REG4 Antibody

What is REG4 and why is it significant in biomedical research?

REG4 (regenerating islet-derived family member 4) is a secreted protein of approximately 18.2 kDa that plays crucial roles in tissue regeneration and inflammatory responses in digestive organs. It's also known as GISP, REG-IV, RELP, or regenerating islet-derived protein 4. REG4 functions as a calcium-independent lectin with mannose-binding specificity that maintains carbohydrate recognition activity in acidic environments .

The significance of REG4 in research stems from its:

• Overexpression in various gastrointestinal cancers, particularly pancreatic ductal adenocarcinoma (PDAC)

• Potential as a serum biomarker for early cancer detection

• Role in promoting cancer cell growth, invasion, and anti-apoptotic functions

• Function in tissue regeneration and inflammatory responses in digestive organs

What types of REG4 antibodies are available for research purposes?

Several types of REG4 antibodies are available for research applications:

Most commercially available antibodies target human REG4, though some cross-react with mouse and rat orthologs based on sequence homology. Both recombinant and conventionally-derived antibodies are available, with applications spanning detection (WB, IHC, ELISA) and functional studies (neutralization) .

How should REG4 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of REG4 antibody activity:

• Store at -20°C for long-term storage (typically stable for one year after shipment)

• For reconstituted antibodies, store at 2-8°C for up to one month under sterile conditions

• For extended storage after reconstitution, store at -20 to -70°C for up to 6 months under sterile conditions

• Avoid repeated freeze-thaw cycles by aliquoting antibodies before freezing

• Most REG4 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some formulations (especially low-volume preparations) may contain 0.1% BSA

These storage conditions apply to most commercial REG4 antibodies, but always verify with manufacturer-specific recommendations.

What are the most validated applications for REG4 antibodies in cancer research?

REG4 antibodies have been extensively validated for several key applications in cancer research:

Western Blot (WB):

• Typical dilutions range from 1:500-1:2000

• Detects REG4 at approximately 18 kDa

• Successfully used with cell lines including SGC-7901 and pancreatic cancer cell lines

• Both cell lysates and concentrated conditioned media can be analyzed

Immunohistochemistry (IHC):

• Recommended dilutions of 1:50-1:500

• Heat-mediated antigen retrieval with citrate buffer (pH 6.0) or TE buffer (pH 9.0)

• Successfully used on FFPE tissue sections of human pancreatic, colon, duodenum, and rectum tissues

• Distinct staining patterns observed in goblet cell-like vesicles or at cell surfaces of cancer cells

ELISA:

• Successfully used in sandwich ELISA format combining monoclonal capture and polyclonal detection antibodies

• Effective for detecting REG4 in human serum and plasma samples

• Has been used for clinical studies assessing REG4 as a potential biomarker

Functional Studies:

• Neutralization of REG4 activity in cell culture

• Inhibition of REG4-induced cell proliferation and invasion

• Assessment of downstream signaling affected by REG4 neutralization

How can I establish a reliable sandwich ELISA for detecting REG4 in patient samples?

To establish a reliable sandwich ELISA for REG4 detection in patient samples:

Materials and Reagents:

• Anti-REG4 monoclonal antibody (for capture)

• Biotinylated anti-REG4 polyclonal antibody (for detection)

• Recombinant human REG4 protein (for standard curve)

• 96-well immuno-module microplate

• HRP-labeled streptavidin

• TMB substrate solution

• Sulfuric acid (0.18 M) as stop solution

Protocol:

• Coat a 96-well microplate with monoclonal antibody to REG4 by incubating overnight at 4°C

• Block for 2 hours at room temperature

• Dilute serum samples 5-fold and add to the plate along with standards

• Incubate with biotinylated anti-REG4 polyclonal antibody for 2 hours

• Wash five times

• Add HRP-labeled streptavidin (approximately 8000-fold diluted) and incubate for 1 hour

• Wash five times

• Add TMB substrate solution and allow to react for 15 minutes

• Stop reaction with 100 μL of 0.18 M sulfuric acid

• Measure color intensity at 450 nm with a reference wavelength of 620 nm

This protocol is based on published methods that successfully detected REG4 in serum of patients with early-stage PDAC .

