RBP4 Antibody

What is RBP4 and why is it important as a research target?

RBP4 is a member of the lipocalin superfamily primarily secreted by the liver. It plays a crucial role in transporting retinol and vitamin A in the bloodstream. Beyond its transport function, RBP4 has emerged as an important biomarker associated with insulin resistance, cardiovascular disease, metabolic syndrome, and various cancers . Studies have shown that RBP4 can promote migration and proliferation of cancer cells through the activation of the RhoA/Rock1 pathway and expression of CyclinD1 . The expression level of RBP4 protein has been found to be closely related to liver damage and plays a significant role in the diagnosis and prognosis of tumors, making it an important research target .

What types of anti-RBP4 antibodies are available for research applications?

Research-grade anti-RBP4 antibodies typically include:

• Monoclonal antibodies (mAbs): Several classes have been developed, including:

  • IgG-type antibodies: Commonly used for various applications including Western blotting and immunohistochemistry

  • IgA-type antibodies: Particularly useful for enzyme-linked immunosorbent sandwich assays (ELISA)

• Polyclonal antibodies: Useful for detecting native RBP4 protein across multiple species and applications

• Antibodies targeting specific epitopes: Research indicates that epitopes in the first 35 amino acids of mature RBP4 may be particularly important for antibody binding

The selection of antibody type should be based on the specific experimental requirements, with monoclonal antibodies offering higher specificity and reproducibility for most research applications .

How are anti-RBP4 antibodies typically produced for research use?

The production of high-quality anti-RBP4 antibodies typically follows these methodological steps:

• Recombinant protein expression: The RBP4 gene is amplified using RT-PCR from normal human liver cell lines (e.g., HL-7702), inserted into expression vectors (e.g., pET-30a), and expressed in prokaryotic systems such as E. coli BL21 (DE3)

• Protein purification: The recombinant RBP4 is purified using chromatography techniques to ensure high purity before immunization

• Immunization protocol: BALB/c mice are typically immunized with the recombinant RBP4 protein through multiple subcutaneous injections at 14-day intervals. The initial dose is usually 80-100 μg/mouse mixed with Freund's complete adjuvant, followed by subsequent immunizations with Freund's incomplete adjuvant

• Hybridoma technology: Spleen cells from immunized mice are fused with myeloma cells to create hybridomas, which are then screened for antibody production using ELISA

• Clone selection and expansion: Positive hybridoma clones are selected and expanded in cell culture or mice to produce the monoclonal antibody

• Antibody purification: The antibodies are typically purified using affinity chromatography (e.g., rProtein G affinity chromatography columns)

This process ensures the development of high-affinity, specific antibodies suitable for various research applications .

What are the primary research applications for anti-RBP4 antibodies?

Anti-RBP4 antibodies are versatile tools employed in multiple research contexts:

• Immunohistochemical analysis: Used to detect RBP4 expression in tissue sections, particularly useful for studying differential expression between disease states and normal tissues (e.g., hepatocellular carcinoma versus adjacent tissues)

• Western blotting: Employed for detecting RBP4 protein in cell lysates, tissue extracts, and biological fluids, providing information about protein size and relative abundance

• ELISA development: Anti-RBP4 antibodies, particularly IgA monoclonal antibodies, are used to develop sensitive sandwich ELISAs for quantitative measurement of RBP4 in serum and other biological samples

• Double indirect immunofluorescence: Used to localize RBP4 in tissues, as demonstrated in studies of alopecia areata where anti-RBP4 antibodies showed localization in the outer root sheath and companion layer of hair follicles

• Biomarker validation studies: Applied in research validating RBP4 as a biomarker for conditions including insulin resistance, metabolic disorders, and various cancers

• Studying protein-protein interactions: Used to investigate how RBP4 interacts with other proteins in metabolic and signaling pathways

The selection of specific application depends on research objectives, with different antibody formats (monoclonal vs. polyclonal) offering advantages for particular techniques .

How do I optimize immunohistochemical (IHC) protocols using anti-RBP4 antibodies?

