CPK19 Antibody

What is Cytokeratin 19 and what is its molecular structure?

Cytokeratin 19 is a 40kDa polypeptide that forms part of the intermediate filament proteins found in epithelial cells. The CK19 antibody specifically recognizes the rod domain of human cytokeratin-19, with the epitope typically mapping between amino acids 312-335. It is structurally characterized as one of the smaller and acidic type I keratins, lacking the carboxy-terminal tail domain common to other cytokeratins. This protein plays a crucial role in maintaining cellular structural integrity and is encoded by the KRT19 gene located on chromosome 17q21.2 .

What cellular and tissue distribution patterns are characteristic of CK19?

Cytokeratin 19 demonstrates a distinctive expression pattern across multiple epithelial tissues. It is abundantly expressed in sweat glands, mammary gland ductal and secretory cells, bile ducts, gastrointestinal tract epithelium, bladder urothelium, oral epithelia, esophagus, and ectocervical epithelium. Notably, CK19 is largely absent in hepatocytes, creating an important differential marker for identifying non-hepatic epithelial cells within liver tissue. This distribution pattern makes CK19 antibodies particularly valuable in distinguishing primary hepatocellular carcinomas from metastatic adenocarcinomas in liver specimens .

How are CK19 antibodies produced and validated for research applications?

CK19 antibodies are typically produced through hybridoma technology or recombinant methods using specific immunogens derived from human CK19 protein. Validation protocols involve demonstrating reactivity against well-characterized cell lines known to express CK19, including MCF-7 (breast cancer), HT-29 (colon adenocarcinoma), DU145 (prostate carcinoma), and HCT-116 (colorectal carcinoma). Western blot analysis confirming detection of a single band at approximately 40kDa provides verification of antibody specificity. Additional validation steps include immunohistochemical staining of known positive and negative tissue controls, with confirmation of expected subcellular localization patterns .

How is CK19 antibody utilized in cancer research and diagnostics?

CK19 antibody serves as a critical tool in cancer research with multiple applications:

The antibody is particularly valuable for identifying disseminated tumor cells in lymph nodes, peripheral blood, and bone marrow, enabling more accurate staging and prognostication in various epithelial malignancies .

What are the methodological considerations for optimizing CK19 immunohistochemistry?

Successful immunohistochemical detection of CK19 requires careful optimization of several parameters:

• Fixation: Standard 10% neutral buffered formalin for 24-48 hours yields optimal results; excessive fixation can mask epitopes

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15-20 minutes at 95-100°C typically provides robust staining

• Antibody dilution: Titration experiments should determine optimal concentration for each specific application

• Detection systems: Polymer-based detection systems generally offer superior sensitivity compared to avidin-biotin methods

• Counterstaining: Light hematoxylin counterstaining prevents obscuring of cytoplasmic CK19 signal

Comparative studies have shown that clone selection significantly impacts staining characteristics, with some clones demonstrating superior sensitivity for detecting low-level CK19 expression in challenging specimens .

What are the current limitations in CK19 antibody research applications?

Despite its utility, researchers should be aware of several limitations when working with CK19 antibodies:

• Heterogeneous expression: CK19 expression can vary within tumors, potentially leading to false-negative results in small biopsy specimens

• Potential cross-reactivity: Some antibody clones may cross-react with other cytokeratins, particularly in poorly preserved specimens

• Technical variability: Pre-analytical variables including fixation time, processing protocols, and storage conditions significantly impact staining results

• Interpretation challenges: Distinguishing weak specific staining from background can be problematic, particularly in tissues with high endogenous biotin

Addressing these limitations requires rigorous validation, inclusion of appropriate controls, and correlation with other epithelial markers .

What controls should be implemented when designing experiments with CK19 antibody?

A robust experimental design for CK19 antibody applications should incorporate multiple control types:

• Positive tissue controls: Include tissues with known CK19 expression (e.g., breast ductal epithelium)

• Negative tissue controls: Include tissues known to lack CK19 (e.g., normal hepatocytes, lymphoid tissue)

• Technical negative controls: Omit primary antibody to assess background staining

• Isotype controls: Use matched isotype antibodies to evaluate non-specific binding

• Absorption controls: Pre-absorb antibody with recombinant CK19 protein to confirm specificity

• Cell line controls: Include well-characterized CK19-positive (MCF-7) and CK19-negative cell lines

These controls help distinguish true positive signals from artifacts and ensure reproducibility across experiments .

How can researchers optimize Western blot protocols for CK19 detection?

