• Sandwich immunoassays: For sensitive detection of TGFB3 in complex biological samples

The specific application should guide antibody selection, as some antibodies work optimally under certain conditions. For example, some TGFB3 antibodies function only under non-reducing conditions , which is a critical consideration for experimental design.

What are the optimal storage conditions for maintaining TGFB3 antibody stability?

For optimal stability and performance of TGFB3 antibodies, researchers should adhere to the following storage guidelines:

• Long-term storage: Keep antibodies at -20°C to -70°C for up to 12 months from the date of receipt as supplied

• After reconstitution:

  • Store at 2-8°C under sterile conditions for up to 1 month

  • For longer storage (up to 6 months), keep at -20°C to -70°C under sterile conditions

It is crucial to use a manual defrost freezer and avoid repeated freeze-thaw cycles, as these can significantly compromise antibody activity and specificity . Aliquoting reconstituted antibodies before freezing is recommended to minimize freeze-thaw cycles when only portions are needed for experiments.

How can researchers validate the specificity of TGFB3 antibodies?

Validation of TGFB3 antibody specificity is essential for reliable research outcomes. A comprehensive validation approach includes:

• Positive and negative controls:

  • Use tissues or cell lines known to express or lack TGFB3

  • Human heart tissue has been documented as a positive control for certain TGFB3 antibodies

• Cross-reactivity testing:

  • Test against related TGFβ family members (TGFB1, TGFB2)

  • Check reactivity across species if performing comparative studies (human, mouse, rat, etc.)

• Antibody neutralization/competition assays:

  • Pre-incubate antibody with recombinant TGFB3 protein

  • If specific, this should eliminate or significantly reduce signal

• Multiple detection methods:

  • Confirm findings using different techniques (e.g., WB and IHC)

  • Use antibodies targeting different epitopes

• Genetic validation:

  • Compare results in TGFB3 knockout/knockdown vs. wild-type samples

What are the key considerations when designing TGFB3 neutralization assays?

When designing TGFB3 neutralization assays, researchers should consider several critical factors:

• Antibody selection:

  • Choose antibodies specifically validated for neutralization activity

  • Determine the Neutralization Dose (ND₅₀), which is typically 0.1-0.3 μg/mL in the presence of 0.1 ng/mL recombinant human TGFB3

• Assay system optimization:

  • For cell-based assays, TGFB3 inhibition can be assessed in conjunction with other cytokines (e.g., IL-4) that have known biological effects

  • Careful titration of both TGFB3 and the neutralizing antibody is essential

• Controls:

  • Include isotype control antibodies to account for non-specific effects

  • Include positive controls (known TGFB3 inhibitors) and negative controls (non-neutralizing TGFB3 antibodies)

• Readout selection:

  • Choose appropriate readouts based on the known biological activities of TGFB3

  • For fibrosis studies, consider measuring collagen production, myofibroblast differentiation, or extracellular matrix gene expression

• Timing considerations:

  • TGFB3 signaling kinetics vary by cell type and context

  • Establish appropriate time points for measuring neutralization effects

How does selective TGFB3 inhibition differ from pan-TGFβ inhibition in fibrotic disease research?

The distinction between selective TGFB3 inhibition and pan-TGFβ inhibition is increasingly important in fibrotic disease research:

Evidence suggests that selective inhibition of TGFB3 signaling may target fibrotic disease pathogenesis while circumventing the adverse events associated with pan-TGFβ or TGFB1 inhibition . Clinical studies have shown that patients receiving the TGFB1-specific inhibitor metelimumab (CAT-192) showed no evidence of clinical activity and had a higher rate of adverse events compared with placebo-treated patients . In contrast, RO7303509, a high-affinity, TGFB3-specific, humanized IgG1 monoclonal antibody, was well tolerated at single subcutaneous doses up to 1200 mg in healthy volunteers with favorable pharmacokinetic data .

What biomarkers can be used to assess TGFB3 pathway activity in research and clinical studies?

