CLDN18 Antibody, FITC conjugated

What is CLDN18.2 and why is it significant as a cancer research target?

CLDN18.2 (Claudin-18 isoform 2) is a tight junction protein normally and specifically expressed in gastric mucosa cells. Its expression is often maintained during malignant transformation, making it an ideal target for cancer therapy. CLDN18.2 is highly expressed in gastric and pancreatic adenocarcinomas while being absent from most normal tissues, which provides a potential therapeutic window against these cancers . The protein has two splice variants: claudin-18.1 (primarily found in lung) and claudin-18.2 (expressed exclusively in the stomach), with the latter being particularly relevant for oncology research .

How does CLDN18.2 expression vary across different cancer types?

Immunohistochemistry (IHC) screening of human cancer tissues has revealed that membrane expression of CLDN18.2 is positive in approximately:

• 58% of gastric adenocarcinomas

• 60% of gastroesophageal junction adenocarcinomas

• 20% of pancreatic adenocarcinomas

This differential expression pattern is crucial for understanding the potential therapeutic applications of CLDN18.2-targeted approaches across various cancer types.

What is the specificity difference between antibodies targeting CLDN18 versus CLDN18.2?

This is a critical distinction in experimental design. Some antibodies target the C terminus of claudin 18 (such as the VENTANA CLDN18 43-14A assay) but do not specifically differentiate isoform 18.2. For isoform-specific applications, researchers must select antibodies like the anti-CLDN EPR19202 kit, which specifically recognizes the CLDN18.2 isoform . This distinction is essential for accurate research interpretation and therapeutic development.

What are the optimal protocols for using FITC-conjugated CLDN18.2 antibodies in flow cytometry?

For flow cytometry applications using FITC-conjugated anti-CLDN18.2 antibodies, researchers should:

• Prepare single-cell suspensions using enzymatic dissociation that preserves surface epitopes

• Block with appropriate buffers containing 1-2% BSA to reduce non-specific binding

• Incubate with the FITC-conjugated CLDN18.2 antibody at 4°C for 30-45 minutes in the dark

• Wash thoroughly with cold buffer (PBS with 0.1% BSA) to remove unbound antibody

• Include appropriate controls: unstained cells, isotype control-FITC, and known CLDN18.2-positive and negative cell lines

This approach has been validated for specific binding to human CLDN18.2 on the surface of CLDN18.2 CHO Recombinant Cell Line . When analyzing results, remember that FITC has excitation and emission peak wavelengths of approximately 495 nm and 519 nm, causing it to fluoresce green .

How can researchers distinguish between CLDN18.1 and CLDN18.2 isoforms in their experiments?

Distinguishing between these isoforms requires:

• Isoform-specific antibodies: Select antibodies validated for specific recognition of CLDN18.2 epitopes not present in CLDN18.1

• Control cell lines: Utilize cell lines expressing only CLDN18.1 (like 293T CLDN18.1) as negative controls alongside CLDN18.2-expressing cells (293T CLDN18.2)

• Competitive binding assays: Perform binding assays with isoform-specific peptides to confirm specificity

• Validation approaches: Sequence-based validation should be performed when developing new detection reagents

Researchers working with phage display technology have successfully screened CLDN18.2-specific peptides by using 293T CLDN18.1 cells to exclude nonspecific binding and CLDN18.1 binding sequences, while using 293T CLDN18.2 cells to select for CLDN18.2-specific binding peptides .

What are the key methodological considerations for immunohistochemical detection of CLDN18.2?

For accurate IHC detection of CLDN18.2:

• Tissue preparation: Use formalin-fixed, paraffin-embedded tissue blocks or prepare 4-micron sections on positively charged slides

• Antigen retrieval: Optimize based on the specific antibody's requirements (typically heat-mediated in citrate buffer)

• Detection system: Use high-sensitivity detection systems appropriate for the primary antibody

• Controls: Include both positive controls (known CLDN18.2-positive gastric cancer tissue) and negative controls

• Scoring criteria: Establish clear membrane staining positivity criteria for interpretation

For diagnostic purposes, validated assays like the VENTANA CLDN18 (43-14A) assay are designed for use with BenchMark IHC/ISH instruments, though this targets the C terminus and doesn't specifically differentiate isoform 18.2 .

How can researchers minimize photobleaching when working with FITC-conjugated CLDN18.2 antibodies?

