IGFBP3 Antibody

What is IGFBP3 and why is it an important research target?

IGFBP3 is a 31.7 kDa protein that serves as the main carrier of insulin-like growth factors (IGFs) in circulation. It plays multifaceted roles in:

• Regulating IGF bioavailability by binding 75-90% of circulating IGF-I in a ternary complex with acid-labile subunit (ALS)

• Modulating IGF-mediated cellular processes including proliferation, differentiation, and apoptosis

• Exhibiting IGF-independent antiproliferative and apoptotic effects through its receptor TMEM219/IGFBP-3R

• Acting as a marker for various pathological conditions including growth disorders, diabetes, and certain cancers

The protein can be identified under alternative names including BP-53, IBP3, IBP-3, and IGF-binding protein 3 .

What applications are IGFBP3 antibodies suitable for?

IGFBP3 antibodies can be utilized across multiple experimental platforms:

When selecting an application, consider the specific biological question and sample type being investigated .

How do I select the appropriate IGFBP3 antibody for my research?

Selection should be based on several critical factors:

• Species reactivity: Determine if the antibody cross-reacts with your target species. Common reactivities include human, mouse, and rat, though some antibodies may recognize canine, porcine, or monkey orthologs .

• Antibody class: Consider whether a monoclonal (higher specificity) or polyclonal (broader epitope recognition) antibody is more appropriate:

  • Monoclonal examples: Anti-IGFBP3 antibody [N2C3], IGFBP3 (B-5) mouse monoclonal

  • Polyclonal examples: IGFBP3 Polyclonal Antibody, IGFBP3 (E6C2E) Rabbit mAb

• Target epitope: Some antibodies target specific regions (e.g., amino acids 113-210 of human IGFBP3) which may affect recognition of modified forms.

• Validated applications: Verify the antibody has been validated for your specific application through published literature or manufacturer data .

• Recognition of variants: Consider whether you need to detect specific post-translationally modified forms, as some antibodies may have differential reactivity to glycosylated versus non-glycosylated IGFBP3 .

What are the best practices for IGFBP3 detection in western blotting?

For optimal western blot detection of IGFBP3:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors to prevent degradation

  • For serum/plasma samples, consider diluting 1:100 as a starting point

  • For conditioned media from cell culture, concentration may be required

• Gel selection:

  • Use 10-12% polyacrylamide gels to effectively resolve the 32-37 kDa IGFBP3 protein

  • Consider gradient gels if detecting both free IGFBP3 and complexed forms

• Transfer conditions:

  • Semi-dry or wet transfer systems are suitable

  • PVDF membranes typically provide better results than nitrocellulose for IGFBP3

• Blocking and antibody incubation:

  • Block with 5% BSA in TBST rather than milk to reduce background

  • Use recommended antibody dilutions (typically 1:200-1:1000)

  • Overnight primary antibody incubation at 4°C often improves signal quality

• Detection considerations:

  • IGFBP3 frequently appears as multiple bands (32-40 kDa) due to glycosylation and proteolytic processing

  • Validated positive controls include HeLa cells, A2780 cells, HepG2 cells, and mouse colon tissue

How should I design immunohistochemistry experiments for IGFBP3 detection in tissue sections?

For successful IGFBP3 immunohistochemistry:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues typically require antigen retrieval

  • Recommended antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

• Protocol optimization:

  • Antibody dilutions typically range from 1:20-1:200 for IHC applications

  • Human liver sections serve as effective positive controls

  • Include negative controls by omitting primary antibody or using isotype controls

• Specificity verification:

  • Confirm specificity by preincubating the antibody with recombinant IGFBP3 to block specific binding

  • Compare staining patterns with published literature

• Visualization systems:

  • DAB (3,3'-diaminobenzidine) is commonly used for colorimetric detection

  • For co-localization studies, consider fluorescent secondary antibodies

• Quantification approaches:

  • Mean fluorescence intensity can be used to quantify IGFBP3 expression levels

  • For example, epithelial IGFBP3 expression has been quantified in asthmatic airways showing 2.2-fold higher intensity in mild asthma and 4.4-fold higher in severe asthma compared to healthy controls

What are the methodological considerations for measuring IGFBP3 levels by ELISA?