Optimizing IHC protocols with anti-RBP4 antibodies requires careful attention to several parameters:

• Tissue fixation and processing:

  • Formalin fixation time should be optimized (typically 24-48 hours)

  • Paraffin embedding should follow standard protocols to preserve RBP4 antigenicity

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective for RBP4

  • Optimization of retrieval time (10-20 minutes) may be necessary for specific tissues

• Antibody dilution optimization:

  • Perform a dilution series (typically starting at 1:100 to 1:1000) to determine optimal signal-to-noise ratio

  • Primary antibody incubation should be conducted overnight at 4°C for best results

• Detection system selection:

  • For low abundance detection, amplification systems like tyramide signal amplification may be beneficial

  • DAB (3,3'-diaminobenzidine) is commonly used as a chromogen for RBP4 visualization

• Control inclusion:

  • Positive controls: Liver tissue is ideal as it exhibits high RBP4 expression

  • Negative controls: Omission of primary antibody and isotype controls

  • RBP4-deficient tissue or knockdown samples where available

• Counterstaining optimization:

  • Light hematoxylin counterstaining typically allows optimal visualization of RBP4 immunoreactivity

Researchers should systematically test these parameters to establish optimal conditions for their specific tissue and research question .

What are the key considerations when developing ELISAs using anti-RBP4 antibodies?

Development of effective ELISA systems for RBP4 detection requires careful consideration of several technical aspects:

• Antibody selection:

  • Use of IgA monoclonal antibodies (such as AG102) has shown superior capture efficiency and specificity compared to traditional IgG antibodies

  • The capture antibody should target stable epitopes of RBP4 that remain accessible in solution

• Protocol optimization:

  • Coating concentration: Typically 0.5-4 μg/mL of capture antibody (optimal determined by checkerboard titration)

  • Sample dilution: Serum samples typically require significant dilution (1:1000 to 1:20,000) due to high RBP4 concentration in serum

  • Detection antibody concentration: Usually 0.1-1 μg/mL for optimal signal-to-noise ratio

• Assay validation:

  • Recovery tests should be performed by spiking known amounts of recombinant RBP4 into samples

  • Cross-reactivity testing with related proteins (other lipocalin family members) is essential

  • Comparison with Western blot quantitation can validate ELISA accuracy (R² values >0.85 indicate good correlation)

• Dynamic range considerations:

  • Commercial RBP4 ELISAs often have limited dynamic range

  • Development of wide dynamic range assays (spanning at least 3 orders of magnitude) is essential for detecting pathological variations in RBP4 levels

• Standardization:

  • Use of well-characterized recombinant RBP4 or RBP4 protein standards (e.g., from R&D Systems) for calibration curves

  • Inclusion of control samples with known RBP4 concentrations in each assay

These considerations are crucial for developing reliable, reproducible ELISA systems for RBP4 quantification in research and potential clinical applications .

How can I validate the specificity of an anti-RBP4 antibody?

Comprehensive validation of anti-RBP4 antibody specificity involves multiple complementary approaches:

• Western blot analysis:

  • Test reactivity against purified recombinant RBP4-His protein and natural RBP4 protein standards

  • Include structurally related proteins as negative controls (e.g., SHBG-His, SAA4-His, NEK2-His) to confirm specificity

  • Test against cell lysates from multiple cell lines with known RBP4 expression levels (e.g., Hep3B, Huh7)

• Cross-reactivity testing:

  • Perform iELISA with the antibody against multiple proteins coated on plates

  • Include BSA and sample diluent as negative controls

  • A specific antibody should show signal only with RBP4 protein

• Immunoprecipitation:

  • Precipitate RBP4 from biological samples and confirm by mass spectrometry

  • This provides definitive identification of the target protein

• Epitope mapping:

  • Serial deletion mutants and internal deletion experiments can identify specific binding regions

  • For RBP4, evidence suggests that important epitopes may be within the first 35 amino acids of the mature protein

• Knockout/knockdown validation:

  • Test antibody against samples from RBP4 knockout models or RBP4-silenced cells

  • The absence of signal in these samples confirms specificity

• Peptide competition assays:

  • Pre-incubate the antibody with excess RBP4 peptide/protein

  • Specific binding should be blocked by this pre-incubation

Thorough validation using multiple approaches ensures reliable results in subsequent experiments using the antibody .

How do I resolve inconsistent results when measuring RBP4 across different assay platforms?