Several biomarkers can be used to assess TGFB3 pathway activity:

• Direct TGFB3 pathway markers:

  • Periostin: A TGFβ pathway-associated biomarker that can be measured in serum samples to provide evidence of TGFB3 activity

  • COMP (Cartilage Oligomeric Matrix Protein): Another TGFβ pathway-associated biomarker that correlates with TGFB3 activity

• Disease-specific markers:

  • In systemic sclerosis, increased expression of TGFB3 is significantly associated with higher levels of biomarkers of disease severity and prognosis

  • These include TGFβ target genes in the skin and systemic COMP and periostin levels

• Analytical methods:

  • Periostin can be tested using the COBAS Elecsys system with proprietary antibodies

  • COMP can be assessed using research-grade platforms such as the ProteinSimple Ella platform

When monitoring TGFB3 pathway inhibition, researchers should collect samples at multiple time points (e.g., baseline, and then at regular intervals after treatment) to track changes in biomarker levels. For example, in clinical studies evaluating RO7303509, samples were collected at baseline on day -1, and after dosing on days 1, 5, 8, 15, 29, and 85 .

What are the optimal protocols for using TGFB3 antibodies in tissue section immunohistochemistry?

For optimal immunohistochemical detection of TGFB3 in tissue sections, researchers should follow these methodological considerations:

• Tissue preparation:

  • Use immersion-fixed, paraffin-embedded tissue sections

  • Heart tissue has been validated for certain TGFB3 antibodies

• Antibody concentration and incubation:

  • Use TGFB3 monoclonal antibody at approximately 25 μg/mL

  • Incubate overnight at 4°C for optimal binding and signal development

• Detection system:

  • For chromogenic detection, use HRP-DAB (horseradish peroxidase-diaminobenzidine) staining systems

  • Counterstain with hematoxylin for nuclear visualization

• Controls and validation:

  • Include known positive and negative control tissues

  • Consider dual staining with cell-type specific markers to identify TGFB3-expressing cell populations

• Optimization steps:

  • Test different antigen retrieval methods (heat-induced, enzymatic)

  • Optimize blocking solutions to reduce background

  • Test a range of antibody concentrations to determine optimal signal-to-noise ratio

A detailed protocol example based on published methods includes:

• Deparaffinize and rehydrate sections

• Perform antigen retrieval (specific method depends on tissue type)

• Block endogenous peroxidase activity with H₂O₂

• Block non-specific binding sites

• Incubate with primary TGFB3 antibody (25 μg/mL) overnight at 4°C

• Apply HRP-conjugated secondary antibody

• Develop with DAB substrate

• Counterstain with hematoxylin

• Dehydrate, clear, and mount

How does TGFB3 expression correlate with disease severity in systemic sclerosis and other fibrotic conditions?

The relationship between TGFB3 expression and fibrotic disease severity has been a focus of recent research:

• Systemic Sclerosis (SSc):

  • Increased expression of TGFB3, but not TGFB1 or TGFB2, is significantly associated with higher levels of biomarkers of SSc disease severity and prognosis

  • TGFB3 expression correlates with TGFβ target genes in the skin and systemic COMP and periostin levels in SSc patients

• Tissue-specific fibrosis:

  • Elevated TGFB3 gene expression has been documented in human lung and liver fibrotic tissue

  • This suggests a potential role as a biomarker for disease progression

• Therapeutic implications:

  • The association between TGFB3 expression and disease severity provides rationale for selective TGFB3 inhibition as a therapeutic strategy

  • RO7303509, a TGFB3-specific antibody, was developed based on this connection and has shown promising results in initial clinical testing

• Monitoring considerations:

  • Serial measurements of TGFB3 expression and associated biomarkers may help track disease progression

  • These measurements could potentially serve as pharmacodynamic indicators in clinical trials of anti-fibrotic therapies

Understanding these correlations has guided the development of more targeted therapeutic approaches for fibrotic diseases, moving away from pan-TGFβ inhibition toward isoform-specific strategies that may offer improved safety profiles while maintaining efficacy.

What experimental conditions affect the detection of TGFB3 in immunoassays?

Several experimental conditions can significantly impact TGFB3 detection in immunoassays:

• Reducing vs. non-reducing conditions:

  • Some TGFB3 antibodies function only under non-reducing conditions

  • This is critical for Western blotting and other protein analysis techniques

• Sample preparation:

  • TGFB3 exists in both latent and active forms in biological samples

  • Acid activation may be required to detect total TGFB3 levels

  • Sample processing methods can affect protein conformation and epitope accessibility

• Antibody selection:

  • Different clones recognize distinct epitopes (e.g., Clone #44922 vs. Clone #20724)

  • Some antibodies may be suitable for multiple applications while others are application-specific

• Cross-reactivity considerations:

  • Antibodies may exhibit varying degrees of cross-reactivity with other TGFβ isoforms

  • Species reactivity varies between antibodies (human, mouse, rat, etc.)