When performing IGFBP3 ELISA:

• Kit selection:

  • Choose between kits measuring total IGFBP3 versus intact IGFBP3 based on research question

  • Consider whether the kit detects IGFBP3/IGF-I complexes

• Sample preparation:

  • For serum/plasma: Initial 1:100 dilution is typically recommended

  • For other biological fluids (e.g., BAL fluid), dilution may need optimization

• Assay procedure (typical for sandwich ELISA):

  • Sample addition: 25 μL of calibrator, controls, and treated unknowns

  • Buffer addition: 100 μL of assay buffer

  • Incubation: 60 minutes at room temperature with shaking (600-800 rpm)

  • Wash steps: 5 washes with 350 μL wash solution

  • Detection: Add enzyme conjugate, substrate, and stopping solution

  • Read at 450 nm with background correction at 630 nm

• Data analysis:

  • Plot log of optical density against log of IGFBP3 concentration

  • Use cubic regression or log vs. log linear regression curve-fit

  • Multiply measured concentrations by the dilution factor (typically 100x)

• Potential interferences:

  • Heterophile antibodies in samples may cause interference

  • High-dose biotin supplements can interfere with measurements (patients should stop biotin consumption 72 hours prior to sample collection)

How can I distinguish between IGF-dependent and IGF-independent functions of IGFBP3 in my research?

To differentiate these mechanisms:

• Experimental approaches:

  • Use recombinant IGFBP3 mutants with reduced IGF binding capacity

  • Compare effects of IGFBP3 in the presence/absence of IGF-I and IGF-II

  • Utilize IGFBP3 receptor (TMEM219) knockdown or blocking strategies

• Mechanistic investigations:

  • For IGF-dependent effects: Focus on IGF receptor signaling pathways (phosphorylation of IGF-1R, IRS proteins, AKT)

  • For IGF-independent effects: Analyze TMEM219 receptor binding and downstream signaling

  • Examine IGFBP3 interaction with retinoid X receptor-alpha (RXRA) for transcriptional effects

• Validation strategy:

  • For IGF-independent mechanisms, research has demonstrated that IGFBP-3 can directly bind to vimentin and induce its degradation through the E3 ligase FBXL14-mediated proteasome machinery

  • Use ecto-TMEM219 (extracellular domain of TMEM219 receptor) to block IGFBP3/TMEM219 interaction and observe if effects persist

What are the critical factors affecting IGFBP3 antibody performance in immunoassays?

Several factors can significantly impact antibody performance:

• Post-translational modifications:

  • Glycosylation: Antibodies may show up to 50% lower reactivity for glycosylated IGFBP3 versus non-glycosylated forms

  • Phosphorylation: Typically has minimal effect on immunoreactivity

  • Proteolysis: Different antibodies may recognize intact IGFBP3 versus proteolytic fragments differently

• Epitope accessibility in different contexts:

  • IGF binding may mask certain IGFBP3 epitopes

  • Complexation with ALS can affect antibody recognition

  • Cellular localization (nuclear versus cytoplasmic) may influence epitope availability

• Assay-specific considerations:

  • In ELISAs, the capture and detection antibodies may target different regions (e.g., C-terminal)

  • For IHC/IF, fixation methods can significantly impact epitope preservation

• Biological sample composition:

  • Different biological fluids show variable IGFBP3 levels detected by the same antibodies:

    • Up to 6-fold differences in measurements were observed between normal adult sera, seminal plasma, pregnancy sera, and amniotic fluid using different ELISA configurations

    • IGFBP3 levels in seminal plasma were undetectable by some antibody combinations

How can IGFBP3 antibodies be applied in studying disease mechanisms?

IGFBP3 antibodies are valuable tools for investigating various pathological conditions:

• Diabetes research:

  • IGFBP3/TMEM219 pathway disruption is observed in both T1D and T2D

  • Elevated IGFBP3 correlates with higher HbA1c, higher fasting plasma glucose, lower insulin sensitivity, and lower insulin secretion rate

  • Research applications include:

    • Immunohistochemical analysis of TMEM219 expression in pancreatic islets

    • Colocalization studies of serum IGFBP3 and TMEM219 in beta cells

    • Immunoprecipitation to confirm IGFBP3-TMEM219 interaction

• Cancer research:

  • IGFBP3 has antimetastatic effects in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC)

  • Silencing IGFBP3 expression elevates migration and invasion in vitro and metastasis in vivo

  • IGFBP3 can serve as a biomarker in pancreatic cancer diagnosis:

    • Decreased plasma IGFBP3 is observed in early-stage pancreatic cancer

    • Functions as a compensatory biomarker for CA19-9

• Respiratory disease research:

  • IGFBP3 levels are increased in airway epithelium of asthma patients

  • IGFBP3 concentration increases in bronchoalveolar lavage fluid after allergen challenge

  • Mean baseline concentration: 0.27 (±0.63) ng/ml

  • Mean post-challenge concentration: 14.85 (±13.64) ng/ml

  • May be involved in allergic airway remodeling through profibrotic effects

What are the current challenges in IGFBP3 antibody-based research and how can they be addressed?