What control samples should be included when validating a new REG4 antibody?

When validating a new REG4 antibody, include the following controls:

Positive Controls:

• Cell lines known to express REG4 (e.g., SUIT-2, SGC-7901 for Western blot)

• Tissues with documented REG4 expression (e.g., colon cancer tissue, duodenum, rectum for IHC)

• Recombinant human REG4 protein

• Conditioned media from REG4-expressing cell lines (concentrated)

Negative Controls:

• Cell lines with minimal or no REG4 expression (e.g., MIAPaCa-2 for functional studies)

• Normal pancreatic tissue (shows faint or no staining in ductal cells)

• Tissues from vital organs not expressing REG4 (heart, lung, kidney, brain)

• Samples treated with REG4 siRNA to knockdown expression

Technical Controls:

• Secondary antibody-only control to assess non-specific binding

• Isotype control antibody (same host species and isotype)

• Blocking peptide competition to confirm specificity

• Cross-reactivity testing with related REG family proteins (REG1, etc.)

How can REG4 antibodies be utilized to investigate the role of REG4 in cancer progression and invasion?

REG4 antibodies can be employed in multiple sophisticated approaches to investigate its role in cancer progression:

Neutralization Studies:

• Treat cancer cell lines expressing endogenous REG4 with neutralizing antibodies (e.g., clone 34-1) to block REG4 function

• Use recombinant human REG4 (rhREG4) with and without neutralizing antibodies to confirm specificity

• Assess effects on proliferation, invasion, and migration using appropriate assays

• Compare with REG4 siRNA knockdown to validate antibody specificity

Signaling Pathway Analysis:

• Use REG4 antibodies to immunoprecipitate native REG4 from conditioned media

• Treat cells with rhREG4 and/or neutralizing antibodies and analyze downstream signaling

• Examine specific phosphorylation events (Akt at Thr308 and Ser473, EGFR at Tyr992 and Tyr1068)

• Assess expression changes in target genes (Bcl-xL, Bcl-2, survivin, MMP-7, MMP-9)

Invasion and Migration Assays:

• Perform Transwell invasion assays comparing:

  • Control conditions

  • Treatment with rhREG4

  • Treatment with conditioned media (CM)

  • Treatment with CM plus REG4 antibody

  • Treatment with CM from REG4 siRNA-transfected cells

Research has demonstrated that REG4 antibody treatment significantly reduces invasive potential of pancreatic cancer cells, with similar effects observed for cell migration .

What experimental approaches can determine whether an anti-REG4 antibody has neutralizing activity?

To determine if an anti-REG4 antibody possesses neutralizing activity:

Binding Affinity Assessment:

• Perform immunoprecipitation using cell culture medium to evaluate the antibody's ability to bind endogenous REG4

• Compare multiple antibody clones to identify those with highest binding affinity

Growth Neutralization Assay:

• Culture REG4-negative cell lines (e.g., PK-45P)

• Add recombinant human REG4 (rhREG4) to stimulate growth

• Add potential neutralizing antibodies at various concentrations

• Measure cell proliferation (e.g., MTT assay) at 24, 48, and 72 hours

• Include control antibodies without neutralizing activity

• Compare growth rates across conditions

Endogenous REG4 Neutralization:

• Culture cell lines expressing high levels of endogenous REG4 (e.g., SUIT-2)

• Treat with varying concentrations of anti-REG4 antibody

• Measure cell growth over time

• Include REG4-negative cell lines (e.g., MIAPaCa-2) as controls

Signaling Pathway Verification:

• Treat cells with rhREG4 to induce Akt phosphorylation

• Add neutralizing antibody

• Assess Akt phosphorylation by Western blot

• Reduction in phosphorylation confirms neutralizing activity

Studies have shown that specific anti-REG4 monoclonal antibodies (e.g., clone 34-1) can completely offset the growth-promoting effects of rhREG4 treatment and suppress Akt phosphorylation, confirming their neutralizing activity .