Inconsistencies in RBP4 measurement across different platforms can be addressed through systematic troubleshooting:

• Sample preparation standardization:

  • Standardize collection methods, processing times, and storage conditions

  • Fresh samples typically yield more reliable results than frozen/thawed specimens

  • Document fasting/non-fasting status as this affects RBP4 levels

• Platform-specific considerations:

  • ELISA: Different commercial kits show significant variability; validate against a reference method

  • Western blot: Denaturation conditions affect epitope accessibility

  • Immunohistochemistry: Fixation methods dramatically impact antigen preservation

• Antibody selection strategy:

  • Use the same validated antibody across platforms where possible

  • For sandwich assays, ensure capture and detection antibodies recognize different, non-overlapping epitopes

  • IgA-type antibodies (like AG102) may show superior recovery from complex biological samples compared to IgG-based assays

• Reference material inclusion:

  • Include identical reference samples across all platforms

  • Use both recombinant RBP4 and natural RBP4 standards in parallel

  • Consider creating an internal laboratory standard from pooled samples

• Method comparison analysis:

  • Perform formal method comparison studies with linear regression analysis

  • Calculate correlation coefficients between methods (R² values)

  • Bland-Altman plots help identify systematic biases between methods

• Biological context consideration:

  • RBP4 exists in multiple forms in circulation (holo-RBP4 bound to retinol, apo-RBP4, RBP4-TTR complex)

  • Different assays may detect these forms with varying efficiency

  • Consider what form of RBP4 is relevant to your research question

Systematic evaluation using these approaches can help reconcile discrepancies and select the most appropriate measurement platform for specific research objectives .

What factors should I consider when studying RBP4 as a biomarker in cancer research?

When investigating RBP4 as a cancer biomarker, several critical factors should be considered:

This multifaceted approach ensures robust and clinically relevant insights when studying RBP4 as a cancer biomarker .

What are common troubleshooting strategies for Western blotting with anti-RBP4 antibodies?

When encountering challenges with Western blotting using anti-RBP4 antibodies, consider these methodological solutions:

• No signal or weak signal:

  • Optimize antibody concentration: For anti-RBP4 mAbs, dilutions typically range from 1:1,000 to 1:10,000

  • Extend incubation time: Consider overnight incubation at 4°C

  • Increase protein loading: RBP4 detection may require 30-50 µg of total protein

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Check sample preparation: Inadequate extraction may reduce RBP4 recovery

• High background:

  • Increase blocking time and concentration (5% non-fat milk or BSA)

  • Add 0.05-0.1% Tween-20 to washing and antibody dilution buffers

  • Ensure antibody specificity through appropriate controls

  • Reduce antibody concentration - over-concentrated antibody is a common cause of background

• Multiple bands or unexpected band size:

  • RBP4 molecular weight is approximately 23 kDa

  • Multiple bands may indicate:

    • Post-translational modifications

    • Proteolytic degradation (add protease inhibitors to samples)

    • Non-specific binding (increase stringency of washing)

  • Use recombinant RBP4-His and RBP4 protein standards as positive controls to confirm correct band size

• Inconsistent results between experiments:

  • Standardize sample collection and processing

  • Use consistent gel percentage (12% SDS-PAGE recommended for RBP4)

  • Employ loading controls appropriate for your sample type

  • Consider using automated Western blot systems for better reproducibility

• Membrane optimization:

  • PVDF membranes are generally recommended for RBP4 detection

  • Optimize transfer conditions: 100V for 60-90 minutes in standard Tris-glycine buffer

  • Confirm transfer efficiency with reversible protein stains before immunodetection

These troubleshooting strategies address the most common challenges when working with anti-RBP4 antibodies in Western blotting applications .

How can I improve detection sensitivity when working with low abundance RBP4 samples?

Enhancing detection sensitivity for low abundance RBP4 samples requires specialized methodological approaches:

• Sample enrichment techniques:

  • Immunoprecipitation to concentrate RBP4 from dilute samples

  • Fractionation of samples to remove high-abundance proteins that may mask RBP4 detection

  • Ultrafiltration to concentrate protein from urine or other dilute biological fluids

• Detection system optimization:

  • Use IgA-based detection systems which have demonstrated superior recovery of RBP4 from dilute samples

  • For Western blots, employ highly sensitive ECL substrates (femtogram detection range)

  • Consider fluorescent secondary antibodies with direct laser scanning for quantitative detection

• ELISA enhancement strategies:

  • Implement sandwich ELISA format with optimized antibody pairs

  • Use amplification systems (biotin-streptavidin, tyramide signal amplification)