Researchers face several technical challenges:

• Antibody cross-reactivity and specificity:

  • Solution: Validate antibodies using IGFBP3 knockout/knockdown controls

  • Published literature shows successful use of siRNA approaches for IGFBP3 knockdown validation

• Detecting specific forms of IGFBP3:

  • Challenge: Distinguishing between free, IGF-bound, and proteolytically processed forms

  • Solution: Use multiple antibodies targeting different epitopes or specific proteolytic fragments

  • Utilize immunoassays capable of differential determination of IGFBP3 variants

• Quantification across different biological samples:

  • Challenge: Significant variation in detected IGFBP3 levels between sample types

  • Solution: Use sample-specific calibration curves and match matrix composition between standards and samples

  • Up to 19-fold variation has been observed between different ELISA configurations when comparing various biological fluids

• Integration with functional studies:

  • Challenge: Connecting IGFBP3 detection to functional outcomes

  • Solution: Combine antibody-based detection with functional assays:

    • Pair Western blot/ELISA quantification with cell migration/invasion assays

    • Correlate immunohistochemical staining with clinical parameters

    • Epithelial cell expression of IGFBP3 has been shown to correlate negatively with FEV₁% predicted (r = −0.54) in respiratory research

What are the normal reference ranges for IGFBP3 in human biological samples?

Reference ranges for IGFBP3 vary by age, sex, and sample type:

For other biological fluids:

• Bronchoalveolar lavage fluid (healthy subjects): approximately 0.27 (±0.63) ng/ml

• Values can increase dramatically in pathological conditions (e.g., post-allergen challenge)

How do I interpret changes in IGFBP3 levels across different experimental conditions?

When analyzing IGFBP3 level changes:

• Consider biological context:

  • IGFBP3 is regulated by growth hormone and produced primarily in the liver

  • Levels may be altered by hepatic dysfunction

  • Expression can be stimulated by mitogenic growth factors such as Bombesin, Vasopressin, PDGF, and EGF

• Account for IGF system interactions:

  • Changes in IGFBP3 may reflect alterations in IGF-I/IGF-II levels

  • The IGFBP3/IGF-I ratio can be more informative than absolute IGFBP3 levels

  • In diabetic conditions, increased IGFBP3/IGF-I ratio has been observed

• Differentiate regulated expression from proteolytic processing:

  • Increased proteolysis of IGFBP3 may decrease detected levels without affecting expression

  • Different immunoassays may yield variable results based on their ability to detect intact versus fragmented IGFBP3

• Correlate with physiological/pathological parameters:

  • In diabetes research, IGFBP3 levels correlate with metabolic parameters (HbA1c, fasting glucose, insulin sensitivity)

  • In respiratory research, epithelial IGFBP3 expression correlates negatively with lung function (FEV₁%)

How can IGFBP3 antibodies contribute to therapeutic research?

IGFBP3 antibodies enable therapeutic research in several areas:

• Target validation:

  • Confirming IGFBP3 as a therapeutic target through visualization and quantification

  • Determining tissue/cellular distribution to predict potential off-target effects

• Therapeutic monitoring:

  • Measuring IGFBP3 levels during recombinant human growth hormone treatment

  • Assessing IGFBP3 response to experimental therapies

• Therapeutic development:

  • IGFBP3/TMEM219 pathway blockade using ecto-TMEM219 has shown protection of beta cells in vitro and prevention of hyperglycemia in murine diabetes models

  • IGFBP3 antibodies can help characterize such novel therapeutic approaches

• Combination therapy research:

  • Investigating how IGFBP3-targeted therapies interact with established treatments

  • Using antibodies to monitor changes in IGFBP3 expression/distribution during combination therapy

What are the latest methodological innovations in IGFBP3 antibody-based research?

Recent advances include:

• Single-cell analysis techniques:

  • Combining IGFBP3 antibody staining with single-cell transcriptomics

  • Analysis of IGFBP3 colocalization with interaction partners at single-cell resolution

• Improved specificity assays:

  • Development of assays capable of differentiating between various IGFBP3 forms

  • Simultaneous detection of IGFBP3 and binding partners (IGF-I, IGF-II, ALS)

• In vivo imaging approaches:

  • Using fluorescently labeled IGFBP3 antibodies for intravital microscopy

  • Development of IGFBP3-targeted probes for molecular imaging

• Multiplex detection systems:

  • Simultaneous quantification of IGFBP3 alongside other biomarkers

  • Integration of IGFBP3 detection into broader IGF system analysis platforms