How can REG4 antibodies be employed to evaluate the relationship between REG4 expression and response to chemotherapy?

To investigate the relationship between REG4 expression and chemotherapy response:

Expression Analysis in Patient Samples:

• Use anti-REG4 antibodies for IHC on tumor tissues from patients before and after chemotherapy

• Correlate REG4 expression levels with treatment response and survival outcomes

• Develop scoring systems based on staining intensity and percentage of positive cells

In Vitro Chemosensitivity Studies:

• Establish cell line panels with varying REG4 expression levels:

  • Parental cells

  • REG4 knockdown cells (siRNA)

  • REG4 overexpressing cells

  • Cells treated with neutralizing REG4 antibody

• Treat cells with chemotherapeutic agents (e.g., 5-FU, gemcitabine)

• Assess:

  • Cell viability

  • Apoptosis markers

  • Changes in Akt signaling pathway components

Mechanistic Investigations:

• Analyze REG4's effect on anti-apoptotic proteins (Bcl-2, Bcl-xL, survivin) in the context of chemotherapy

• Examine changes in EGFR and Akt signaling pathways when combining REG4 antibody treatment with chemotherapy

• Assess whether REG4 neutralization enhances chemotherapy-induced apoptosis

Research has shown that REG4 antibody treatment can significantly enhance chemosensitivity of cancer cells to 5-FU, and REG4 silencing causes loss of chemoresistance, suggesting a potential combination therapy approach .

What are the most common technical challenges when using REG4 antibodies for immunohistochemistry?

Common challenges and solutions when using REG4 antibodies for IHC include:

Antigen Retrieval Optimization:

• Challenge: Inadequate exposure of epitopes leading to weak staining

• Solution: Compare different retrieval methods; published protocols recommend heat-mediated retrieval with either:

  • Citrate buffer (pH 6.0)

  • TE buffer (pH 9.0)

Signal Specificity:

• Challenge: Non-specific background staining

• Solutions:

  • Optimize primary antibody dilution (typically 1:50-1:500)

  • Extend blocking step (10% normal goat serum for 15+ minutes)

  • Include appropriate negative controls (normal tissues, isotype controls)

  • Use antigen competition controls

Differential Expression Patterns:

• Challenge: Interpreting variable staining patterns across tissue types

• Solution: Be aware that REG4 has been observed in:

  • Goblet cell-like vesicles in PDAC

  • Cell surface staining in some cancer cells

  • Mucus of goblet cells in duodenum

  • Cytoplasm of glandular cells in rectum

  • Each pattern may have different biological significance

Tissue-Specific Considerations:

• Challenge: Varying fixation requirements for different tissues

• Solution: Optimize fixation time and conditions for each tissue type; published protocols typically use formalin-fixed, paraffin-embedded tissues with 3 min autoclaving in citrate buffer

When attempting to determine REG4 levels in serum samples, what factors might affect assay performance?

Several factors can impact REG4 serum assay performance:

Pre-analytical Variables:

• Sample collection method (serum vs. plasma)

• Storage conditions and freeze-thaw cycles

• Processing time between collection and analysis

• Patient fasting status

Assay Design Considerations:

• Antibody pair selection is critical - research shows optimal results using a mouse monoclonal capture antibody with a biotinylated rabbit polyclonal detection antibody

• Sample dilution factor (typically 5-fold for serum samples)

• Incubation times and temperatures (2-hour incubation at room temperature is commonly used)

• Washing efficiency between steps

Analytical Challenges:

• Hook effect at high REG4 concentrations

• Matrix effects from serum proteins

• Cross-reactivity with other REG family members

• Interfering heterophilic antibodies

Data Interpretation:

• Establish appropriate reference ranges for various clinical contexts

• Account for demographic factors (age, sex)

• Consider comorbidities that might affect REG4 levels

• Evaluate along with other biomarkers for improved diagnostic accuracy

Research has successfully used sandwich ELISA to detect significant REG4 elevation in serum of patients with early-stage PDAC, demonstrating its potential as a diagnostic biomarker when these factors are properly controlled .

How should discrepancies between REG4 protein detection and mRNA expression be investigated and resolved?