• Detection sensitivity:

  • Sandwich immunoassays typically offer higher sensitivity than direct detection methods

  • Signal amplification strategies may be necessary for low-abundance samples

Researchers should carefully validate their experimental protocols and consider these factors when designing TGFB3 detection assays to ensure reliable and reproducible results.

How can researchers troubleshoot cross-reactivity between TGF-beta isoforms?

Cross-reactivity between TGF-beta isoforms presents a significant challenge in isoform-specific research. Effective troubleshooting approaches include:

• Antibody selection strategies:

  • Choose antibodies raised against unique regions of TGFB3 not conserved in other isoforms

  • Review validation data showing specificity testing against related isoforms

  • Consider using antibodies targeting the middle region of TGFB3 for higher specificity

• Validation techniques:

  • Perform competitive binding assays with recombinant TGFB1, TGFB2, and TGFB3

  • Use knockout/knockdown models for each isoform as definitive controls

  • Implement Western blot analysis to confirm single-band detection at the appropriate molecular weight

• Assay optimization:

  • Adjust antibody concentration to minimize non-specific binding

  • Optimize blocking conditions to reduce background

  • Consider using more stringent washing steps in immunoassays

• Complementary approaches:

  • Corroborate antibody-based findings with nucleic acid-based detection (RT-PCR, RNA-seq)

  • Use multiple antibodies targeting different epitopes of TGFB3

  • Implement proximity ligation assays for improved specificity

• Data interpretation:

  • Be aware of the sequence homology between TGF-beta isoforms when interpreting results

  • Consider quantitative analysis of cross-reactivity and adjust findings accordingly

By employing these strategies, researchers can minimize the impact of cross-reactivity and obtain more reliable isoform-specific data in their TGFB3 studies.

What is the current state of TGFB3-targeted therapeutics in fibrotic disease research?

The development of TGFB3-targeted therapeutics represents an evolving approach in fibrotic disease treatment:

• Rationale for selective targeting:

  • Pan-TGFβ inhibitors have shown unacceptable toxicities in clinical trials

  • Selective inhibition of TGFB3 may target fibrotic disease pathogenesis while reducing adverse effects

  • Increased expression of TGFB3 is associated with markers of disease severity in systemic sclerosis

• Clinical development status:

  • RO7303509 (MTBT1466A): A high-affinity, TGFB3-specific, humanized IgG1 monoclonal antibody

  • Phase 1a results: Well tolerated at single subcutaneous doses up to 1200 mg in healthy volunteers

  • Favorable pharmacokinetic data that appeared to increase dose-proportionally

  • No subjects developed anti-drug antibodies (ADAs) at baseline; only one subject (2.8%; 50 mg IV) tested positive for ADAs at a single time point (day 15)

• Pharmacokinetic characteristics:

  • Serum concentrations of RO7303509 were best characterized by a two-compartment model plus a depot compartment with first-order SC absorption kinetics

  • Maximum serum concentrations (Cmax) and area under the concentration-time curve (AUC) values appeared to increase dose-proportionally across all doses tested

• Safety profile:

  • Most frequent adverse events related to TGFB3-specific antibody treatment were injection site reactions and infusion-related reactions

  • This contrasts with the higher rate of adverse events observed with TGFB1-specific inhibitors like metelimumab (CAT-192)

• Comparative approaches:

  Therapeutic Approach	Example	Development Status	Key Findings
  TGFB3-specific inhibition	RO7303509	Phase 1a completed	Well tolerated, favorable PK
  TGFB1-specific inhibition	Metelimumab (CAT-192)	Clinical trials	No evidence of clinical activity, higher rate of AEs
  TGFB1/TGFB3 inhibition	AVID200	Clinical trials	Improvements in skin fibrosis, acceptable safety, but grade 2/3 anemia observed

These findings support the further development of TGFB3-specific inhibition as a potential therapeutic strategy for fibrotic diseases, with ongoing research focusing on efficacy in disease-specific contexts.

How are TGFB3 antibodies used in preclinical models of fibrosis?

