SCGB2A2 Antibody

What is SCGB2A2 and what are its key characteristics?

SCGB2A2 (Secretoglobin Family 2A Member 2) is a protein-coding gene that belongs to the secretoglobin superfamily. It encodes Mammaglobin A, a 93-amino acid protein with a calculated molecular weight of approximately 10.5 kDa . Previous HGNC symbols for this gene include MGB1 and PSBP1 . It has highly specific expression in breast cancer cells and has become one of the most specific markers for breast cancer detection and for identifying circulating tumor cells (CTCs) and micrometastases .

Key characteristics of SCGB2A2:

• Gene ID (NCBI): 4250

• UniProtKB/Swiss-Prot: Q13296

• OMIM: 605562

• Subcellular location: Secreted protein

• Predicted molecular mass: 10-13 kDa (though observed between 6-10 kDa in some studies)

• Forms a covalent complex with lipophilin B (SCGB1D2)

How specific is SCGB2A2 expression in breast tissue versus other tissues?

• SCGB2A2 is predominantly expressed in breast tissue, both normal and malignant

• High expression is observed in most breast cancers (approximately 80% sensitivity)

• Low-level expression has been detected in some normal tissues beyond the breast

• Up to 15% of non-breast carcinomas may show focal positivity, including tumors from stomach, lung, colon, hepatobiliary, thyroid, ovarian, and urothelial origins

In a study using multiple tissue northern blots and cancer profiling arrays, researchers found that while SCGB2A2 expression is highest in breast tissue, it is not absolutely breast-specific . Quantitative RT-PCR confirmed expression in the ZR-75.1 breast cancer cell line but not in five other breast cancer cell lines or five non-breast cancer cell lines tested .

What types of SCGB2A2 antibodies are commercially available for research applications?

Various types of SCGB2A2 antibodies are available for research, including:

Each antibody has been validated for specific applications and may have different sensitivities and specificities.

What are the optimal conditions for SCGB2A2 antibody use in immunohistochemistry?

Optimal conditions for SCGB2A2 antibody use in immunohistochemistry include:

Antigen retrieval methods:

• TE buffer pH 9.0 (suggested primary method)

• Alternatively, citrate buffer pH 6.0 can be used

Recommended dilutions:

• Polyclonal antibodies: 1:20-1:200 or 5-20 μg/mL

• Monoclonal antibodies: 1:100-1:400 or 1:200-1:1000

Detection systems:

• HRP-linked secondary antibodies work well with DAB staining

• For human breast cancer tissue samples, an example protocol used:

  • Primary antibody: 10 μg/ml Rabbit Anti-Human SCGB2A2 Antibody

  • Secondary antibody: 2 μg/mL HRP-Linked Caprine Anti-Rabbit IgG Polyclonal Antibody

Positive control tissues:

• Human breast cancer tissue

• Human mammary gland tissue

How can I design a quantitative RT-PCR experiment to accurately measure SCGB2A2 expression?

For quantitative RT-PCR measurement of SCGB2A2 expression, consider the following experimental design:

Primer and probe design:
Use validated primers and probes such as:

RNA extraction and cDNA synthesis:

• Extract total RNA using standard methods

• For each cDNA synthesis, use 1 μg of RNA

• Reverse transcribe using a reliable system (e.g., Superscript II Reverse Transcription System)

Controls:

• Include GAPDH as an internal control for normalization

• Use ZR-75.1 cells as a positive control (known to express SCGB2A2)

• Include negative controls (cell lines known not to express SCGB2A2)

• Consider analyzing both normal and malignant breast tissue samples for comparison

Data analysis:

• Normalize SCGB2A2 expression to the internal control gene

• Use the comparative CT method (2^-ΔΔCT) for relative quantification

• Statistically analyze differences between sample groups

What approaches can I use to validate the specificity of a SCGB2A2 antibody?