Western blot detection of CK19 requires specific optimization strategies:

• Sample preparation: Use RIPA or NP-40 buffers containing protease inhibitors; avoid harsh detergents that may disrupt epitope structure

• Protein loading: Load 20-30μg total protein per lane for cell lines; higher amounts may be needed for tissue samples

• Gel percentage: 10-12% SDS-PAGE gels provide optimal resolution for the 40kDa CK19 protein

• Transfer conditions: Semi-dry transfer at 15V for 45 minutes or wet transfer at 100V for 1 hour in Tris-glycine buffer

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature minimizes background

• Antibody dilution: Typically 1:1000-1:2000 for primary antibody incubation overnight at 4°C

• Detection: HRP-conjugated secondary antibodies with enhanced chemiluminescence provide sensitive detection

Researchers should validate these parameters for their specific experimental conditions .

What approaches can address data inconsistencies when working with CK19 antibodies?

When confronted with inconsistent CK19 antibody results, researchers should systematically investigate:

• Antibody batch effects: Different lots may show variability; maintain consistent lot numbers for critical experiments

• Sample processing variables: Standardize fixation times, processing protocols, and storage conditions

• Epitope masking: Evaluate alternative antigen retrieval methods if standard protocols yield weak staining

• Clone selection: Compare multiple CK19 antibody clones if results with one clone are suboptimal

• Multiplexing: Incorporate additional epithelial markers (e.g., EpCAM, pan-cytokeratin) to corroborate CK19 findings

• Quantitative validation: Confirm immunohistochemistry results with quantitative methods like RT-PCR for CK19 mRNA

This systematic approach helps identify sources of variability and establish more reproducible protocols .

How is CK19 antibody being utilized in circulating tumor cell research?

CK19 antibody has become instrumental in circulating tumor cell (CTC) research:

• CTC enrichment: Anti-CK19 antibodies coupled to magnetic beads allow positive selection of epithelial CTCs from blood samples

• CTC identification: Immunofluorescence detection of CK19 helps distinguish CTCs from leukocytes in microfluidic devices

• Molecular characterization: Combining CK19 detection with other molecular markers enables phenotypic characterization of CTCs

• Prognostic applications: Quantification of CK19-positive CTCs provides valuable prognostic information in breast, colorectal, and lung cancers

• Treatment monitoring: Serial assessment of CK19-positive CTCs can serve as a surrogate marker for treatment response

The high specificity of CK19 for epithelial cells makes it particularly valuable for identifying rare CTCs in peripheral blood samples .

What is the significance of CK19 fragmentation in research applications?

Research has revealed important implications of CK19 fragmentation:

• CYFRA 21-1: The soluble fragment of CK19 (CYFRA 21-1) serves as a serum tumor marker in lung, breast, and gastrointestinal cancers

• Fragment detection: Specific antibodies recognizing CK19 fragments can complement intact CK19 detection in liquid biopsies

• Apoptosis marker: CK19 fragmentation correlates with apoptotic processes in certain epithelial tumors

• Enzyme activity: Patterns of CK19 cleavage reflect the activity of specific proteases in the tumor microenvironment

• Assay development: Understanding fragmentation patterns has enabled the development of sensitive ELISA assays for detecting CK19 fragments in serum

These applications extend the utility of CK19 beyond conventional tissue-based detection to liquid biopsy applications .

How do CK19 expression patterns correlate with tumor molecular subtypes?

Emerging research has identified important correlations between CK19 expression and molecular subtypes:

These correlations provide additional layers of diagnostic and prognostic information beyond simple presence/absence of CK19 expression .

What are common technical challenges with CK19 immunohistochemistry and their solutions?

Researchers frequently encounter several technical challenges when performing CK19 immunohistochemistry:

Systematic optimization addressing these challenges significantly improves staining quality and reproducibility .

How should researchers interpret equivocal CK19 immunostaining results?

Interpretation of borderline CK19 staining patterns requires careful consideration:

• Quantitative thresholds: Establish clear criteria for positivity (e.g., >5% of cells showing intensity above background)

• Pattern analysis: Distinguish cytoplasmic (specific) from membranous or nuclear (potentially non-specific) staining

• Comparative evaluation: Assess multiple fields across the specimen to account for heterogeneity

• Sequential sections: Examine sequential sections stained with alternative epithelial markers

• Complementary methods: Confirm equivocal results with RT-PCR or other molecular methods

• Blinded review: Implement independent evaluation by multiple observers

This systematic approach reduces subjectivity and increases confidence in borderline results .

What quality control measures ensure reliable CK19 antibody performance over time?

Maintaining consistent CK19 antibody performance requires rigorous quality control:

• Reference standards: Maintain well-characterized positive control specimens with known staining intensity

• Lot testing: Validate each new antibody lot against the previous lot before implementation

• Stability monitoring: Perform periodic testing of antibody aliquots to detect potential degradation

• Environmental controls: Store antibodies at manufacturer-recommended temperatures; avoid freeze-thaw cycles

• Calibration slides: Include standardized control slides with each batch of samples

• Performance metrics: Track and analyze staining quality indicators (signal-to-noise ratio, intensity, etc.)

These measures ensure consistency across experiments and facilitate meaningful comparison of results over time .