18 kDa cell wall Antibody

What are the major types of 18 kDa proteins used as targets for antibody development in research?

The 18 kDa protein family encompasses several biologically significant targets across diverse organisms:

• Cytokeratin 18 (K18): A 44-48 kDa Class I (acidic pH) intermediate filament protein that heterodimerizes with 50-52 kDa class II KRT-8 to form 8-10 nm filaments in single strata and hepatic epithelia

• Translocator Protein 18 kDa (TSPO): A stress response protein upregulated in microglia and astrocytes during neuroinflammation that associates with NADPH oxidase complexes

• Mycobacterium leprae 18 kDa protein: A species-specific antigen recognized by monoclonal antibody L5, with restricted distribution confined to M. leprae

• Taenia pisiformis 18 kDa oncosphere antigen (TPO18): A protein expressed in the oncosphere stage that plays a role in tissue invasion

• Vibrio cholerae 18 kDa outer-membrane antigen: A major antigen (cholera protective antigen) that reacts with human sera

How is specificity assessed when developing antibodies against 18 kDa cell wall proteins?

Specificity assessment involves multifaceted validation approaches:

• Cross-reactivity testing: Evaluating reactivity against related species/proteins. For example, anti-TPO18 monoclonal antibodies are validated against both target and non-target tissues

• Positive/negative cell line validation: Testing antibody reactivity in positive cell lines (e.g., A431 human epithelial carcinoma for Cytokeratin 18) versus negative cell lines (e.g., MOLT-4 acute lymphoblastic leukemia lines)

• Western blot analysis: Confirming specific binding at the expected molecular weight (45-48 kDa for Cytokeratin 18)

• Immunohistochemical mapping: Determining spatial distribution of the target protein, as demonstrated with TPO18 being localized to the drape and wall of uteri in adult parasites

• Loading controls: Using established markers like GAPDH to normalize protein expression levels

What are the optimal antibody dilutions for different experimental applications involving 18 kDa proteins?

The optimal dilution should be determined experimentally for each application, as factors such as sample type, fixation method, and detection system significantly influence results .

What protocols are recommended for immunohistochemical detection of 18 kDa cell wall proteins?

A methodical approach to immunohistochemistry includes:

• Tissue preparation:

  • For paraffin sections: Standard fixation in formalin, embedding in paraffin, and sectioning

  • For frozen sections: Rapid freezing and cryosectioning

• Antigen retrieval:

  • Heat-induced epitope retrieval often necessary for formalin-fixed tissues

  • Buffer selection based on target protein (citrate or EDTA-based)

• Blocking and antibody incubation:

  • Blocking with appropriate serum (e.g., 5% normal goat serum)

  • Primary antibody incubation at optimal dilution (e.g., 15 μg/mL overnight at 4°C for Cytokeratin 18)

  • Secondary antibody conjugated to detection system (e.g., HRP-DAB Cell & Tissue Staining Kit)

• Detection and counterstaining:

  • Development with chromogenic substrate (DAB) for brightfield

  • Fluorescent secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Mouse IgG) for fluorescence

  • Counterstaining with hematoxylin for brightfield or DAPI for fluorescence

• Controls:

  • Positive control tissues with known expression

  • Negative controls (primary antibody omission)

  • Isotype controls to assess non-specific binding

What are common challenges when working with antibodies against 18 kDa cell wall proteins, and how can they be addressed?

How should researchers properly validate newly developed antibodies against 18 kDa cell wall proteins?

A comprehensive validation protocol includes:

• Initial screening:

  • ELISA assays against purified target protein

  • Fusion protein expression and purification as exemplified in TPO18 studies

• Clone selection and characterization:

  • Subcloning to establish stable cell lines (e.g., hybridomas 4E8, 5G5, 7E8 for TPO18)

  • Isotype determination (e.g., IgG2b heavy chain and kappa light chain for anti-TPO18)

  • Titer determination through serial dilution assays

• Specificity testing:

  • Western blot against recombinant and native proteins

  • Cross-reactivity assessment with related proteins

  • Immunohistochemistry across various tissues and species

• Functional validation:

  • Immunoprecipitation to confirm target binding

  • Neutralization assays if applicable

  • Knockout/knockdown validation in appropriate systems

• Reproducibility assessment:

  • Inter-lab validation

  • Batch-to-batch consistency evaluation

  • Performance across multiple applications

How can antibodies against 18 kDa proteins be utilized for studying protein-protein interactions?