To validate the specificity of a SCGB2A2 antibody, implement multiple complementary approaches:

1. Cross-reactivity testing:

• Create dot blots with different concentrations of SCGB2A2 and closely related proteins (e.g., lipophilin B)

• Spot 1 ng, 100 pg, 10 pg, and 1 pg of each cDNA or protein

• Probe with your SCGB2A2 antibody to ensure it only binds to SCGB2A2 and not to related proteins

2. Western blot analysis:

• Use recombinant SCGB2A2 protein as a positive control

• Include lysates from cells known to express SCGB2A2 (e.g., ZR-75.1) and those that don't

• Verify the expected molecular weight (approximately 10-11 kDa)

3. Immunohistochemistry controls:

• Perform IHC on tissues known to express SCGB2A2 (breast cancer tissues)

• Include negative control tissues (non-breast tissues)

• Use blocking peptides to confirm binding specificity

• Perform parallel staining with different SCGB2A2 antibody clones

4. RNA-protein correlation:

• Compare protein detection by your antibody with RNA expression measured by qRT-PCR

• Concordance between protein and RNA levels supports antibody specificity

How can SCGB2A2 antibodies be used to detect circulating tumor cells and micrometastases in breast cancer patients?

SCGB2A2 antibodies have proven valuable for detecting circulating tumor cells (CTCs) and micrometastases in breast cancer patients through several methodological approaches:

Bone marrow micrometastasis detection:
In a study examining bone marrow samples from breast cancer patients, SCGB2A2 (mammaglobin-1) immunostaining demonstrated superior sensitivity compared to traditional methods:

• SCGB2A2 immunostaining detected micrometastases in 16/30 patients (53.3%)

• Standard H&E staining of bone marrow biopsies detected only 5/30 patients (16.7%)

• Bone marrow aspirate analysis detected 0 cases (0%)

This indicates that SCGB2A2 immunostaining can detect micrometastatic cells that would be missed by conventional histological methods.

Protocol considerations:

• Use optimized antigen retrieval methods for bone marrow specimens

• Include appropriate positive and negative controls

• Consider correlation with other markers such as CA15-3 in serum and bone marrow plasma

• Validate positive findings using additional breast cancer-specific markers

Peripheral blood CTC detection:
The mammaglobin A/lipophilin B complex has been observed in the peripheral circulation of breast cancer patients, suggesting its potential as a serological marker for breast cancer . For peripheral blood CTC detection:

• Process samples promptly after collection

• Consider immunomagnetic enrichment of CTCs prior to staining

• Use highly specific antibody clones to avoid false positives

• Implement multi-marker approaches (combining SCGB2A2 with other breast cancer markers)

What is the relationship between SCGB2A2 and lipophilin B (SCGB1D2) in breast cancer?

SCGB2A2 (Mammaglobin A) and lipophilin B (SCGB1D2) have a significant biological relationship in breast cancer:

Physical interaction:

• SCGB2A2 and lipophilin B proteins form a covalent complex in breast tissue

• This complex has been observed in both normal and malignant breast tissues

• The complex can also be detected in the peripheral circulation of breast cancer patients

Co-expression patterns:
Expression analysis using northern blot and cancer profiling arrays revealed:

• SCGB2A2 and lipophilin B are co-expressed in the same breast tumor samples

• Transcripts of approximately 600 bp in size for both genes were expressed with similar intensity in breast cancer samples

• Their co-expression is not restricted to breast tumors but is also present in normal breast tissue

Cell line expression:

• In cell line studies, SCGB2A2 was expressed only in the ZR-75.1 breast cancer cell line

• Lipophilin B was expressed in ZR-75.1 as well as in T-47D (breast cancer) and LnCaP (prostate cancer) cell lines

• This suggests that while often co-expressed, lipophilin B has a broader expression pattern than SCGB2A2

The SCGB2A2/lipophilin B complex may have functional significance in breast cancer biology and potentially serves as a more specific diagnostic marker than either protein alone.

How does SCGB2A2 expression correlate with breast cancer subtypes and clinical outcomes?

Research indicates that SCGB2A2 expression varies across breast cancer subtypes and may have implications for prognosis:

Expression across breast cancer subtypes:

Clinical correlation:

• SCGB2A2 expression has been found to be a marker of tumor progression

• Evidence supports its association with lymph node involvement and distant metastasis

• The presence of SCGB2A2-positive cells in bone marrow correlates with serum and bone marrow plasma CA15-3 levels, which are established markers of tumor burden

Diagnostic utility in combination with other markers:
For optimal diagnostic utility, SCGB2A2 is often used in a panel approach:

• Common panel components include GCDFP-15 and Estrogen receptor alpha

• This multimarker approach improves sensitivity while maintaining specificity for breast origin

More research is needed to fully elucidate the prognostic value of SCGB2A2 expression in different breast cancer molecular subtypes and treatment response prediction.