When facing discrepancies between REG4 protein and mRNA levels:

Technical Validation:

• Verify antibody specificity using multiple approaches:

  • Western blot with recombinant REG4 protein

  • Testing in cell lines with confirmed high and low expression

  • Knockdown studies using REG4 siRNA

  • Comparison of multiple antibodies targeting different epitopes

• Validate mRNA detection methods:

  • Use multiple primer sets targeting different regions

  • Compare different RT-PCR methodologies

  • Consider digital PCR for absolute quantification

Biological Explanations to Investigate:

Cell Type-Specific Regulation:

• REG4 is often secreted by cells, so analyze both cell lysates and concentrated conditioned media

• Consider that REG4 can be produced by one cell type and act on another in a paracrine manner

• Perform immunohistochemistry to identify specific cell types expressing REG4 within heterogeneous samples

Research has shown that REG4 protein is often detected in goblet cell-like vesicles and at cell surfaces, suggesting active secretion processes that might explain discrepancies between cellular mRNA and protein levels .

What emerging research areas might benefit from REG4 antibody applications beyond cancer studies?

Several promising research areas beyond cancer could benefit from REG4 antibody applications:

Inflammatory Bowel Disease (IBD):

• REG4 serves as a protective factor in intestinal inflammation

• Antibodies could help monitor REG4 expression during disease progression and treatment response

• Potential to develop prognostic markers for IBD complications

Tissue Regeneration and Stem Cell Research:

• REG4 has been used as a marker for unique populations of secretory progenitor cells in intestinal stem cell-derived organoid cultures

• Antibodies can help track differentiation in intestinal organoids

• Applications in studying epithelial regeneration after injury

Gastrointestinal Infection Response:

• Investigate REG4's role in response to bacterial or viral challenges

• Monitor epithelial protective mechanisms during infection

• Study REG4 as part of the innate immune response

Diabetes and Pancreatic Islet Biology:

• Examine REG4's relationship to other REG family members known to affect islet regeneration

• Investigate potential protective roles in beta cell function

• Explore connections between inflammation, islet function and REG4 expression

Diagnostic Development:

• Multi-marker panels including REG4 for early disease detection

• Point-of-care testing applications

• Companion diagnostics for treatments targeting REG4-dependent pathways

These applications leverage REG4's roles in tissue protection, regeneration during damage, and inflammatory responses beyond its established functions in cancer biology .

How might computational approaches and machine learning enhance the interpretation of REG4 antibody-based imaging results?

Computational approaches and machine learning can revolutionize REG4 antibody-based imaging analysis:

Automated Image Analysis:

• Develop algorithms to quantify REG4 staining intensity and distribution patterns

• Machine learning models to classify different staining patterns (goblet cell-like vesicles, cell surface, cytoplasmic)

• Deep learning networks trained to recognize subtle expression differences between normal, precancerous, and malignant tissues

Predictive Modeling:

• Correlate REG4 expression patterns with clinical outcomes

• Generate predictive models for treatment response based on REG4 distribution

• Identify novel morphological features associated with REG4 expression that human observers might miss

Multiparameter Analysis:

• Integrate REG4 expression data with other biomarkers in multiplexed immunofluorescence

• Create comprehensive tissue "fingerprints" combining REG4 with markers of proliferation, apoptosis, and invasion

• Identify specific cellular niches and microenvironmental features associated with REG4 expression

Digital Pathology Integration:

• Whole-slide imaging analysis of REG4 expression across entire tissue sections

• Spatial analysis of REG4 distribution relative to tissue architecture

• Correlation of REG4 with tumor invasion fronts and metastatic potential

These computational approaches could significantly enhance the diagnostic and prognostic value of REG4 antibody-based imaging beyond what is possible with traditional pathological assessment .

What experimental designs would be most effective for investigating the therapeutic potential of anti-REG4 neutralizing antibodies?