Antibodies serve as powerful tools for investigating protein-protein interactions through multiple approaches:

• Co-immunoprecipitation studies:

  • Example: TSPO antibodies have been used to investigate interactions with gp91^phox, a component of the NADPH oxidase complex in microglia

  • Both traditional and crosslinking IP protocols can be employed

• Proximity ligation assays (PLA):

  • Enables visualization of protein interactions in situ with subcellular resolution

  • Particularly useful for transient or weak interactions

• Pull-down assays:

  • Recombinant tagged proteins combined with specific antibodies

  • Example: GST-TPO18 has been used in pull-down experiments to study binding partners

• Bimolecular fluorescence complementation (BiFC):

  • When combined with epitope tagging, allows real-time visualization of interactions

• FRET/FLIM analysis:

  • Antibody-conjugated fluorophores enable energy transfer studies

  • Provides spatial information about interaction dynamics

What is the significance of using 18 kDa proteins as biomarkers in disease research?

18 kDa proteins serve as valuable biomarkers across multiple disease contexts:

• Cancer diagnostics and classification:

  • Cytokeratin 18 is expressed in adenocarcinomas of the breast, lung, ovary, and gastrointestinal tract

  • K18 expression patterns help distinguish different epithelial tumor types

  • Detected in squamous cell carcinoma using immunohistochemistry

• Infectious disease diagnostics:

  • The 18-kDa protein of M. leprae shows species-specific distribution, making it valuable for leprosy diagnosis

  • Antibody responses to M. leprae 18-kDa protein differ between leprosy and tuberculosis patients

  • TPO18 antibodies may contribute to diagnosis of parasitic infections like cysticercosis pisiformis

• Neuroinflammation assessment:

  • TSPO (18 kDa) serves as a biomarker for neuroinflammation in preclinical and clinical neuroimaging studies

  • Upregulated in microglia and astrocytes during diverse CNS pathologies

• Vaccine development:

  • The 18-kDa outer-membrane antigen of Vibrio cholerae is a major target of the human immune response, with potential as a cholera protective antigen

How can researchers optimize sample preparation protocols for detection of 18 kDa cell wall proteins?

Sample preparation must be tailored to the specific protein and application:

• Cell lysis considerations:

  • For cytoskeletal proteins like Cytokeratin 18, use buffers containing detergents that preserve filamentous structures

  • Example protocol: HeLa and A431 cell lysates for Cytokeratin 18 detection prepared under reducing conditions using specific immunoblot buffer groups

• Tissue-specific optimizations:

  • Epithelial tissues: Cytoplasmic intermediate filaments require preservation of cytoskeletal integrity

  • Parasitic tissues: Specific fixation methods needed for TPO18 detection in T. pisiformis adults and cysticerci

  • Brain tissues: Special considerations for microglia preparation when studying TSPO

• Subcellular fractionation:

  • Membrane proteins (like TSPO or bacterial outer membrane proteins) require specific extraction methods

  • Detergent selection critical for maintaining native conformation

• Protein quantification and loading:

  • Standardization using loading controls (e.g., GAPDH)

  • Protein concentration determination before immunoblotting

  • Typical working range: 0.5 mg/mL for A431 cell lysates in Simple Western assays

• Preservation of post-translational modifications:

  • Use of phosphatase/protease inhibitors during extraction

  • Appropriate storage conditions to prevent degradation

How are antibodies against 18 kDa proteins being utilized in diagnostic development?

Current diagnostic applications show promising developments:

• Serodiagnostic assays:

  • Anti-18 kDa protein antibodies used in ELISA and immunoblot techniques for detecting patient immune responses to pathogens

  • Example: Detection of antibody responses to M. leprae 18-kDa protein in leprosy patients

• Immunohistochemical markers:

  • Cytokeratin 18 antibodies widely employed for tumor classification

  • Help differentiate between epithelial tumor types and determine tissue origin

• Multiplex detection systems:

  • Combination with other biomarkers improves sensitivity and specificity

  • Particularly valuable for infectious disease diagnosis

• Point-of-care development:

  • Adaptation of laboratory techniques to field-applicable formats

  • Example: Potential application of anti-TPO18 MAbs for parasitic infection detection

What novel technical approaches are being explored for studying 18 kDa protein interactions?

Cutting-edge methodologies include:

• Advanced imaging techniques:

  • Super-resolution microscopy allows visualization of protein complexes beyond diffraction limit

  • Example: Cytokeratin 18 localization to intermediate filaments in cytoplasm visualized with immunofluorescence

• Mass spectrometry-based interactomics:

  • Antibody-based purification coupled with MS identification

  • Enables unbiased discovery of novel interacting partners

• CRISPR-based functional screens:

  • Systematic investigation of genes affecting 18 kDa protein expression/function

  • Validation of antibody specificity in knockout systems

• Single-cell analysis:

  • Antibody-based detection of 18 kDa proteins at single-cell resolution

  • Reveals heterogeneity in expression patterns within tissues

• Structural biology integration:

  • Epitope mapping combined with structural data

  • Provides insights into functional domains and interaction interfaces