  • Optimize incubation temperatures and times (extended incubations at 4°C may improve sensitivity)

  • Select high-binding ELISA plates specifically designed for maximum protein binding

• Signal amplification methods:

  • Polymeric detection systems that carry multiple enzyme molecules per binding event

  • Rolling circle amplification for enhanced sensitivity

  • Quantum dot conjugated antibodies for improved signal stability and sensitivity

• Specialized instrumentation:

  • Consider digital ELISA platforms (e.g., Simoa technology) for ultra-sensitive detection

  • Use cooled CCD camera systems for improved signal detection in Western blots

  • Flow cytometry-based bead assays may offer superior sensitivity for some applications

• Assay validation parameters:

  • Establish lower limit of detection (LLOD) and lower limit of quantification (LLOQ)

  • Compare quantitative Western blot results with ELISA measurements to validate findings

  • Include appropriate dilution series of standards covering the expected low range of detection

These approaches can significantly improve detection of RBP4 in challenging samples with low abundance .

What controls should be included when using anti-RBP4 antibodies in research?

A comprehensive control strategy ensures reliable and interpretable results when working with anti-RBP4 antibodies:

• Positive controls:

  • Recombinant RBP4-His protein at known concentrations

  • Commercial RBP4 protein standards (e.g., R&D Systems)

  • Liver tissue or hepatocyte cell lines (high endogenous RBP4 expression)

  • Serum samples with characterized RBP4 levels

• Negative controls:

  • Structurally related proteins to assess specificity:

    • SHBG-His (Sex hormone-binding globulin)

    • SAA4-His (Serum amyloid A-4)

    • NEK2-His (Never in mitosis gene-A related expressed kinase 2)

  • BSA or other unrelated proteins to confirm absence of non-specific binding

  • Sample diluent alone as reagent blank

• Assay validation controls:

  • Dilution linearity series to confirm accurate quantitation across concentration ranges

  • Spike-and-recovery experiments with known amounts of recombinant RBP4

  • Inter-assay and intra-assay calibrators to monitor reproducibility

  • Comparison with established reference methods (mass spectrometry)

• Procedural controls:

  • For IHC: Isotype control antibodies matched to primary antibody class and species

  • For Western blots: Loading controls appropriate to sample type

  • For ELISA: Standard curves with 7-8 points covering the expected range of detection

  • No primary antibody controls to assess secondary antibody non-specific binding

• Biological controls:

  • Samples from multiple individuals/patients to account for biological variation

  • Time course samples to assess stability of RBP4 detection

  • For disease studies: confirmed positive and negative cases with established diagnoses

Implementation of these controls provides a robust framework for validating experimental findings and troubleshooting potential issues in RBP4 antibody-based research .

How can I correlate RBP4 expression with immune cell infiltration in cancer research?

To effectively correlate RBP4 expression with immune cell infiltration in cancer research, implement this methodological workflow:

• Sample collection and processing:

  • Obtain matched tumor and adjacent normal tissues

  • Process for both protein extraction (for RBP4 quantification) and immunophenotyping

  • Consider tissue microarrays for high-throughput analysis

• RBP4 expression analysis:

  • Quantify RBP4 expression using validated immunohistochemistry protocols

  • Alternative approaches include RT-qPCR for mRNA expression and Western blotting

  • Score expression using standardized methods (H-score, Allred score)

• Immune cell characterization:

  • Implement multiplex immunohistochemistry to simultaneously detect RBP4 and immune cell markers

  • Flow cytometry of dissociated tumor samples for detailed immune profiling

  • Consider spatial transcriptomics for localized expression patterns

• Bioinformatic approaches:

  • Use computational tools like "xCell" to analyze the relationship between RBP4 expression and immune cell types

  • Correlation analysis between RBP4 expression and 37 types of immune cells across cancer types

  • Utilize R packages like "ggplot2", "ggpubr", and "corrplot" for visualization

• Integration with clinical data:

  • Correlate findings with patient outcomes (survival, treatment response)

  • Use multivariate analysis to control for confounding variables

  • Kaplan-Meier survival analyses with log-rank tests to assess prognostic significance

• Checkpoint correlation analysis:

  • Perform co-expression analysis between RBP4 and immune checkpoint genes

  • Visualize results using established bioinformatics platforms

  • This may reveal potential therapeutic implications for immunotherapy

This integrated approach provides a comprehensive analysis of how RBP4 expression relates to the immune microenvironment in cancer, potentially revealing new therapeutic targets or prognostic indicators .

