Liquid-phase binding assay of human chorionic gonadotropin using high-performance liquid chromatography

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Characterizing liquid-phase antigen-antibody reactions is crucial for accurate immunoassays.
• Traditional methods may face limitations in speed and quantitative precision for these reactions.

Purpose of the Study:

• To introduce a new enzyme immunoassay technique, the liquid-phase binding assay (LBA).
• To demonstrate the utility of LBA for examining liquid-phase antigen-antibody reaction characteristics.
• To establish a method for accurate antigen concentration determination.

Main Methods:

• Developed a liquid-phase binding assay (LBA) using human chorionic gonadotropin (hCG) and peroxidase (POD)-labeled anti-hCG monoclonal antibody (Fab'-POD).
• Analyzed reaction mixtures using gel filtration high-performance liquid chromatography (HPLC) with postcolumn enzyme activity measurement.
• Separated bound and free enzyme-labeled antibodies by molecular mass and quantified POD activity fluorophotometrically.

Main Results:

• The LBA system successfully separated bound (hCG-Fab'-POD) and free (Fab'-POD) enzyme-labeled antibodies.
• Demonstrated a stoichiometric relationship where hCG could be measured via a single POD molecule's activity.
• Confirmed that liquid-phase antibody reactions were rapid, quantitative, and easily influenced by reaction conditions.

Conclusions:

• The LBA is a simple and convenient method for examining antigen-antibody reactions.
• The assay allows for the straightforward determination of equilibrium/rate constants, optimal pH, and temperature effects.
• LBA is applicable for antigen assays requiring precise concentration definition, including hCG measurement.