TGFB3 antibodies serve as valuable tools in preclinical fibrosis research:

• Mechanistic studies:

  • TGFB3-specific antibodies help delineate the distinct roles of TGFβ isoforms in fibrotic processes

  • Neutralizing antibodies can block TGFB3 activity to assess its contribution to fibrogenesis

  • Antibodies are used to track TGFB3 expression and localization during disease progression

• Animal models:

  • In preclinical mouse studies, murine surrogates of TGFB3-specific antibodies (like RO7303509) significantly inhibited endogenous activation of TGFβ target genes in models of fibrotic lung disease

  • These models provide proof-of-concept for the therapeutic potential of TGFB3 inhibition

• Biomarker validation:

  • TGFB3 antibodies are employed to correlate TGFB3 expression with disease biomarkers such as periostin and COMP

  • These correlations help establish the predictive and prognostic value of TGFB3 in fibrotic conditions

• Target validation approaches:

  • Dose-response studies with TGFB3 antibodies in preclinical models help establish the relationship between target inhibition and antifibrotic effects

  • Combination studies with other antifibrotic agents assess potential synergistic effects

• Translational research applications:

  • Findings from antibody-based preclinical studies inform clinical trial design

  • Biomarker strategies validated in animal models using TGFB3 antibodies are adapted for human studies

These applications collectively contribute to our understanding of TGFB3's role in fibrosis and facilitate the development of targeted therapeutic strategies with improved efficacy and safety profiles.

What emerging technologies are advancing TGFB3 antibody research?

Several cutting-edge technologies are transforming TGFB3 antibody research:

• Advanced antibody engineering:

  • Development of bispecific antibodies targeting TGFB3 and complementary fibrotic pathway components

  • Creation of antibody fragments with improved tissue penetration for enhanced efficacy in fibrotic tissues

  • Humanized and fully human antibodies with reduced immunogenicity for therapeutic applications

• High-throughput screening platforms:

  • Microfluidic systems for rapid evaluation of antibody binding characteristics

  • Automated cell-based assays for functional screening of TGFB3 neutralizing antibodies

  • Computational approaches to predict antibody-antigen interactions and optimize binding properties

• Single-cell analysis technologies:

  • Integration of TGFB3 antibodies with single-cell RNA sequencing to map TGFB3 activity at cellular resolution

  • Imaging mass cytometry combining TGFB3 antibodies with spatial tissue analysis

  • Multiplexed immunofluorescence to simultaneously visualize TGFB3 and downstream signaling components

• In vivo imaging techniques:

  • Labeled TGFB3 antibodies for non-invasive tracking of TGFB3 expression in preclinical models

  • Multimodal imaging approaches combining antibody-based detection with functional assessment of fibrotic processes

• Biomarker discovery platforms:

  • Proteomic approaches to identify novel TGFB3-associated biomarkers for improved patient stratification

  • Integration of TGFB3 antibody-based assays with machine learning algorithms for predictive biomarker development

These technological advances are expected to accelerate research on TGFB3's role in fibrotic diseases and facilitate the development of more effective diagnostic and therapeutic strategies.

What are the key areas for future investigation in TGFB3 biology and antibody development?

Several critical research gaps and opportunities exist in TGFB3 biology and antibody development:

• Isoform-specific signaling pathways:

  • Further elucidation of TGFB3-specific signaling mechanisms distinct from TGFB1 and TGFB2

  • Investigation of tissue-specific functions of TGFB3 in different fibrotic conditions

  • Characterization of TGFB3-specific receptor complexes and downstream signaling events

• Therapeutic optimization strategies:

  • Development of combination approaches targeting TGFB3 alongside other fibrotic pathways

  • Exploration of tissue-targeted delivery systems for TGFB3 antibodies to enhance local efficacy

  • Investigation of potential biomarkers for patient selection in TGFB3-targeted therapies

• Clinical translation challenges:

  • Assessment of long-term safety profiles of TGFB3-specific inhibition

  • Development of companion diagnostics to identify patients most likely to benefit from TGFB3-targeted therapies

  • Comparative studies between different TGFB3 antibody candidates to optimize therapeutic potential

• Disease-specific investigations:

  • Expanded studies of TGFB3's role in various fibrotic conditions beyond systemic sclerosis

  • Investigation of TGFB3 contributions to fibrosis in different organ systems (lung, liver, kidney, heart)

  • Exploration of potential applications in non-fibrotic conditions where TGFB3 signaling is dysregulated

• Technical advances in antibody development:

  • Development of next-generation TGFB3 antibodies with enhanced specificity and functional properties

  • Exploration of alternative antibody formats (single-domain antibodies, nanobodies) for improved tissue penetration

  • Creation of multispecific antibodies targeting TGFB3 alongside complementary therapeutic targets