To investigate the therapeutic potential of anti-REG4 neutralizing antibodies, consider these experimental designs:

In Vitro Studies:

• Antibody Characterization:

  • Screen multiple antibody clones for neutralizing activity

  • Determine specificity, affinity, and epitope mapping

  • Assess ability to block REG4-induced signaling (Akt/EGFR pathways)

• Functional Assays:

  • Measure effects on cell proliferation, invasion, and migration

  • Combine with chemotherapeutic agents to assess synergistic potential

  • Evaluate impact on apoptosis markers and resistance mechanisms

In Vivo Preclinical Models:

• Xenograft Studies:

  • Establish xenografts using cell lines with varying REG4 expression

  • Treat with anti-REG4 antibodies alone or in combination with standard therapies

  • Monitor tumor growth, metastasis, and survival

  • Include pharmacokinetic and biodistribution analyses

• Patient-Derived Xenograft (PDX) Models:

  • Select PDX models with confirmed REG4 expression

  • Test antibody efficacy across a spectrum of patient-derived tumors

  • Correlate response with REG4 expression levels

Combination Therapy Evaluation:

• Test combinations with:

  • Conventional chemotherapy (5-FU, gemcitabine)

  • Targeted therapies (EGFR inhibitors)

  • Immunotherapies

• Identify optimal sequencing and dosing regimens

Antibody Optimization:

• Compare various antibody formats (full IgG, Fab, scFv)

• Evaluate potential for antibody-drug conjugates

• Explore bispecific antibodies targeting REG4 and other relevant targets

These designs build on existing research showing that anti-REG4 monoclonal antibodies can neutralize REG4's growth-promoting effects, inhibit invasion, and enhance chemosensitivity of cancer cells .

What quality control measures should be implemented when validating REG4 antibodies for critical research applications?

Comprehensive quality control for REG4 antibodies should include:

Antibody Characterization:

• Determine specificity via Western blot against recombinant REG4 and related family members

• Verify recognition of native vs. denatured protein

• Confirm epitope location and accessibility

• Assess lot-to-lot consistency with standardized assays

Application-Specific Validation:

• For WB: Verify expected molecular weight (18 kDa) and band pattern

• For IHC: Confirm specific staining pattern in known positive tissues

• For ELISA: Establish detection limits, linear range, and precision

• For neutralization: Quantify dose-dependent inhibition of REG4 function

Control Samples Matrix:

Documentation Requirements:

• Record complete validation methods

• Document lot numbers and specific conditions

• Maintain detailed antibody characterization data

• Create standardized protocols for each application

How can researchers distinguish between technical artifacts and true biological variations when interpreting REG4 staining patterns?

To distinguish between technical artifacts and true biological variations in REG4 staining:

Systematic Controls and Replication:

• Include positive and negative tissue controls in every staining run

• Run technical replicates using the same protocol

• Test biological replicates to confirm patterns

• Compare results using different antibody clones targeting different epitopes

Characteristic REG4 Staining Patterns to Recognize:

• Authentic REG4 patterns documented in literature include:

  • Goblet cell-like vesicles in PDAC

  • Cell surface staining in cancer cells

  • Cytoplasmic staining in glandular cells

  • Mucus of goblet cells in duodenum

Common Artifact Patterns to Recognize:

• Edge artifacts (staining only at tissue margins)

• Nuclear staining (REG4 is primarily cytoplasmic/secreted)

• Uniform staining across all tissue types (suggests non-specific binding)

• Precipitate artifacts (granular, irregular distribution not associated with cellular structures)

Validation Approaches:

• Correlative Analysis:

  • Compare antibody staining with mRNA expression in the same samples

  • Verify REG4 protein expression using alternative methods (Western blot)

  • Confirm secretion by analyzing conditioned media

• Functional Correlation:

  • Associate staining patterns with known REG4 biological activities

  • Correlate with downstream signaling markers (phospho-Akt)

  • Relate to clinical outcomes or phenotypes

• Cross-validation:

  • Compare results across different fixation and preparation methods

  • Use orthogonal detection methods (IF vs. IHC)

  • Verify with other members of the research community

Research has established that true REG4 expression is often associated with well-differentiated PDAC (G1) more frequently than less-differentiated PDAC (G2, G3, G4), providing a biological correlation that can help distinguish true signal from artifacts .