Using microparticle labeling and counting for attomole-level detection in heterogeneous immunoassay

Area of Science:

• Biochemistry
• Immunology
• Nanotechnology

Background:

• Immunoassays are crucial for detecting biomolecules.
• High sensitivity is often limited by detection methods.
• Microparticle-based assays offer potential for enhanced sensitivity.

Purpose of the Study:

• To develop a novel heterogeneous immunoassay with microparticle labels for high-sensitivity antigen detection.
• To optimize microparticle preparation and antibody conjugation for improved assay performance.
• To establish the lower detection limit and immunoreaction efficiency of the developed assay.

Main Methods:

• A "sandwich" immunoassay format was employed using antibody-functionalized microparticles.
• Antigen was captured on a microplate, followed by reaction with microparticle-bound antibodies.
• Carboxylated microparticles were conjugated with antibodies using carbodiimide chemistry.
• Detection and counting of antigen-bound microparticles were performed using an inverted optical microscope.

Main Results:

• The assay demonstrated high sensitivity due to the detection of single microparticle-bound antigen molecules.
• Optimization of microparticle preparation and antibody conjugation yielded approximately 1100 antibody molecules per microparticle.
• An immunoreaction efficiency of 18% was achieved at an antigen concentration of 1 x 10(-13) mol/liter.
• A lower detection limit of 3.1 x 10(-14) mol/liter (1.6 amol) was established using human alpha-fetoprotein.

Conclusions:

• The developed microparticle-based immunoassay offers a highly sensitive method for antigen detection.
• The protocol for antibody conjugation and microparticle preparation is critical for achieving high sensitivity.
• This assay has potential applications in diagnostics and biomedical research requiring sensitive biomolecule quantification.