Human CCDC47 sandwich immunoassay development with electrochemiluminescence technology.

Area of Science:

• Biochemistry
• Cell Biology
• Immunology

Background:

• Coiled-Coil Domain Containing 47 (CCDC47) is an endoplasmic reticulum (ER) transmembrane protein.
• CCDC47 plays roles in calcium signaling and endoplasmic reticulum-associated degradation (ERAD).
• CCDC47 is involved in ER stress relief mechanisms.

Purpose of the Study:

• To develop human CCDC47 monoclonal antibodies.
• To establish a sensitive sandwich immunoassay for CCDC47 quantification.
• To compare traditional ELISA with electrochemiluminescence (ECL) for enhanced analytical sensitivity.

Main Methods:

• Development and validation of monoclonal antibodies against human CCDC47.
• Specificity confirmation using immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Comparison of colorimetric ELISA and ECL immunoassay technologies.
• Development and performance evaluation of a CCDC47 sandwich ECL assay.

Main Results:

• Successfully developed and validated human CCDC47 monoclonal antibodies.
• Confirmed antibody specificity through immunoblot and LC-MS/MS.
• Achieved a 3-log increase in analytical sensitivity using ECL compared to traditional ELISA.
• Developed and characterized a robust CCDC47 sandwich ECL assay for biological matrices.

Conclusions:

• Human CCDC47 monoclonal antibodies and a sandwich ECL immunoassay have been successfully developed.
• The ECL assay offers significantly enhanced sensitivity for CCDC47 measurement.
• The developed assay is suitable for quantifying CCDC47 in biological samples, with characterized performance and stability.