Photobleaching is a common challenge with FITC conjugates. Researchers should:

• Minimize exposure to light during all preparation steps

• Store antibody solutions in dark containers at recommended temperatures

• Use anti-fade mounting media for microscopy applications

• Consider adding protein stabilizers to buffer solutions

• Work efficiently during flow cytometry to minimize laser exposure time

• If extended analysis is needed, consider alternative more photostable fluorophores for conjugation

The specific storage buffer for BPS Bioscience's FITC-labeled anti-CLDN18.2 antibody (8 mM Phosphate, pH 7.4, 110 mM NaCl, 2.2 mM KCl, 0.09% Sodium Azide, 0.2% BSA, and up to 20% glycerol) includes protein stabilizers to help maintain functionality .

What are common sources of false positivity/negativity in CLDN18.2 detection assays?

Researchers should validate their assays using positive and negative controls with known CLDN18.2 expression status to establish assay performance characteristics .

How does CLDN18.2 expression correlate with responses to immunotherapy in gastric cancer?

Recent research indicates significant associations between CLDN18.2 expression and specific molecular features in gastric cancer:

• CLDN18.2 expression is significantly and positively associated with Epstein-Barr virus (EBV) status in gastric cancer

• CLDN18.2-positive tumors show higher PD-L1 expression compared to CLDN18.2-negative tumors

• Both CLDN18.2 expression and tumor-infiltrating NK cells are significantly higher in EBV-associated gastric cancer compared to other molecular subtypes

What are the mechanisms of action for CLDN18.2-targeted antibody therapies?

CLDN18.2-directed monoclonal antibodies operate through multiple mechanisms:

• Antibody-dependent cellular cytotoxicity (ADCC): CLDN18.2-specific antibodies like CLDN18.2-307-mAb bind with high affinity to CLDN18.2-positive cells and induce ADCC, activating immune cells like NK cells to attack tumor cells

• Direct growth inhibition: Treatment with CLDN18.2-mAb has been shown to block the growth of CLDN18.2-positive gastric and pancreas cancer cell line xenograft models

• Antibody-drug conjugate (ADC) delivery: Upon binding to the extracellular domain of CLDN18.2, ADCs like CLDN18.2-307-ADC/CLDN18.2 are internalized and localized to the lysosomal compartment, releasing cytotoxic payloads (e.g., MMAE) that induce tumor regressions

Understanding these mechanisms is crucial for designing experiments to evaluate novel CLDN18.2-targeting approaches and analyzing potential resistance mechanisms.

How can FITC-conjugated CLDN18.2 antibodies be used to develop novel imaging approaches?

FITC-conjugated CLDN18.2 antibodies enable several advanced imaging applications:

• In vivo tumor visualization: Conjugation methodologies like FluoSite allow site-specific attachment of fluorophores away from antigen-binding domains, preserving functionality while enabling sensitive detection

• Multiplex imaging: FITC-labeled antibodies can be combined with other fluorophore-labeled markers for multiplexed analysis of tumor microenvironments

• Model system development: Similar conjugation approaches have led to development of radiolabeled peptides (e.g., 68Ga-DOTA-conjugated peptides) for PET imaging of CLDN18.2-expressing tumors

These applications support translational research bridging laboratory findings to clinical applications for patient selection and treatment monitoring.

What are the established cutoff values for CLDN18.2 positivity in patient selection for targeted therapies?

While standardization is still evolving, current clinical trials and studies use various thresholds:

• For eligibility in zolbetuximab (VYLOY) treatment, patients typically need to demonstrate CLDN18.2 expression by immunohistochemistry

• Different scoring systems exist, with some studies defining positivity as membrane staining in ≥75% of tumor cells, while others use lower thresholds

• Both staining intensity and percentage of positive cells are considered in most evaluation systems

Researchers should be aware that these cutoffs may evolve as more clinical data becomes available, and standardization efforts are ongoing to establish consensus criteria for CLDN18.2 positivity.

How does the method of CLDN18.2 detection influence research outcomes and therapeutic decisions?

Different detection methods have important implications:

• IHC methods: Currently, approved companion diagnostic IHC tests like VENTANA CLDN18 (43-14A) assay are designed for clinical use but don't specifically differentiate isoform 18.2

• Flow cytometry: FITC-conjugated antibodies provide quantitative assessment of surface expression but require viable cells, making them more suitable for research than diagnostics

• Molecular methods: RT-PCR or RNA-seq provide transcript-level data but may not reflect functional protein expression at the cell surface

Researchers should be cautious when comparing results obtained using different methodologies and understand the specific limitations of each approach when designing studies and interpreting results.