Tissue polypeptide specific antigen (TPS) detects a specific epitope structure on human cytokeratin

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• The TPS assay utilizes the M3 monoclonal antibody to detect tumor markers.
• Understanding the precise target of the M3 antibody is crucial for assay validation and interpretation.
• Cytokeratin 18 (CK18) is an intermediate filament protein expressed in various epithelial cells, including those in colon adenocarcinoma.

Purpose of the Study:

• To identify the specific protein recognized by the M3 monoclonal antibody.
• To determine the exact location of the M3 epitope on its target protein.
• To characterize the TPS assay's reactive component at a molecular level.

Main Methods:

• Purification of a TPS-reactive protein fragment from a human colon adenocarcinoma cell line.
• N-terminal sequence analysis to identify the purified protein fragment.
• Screening of an expression library using the M3 antibody.
• PCR amplification and DNA sequencing of reactive phage clones.
• Expression of engineered fusion proteins to map the epitope.

Main Results:

• A 13 kDa protein fragment, reactive with the M3 antibody, was purified.
• N-terminal sequencing identified this fragment as human cytokeratin 18 (CK18).
• DNA sequencing confirmed the presence of a CK18 nucleotide fragment in M3-reactive clones.
• The M3 epitope was precisely localized to amino acid residues 322-340 of CK18.

Conclusions:

• The M3 monoclonal antibody specifically recognizes a fragment of human cytokeratin 18.
• The M3 epitope is located within residues 322-340 of CK18.
• This precise epitope mapping enhances the understanding of the TPS assay's molecular basis and CK18 as a potential biomarker.