What are common issues when using SCGB2A2 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with SCGB2A2 antibodies:

Issue 1: Unexpected molecular weight in Western blots

• Expected molecular weight: 10.5-12.2 kDa

• Observed molecular weight: Often between 6-10 kDa

• Resolution: This discrepancy may be due to post-translational modifications or protein processing. Verify using recombinant SCGB2A2 protein as a positive control and consider using gradient gels to better resolve small proteins.

Issue 2: Weak or variable staining in IHC

• Resolution: Optimize antigen retrieval methods (try both pH 9.0 TE buffer and pH 6.0 citrate buffer)

• Test different antibody concentrations (1:20-1:1000 depending on the antibody)

• Increase incubation time or temperature

• Use signal amplification systems if necessary

Issue 3: Cross-reactivity with other secretoglobins

• Resolution: Validate antibody specificity using dot blots with recombinant proteins

• Conduct blocking experiments with specific peptides

• Select antibodies raised against unique epitopes of SCGB2A2 rather than conserved regions of the secretoglobin family

Issue 4: Storage and stability issues

• Resolution: Store antibodies according to manufacturer recommendations (typically at -20°C)

• Aliquot to avoid repeated freeze/thaw cycles

• Add stabilizers like glycerol (50%) for long-term storage

• Check expiration dates and look for visible precipitation before use

Issue 5: Unexpected negative results in known positive samples

• Resolution: Include positive controls in each experiment (breast cancer tissue or ZR-75.1 cells)

• Verify sample quality and fixation/processing methods

• Consider using multiple SCGB2A2 antibody clones recognizing different epitopes

How should I interpret contradictory SCGB2A2 expression data from different detection methods?

When faced with contradictory SCGB2A2 expression data from different detection methods, consider the following interpretation framework:

1. Method-specific considerations:

2. Technical reasons for discrepancies:

• Different antibodies may recognize different epitopes or isoforms

• Post-translational modifications might affect antibody binding

• Fixation methods can impact epitope accessibility in IHC

• Sensitivity thresholds vary between methods

3. Biological explanations:

• Discrepancy between mRNA and protein levels due to post-transcriptional regulation

• Heterogeneous expression within tumors may lead to sampling variability

• Expression may change during disease progression

4. Resolution approaches:

• Validate findings using multiple detection methods

• Use multiple antibodies targeting different epitopes

• Correlate with functional assays

• Consider the biological context when interpreting results

• Use larger sample sizes to account for heterogeneity

What factors should be considered when designing experiments to correlate SCGB2A2 expression with clinical parameters?

When designing experiments to correlate SCGB2A2 expression with clinical parameters, researchers should consider several critical factors:

1. Sample considerations:

• Select appropriate cohorts with sufficient statistical power

• Include diverse breast cancer subtypes (luminal, HER2+, triple-negative)

• Ensure balanced representation of different stages and grades

• Include matched normal breast tissue when possible

• Consider collecting longitudinal samples (pre-treatment, post-treatment, recurrence)

2. Experimental design factors:

• Use validated SCGB2A2 antibodies with established specificity

• Implement standardized staining and scoring protocols

• Consider multiplexed approaches to simultaneously detect SCGB2A2 and other markers

• Include appropriate technical and biological controls

• Use digital pathology tools for quantitative assessment when possible

3. Clinical correlation parameters:

4. Statistical approaches:

• Define appropriate statistical methods before data collection

• Consider multivariate analyses to adjust for confounding factors

• Use stringent criteria for determining significance in multiple comparisons

• Validate findings in independent cohorts when possible

• Calculate sample sizes needed for adequate statistical power

5. Methodological considerations:

• Consider using multiple detection methods (IHC, qRT-PCR, ELISA)

• For circulating markers, standardize sample collection and processing times

• Document pre-analytical variables that might affect results

What are the latest research developments regarding SCGB2A2 as a biomarker for breast cancer?

Recent research developments regarding SCGB2A2 as a breast cancer biomarker include:

Bone marrow micrometastasis detection:
A 2020 study demonstrated that mammaglobin-1 (SCGB2A2) immunostaining in bone marrow biopsies significantly improved detection of micrometastases compared to traditional H&E staining (53.3% vs. 16.7% detection rate) . This provides initial evidence for using SCGB2A2 immunostaining as a tool to investigate early bone marrow micrometastases in breast cancer patients.