How can DNA methylation analysis enhance understanding of RBP4 expression patterns?

DNA methylation analysis provides crucial insights into epigenetic regulation of RBP4 expression:

• Methodological approach:

  • Utilize bisulfite conversion methods to identify methylated cytosines in CpG islands

  • Perform methylation-specific PCR or pyrosequencing for targeted analysis of the RBP4 promoter

  • The UCSC Xena database can be searched to explore DNA methylation levels of the RBP4 promoter in specific cancers

• Technical implementation:

  • Annotate methylation chip data using the R package "biomaRt"

  • Analyze the distribution of methylation probes across RBP4-related chromosomal regions

  • Compare methylation patterns between tumor and normal tissues to identify disease-specific changes

• Correlation analysis:

  • Perform Spearman's correlation test to evaluate the association between:

    • RBP4 expression and DNA methylation levels

    • Methylation status and clinical characteristics

    • Changes in methylation and disease progression

• Integrated multi-omics analysis:

  • Combine methylation data with:

    • Transcriptomic data (RNA-seq)

    • Genomic information (mutations, copy number variations)

    • Proteomic data (RBP4 protein expression)

  • This integration provides a comprehensive view of RBP4 regulation mechanisms

• Epigenetic therapy implications:

  • Identify potential targets for demethylating agents

  • Assess how altered methylation impacts RBP4 expression in different diseases

  • Evaluate whether epigenetic modifications of RBP4 could serve as therapeutic targets

• Experimental validation:

  • Treat cell lines with demethylating agents (e.g., 5-azacytidine) to confirm methylation-dependent regulation

  • Perform reporter assays with methylated and unmethylated RBP4 promoter constructs

  • Use CRISPR-based epigenetic editing to directly modify methylation at specific sites

This comprehensive approach to methylation analysis provides deeper insights into the epigenetic mechanisms controlling RBP4 expression, which may reveal new diagnostic biomarkers or therapeutic targets .

What methodological approaches are recommended for studying RBP4 in metabolic disorders?

For investigating RBP4 in metabolic disorders, implement these specialized methodological approaches:

• Patient cohort design:

  • Stratify subjects based on metabolic parameters (insulin sensitivity, HOMA-IR, glucose tolerance)

  • Include appropriate matching for age, sex, BMI, and comorbidities

  • Collect comprehensive clinical data including anthropometrics, blood pressure, and lipid profiles

• Sample collection and processing:

  • Standardize fasting conditions (8-12 hours) prior to blood collection

  • Process samples consistently with minimal delay to prevent degradation

  • Consider collecting both serum and plasma to account for matrix-specific effects

• RBP4 quantification strategies:

  • Implement IgA-based ELISA systems which have demonstrated superior performance for serum RBP4 quantification

  • Consider Western blotting with quantitative analysis as a complementary approach

  • Mass spectrometry for absolute quantification and identification of RBP4 isoforms

• Functional assessments:

  • Correlate RBP4 levels with:

    • Glucose tolerance tests (OGTT, IVGTT)

    • Hyperinsulinemic-euglycemic clamp studies (gold standard for insulin sensitivity)

    • Measures of adiposity (DXA, MRI) and fat distribution

• Multivariate analysis:

  • Account for potential confounders including:

    • Kidney function (RBP4 is influenced by renal clearance)

    • Vitamin A status (affects RBP4-retinol complex formation)

    • Inflammatory markers (CRP, IL-6) which may alter RBP4 metabolism

• Integrative approaches:

  • Assess RBP4 in conjunction with other adipokines (adiponectin, leptin)

  • Investigate related molecular pathways using transcriptomics or proteomics

  • Consider tissue-specific expression in liver and adipose biopsies when feasible

• Longitudinal design elements:

  • Measure RBP4 changes in response to interventions (weight loss, exercise, medications)

  • Assess predictive value for disease progression

  • Evaluate treatment responses in relation to baseline RBP4 levels

These methodological considerations ensure robust and clinically relevant investigation of RBP4 in metabolic disorders, accounting for the complex nature of metabolic disease pathophysiology .