Comprehensive tissue expression analysis:
A pioneering study conducted in 2023 examined the tissue expression signature of mammaglobin protein across more than 16,328 samples derived from various malignancies . This large-scale analysis has helped better define the specificity profile of SCGB2A2 across different cancer types.

Circulating tumor cell detection:
The mammaglobin A/lipophilin B complex has been observed in the peripheral circulation of breast cancer patients, indicating its potential utility as a serological marker . This suggests possibilities for liquid biopsy applications.

Diagnostic specificity refinement:
While SCGB2A2 was initially thought to be exclusively expressed in breast tissue, newer research has identified that up to 15% of non-breast carcinomas may show focal positivity . This improved understanding of expression patterns helps researchers better interpret SCGB2A2 findings in diagnostic contexts.

How can SCGB2A2 antibodies be integrated into multiplexed immunoassays for comprehensive cancer profiling?

Integration of SCGB2A2 antibodies into multiplexed immunoassays offers significant advantages for comprehensive cancer profiling:

Multiplexed IHC/IF approaches:

• SCGB2A2 can be combined with other breast cancer markers (GCDFP-15, ER, PR, HER2)

• Use spectrally distinct fluorophores or chromogens for simultaneous detection

• Implement automated multispectral imaging systems for quantitative analysis

• Consider tyramide signal amplification for detecting low-abundance targets

Methodological considerations:

• Validate antibody compatibility in multiplexed formats

• Optimize staining conditions for each antibody in the panel

• Test for potential cross-reactivity between detection systems

• Ensure antibodies are raised in different host species to avoid cross-reactivity

• Consider sequential staining approaches for challenging combinations

Panel design strategies:

• Include SCGB2A2 in panels targeting:

  • Breast cancer subtyping (with ER, PR, HER2, Ki-67)

  • Metastatic site identification (with organ-specific markers)

  • Tumor microenvironment characterization (with immune cell markers)

  • Epithelial-mesenchymal transition assessment (with E-cadherin, vimentin)

Digital pathology integration:

• Implement machine learning algorithms for automated scoring

• Develop spatial analysis tools to assess marker co-localization

• Create standardized reporting formats for multiplexed data

• Integrate with clinicopathological data for comprehensive analysis

Quality control measures:

• Include tissue microarrays with known expression patterns as controls

• Use spike-in controls for assay performance monitoring

• Implement batch correction algorithms for multi-center studies

• Standardize image acquisition parameters

What experimental approaches could advance our understanding of SCGB2A2 function in breast cancer biology?

Despite its established role as a breast cancer marker, the precise function of SCGB2A2 in breast cancer biology remains incompletely understood. Several experimental approaches could advance our understanding:

Functional genomics approaches:

• CRISPR/Cas9-mediated knockout or knockdown of SCGB2A2 in breast cancer cell lines

• Overexpression studies to assess effects on proliferation, migration, and invasion

• Single-cell RNA sequencing to characterize SCGB2A2-expressing cell populations

• Spatial transcriptomics to understand SCGB2A2 expression in the tumor microenvironment

Protein interaction studies:

• Investigate the SCGB2A2/lipophilin B complex formation and function

• Perform co-immunoprecipitation followed by mass spectrometry to identify novel binding partners

• Use proximity ligation assays to verify protein interactions in situ

• Develop structural biology approaches to understand the complex architecture

In vivo models:

• Generate transgenic mouse models with tissue-specific SCGB2A2 expression

• Develop patient-derived xenografts from tumors with varying SCGB2A2 expression levels

• Use in vivo imaging with labeled SCGB2A2 antibodies to track metastatic spread

• Test SCGB2A2-targeted therapies in relevant animal models

Translational approaches:

• Develop SCGB2A2-based liquid biopsy assays for early detection and monitoring

• Explore SCGB2A2 as a target for antibody-drug conjugates

• Investigate SCGB2A2 as a potential immunotherapy target

• Assess SCGB2A2 expression changes in response to standard treatments

Multi-omics integration:

• Correlate SCGB2A2 expression with genomic alterations, methylation patterns, and proteomics data

• Identify potential regulatory mechanisms controlling SCGB2A2 expression

• Map SCGB2A2 into breast cancer molecular pathways

• Develop predictive models incorporating SCGB2A2 expression data