A general affinity method to purify peroxidase-tagged antibodies

Area of Science:

• Biochemistry
• Immunology
• Biotechnology

Background:

• Enzyme-labelled antibodies, such as those with horseradish peroxidase (HRPO), are vital for immunoassays and immunotherapies.
• Purifying these antibody conjugates is a significant challenge, limiting their specific activity and utility.
• Existing methods often face difficulties in achieving high purity and specific activity.

Purpose of the Study:

• To develop an efficient purification strategy for enzyme-labelled antibodies, specifically HRPO conjugates.
• To overcome the obstacle of antibody purification in creating high-specific-activity immunoprobes.
• To establish a method for purifying various antibody types (polyclonal, monoclonal, bispecific) conjugated with enzymes.

Main Methods:

• Utilized benzhydroxamic acid-agarose affinity chromatography for purification.
• Precipitated antibodies using ammonium sulfate and dialyzed them.
• Incubated antibody fractions with HRPO to form immune complexes before affinity purification.
• Eluted labelled antibodies under mild conditions (borate buffer, pH 9.0).

Main Results:

• Successfully purified bispecific antibody-HRPO complexes with high specific activity.
• Achieved an effective yield of 30 assay plates or 3000 wells for the bispecific antibody-HRPO complex.
• Demonstrated successful co-purification of covalent polyclonal-HRPO conjugates and HRPO-labelled streptavidin.
• Obtained enzyme-labelled probes with high specific activities for diverse applications.

Conclusions:

• Benzhydroxamic acid-agarose is an effective affinity matrix for purifying HRPO-labelled antibodies.
• This method circumvents common purification challenges, yielding high-specific-activity immunoprobes.
• The strategy is versatile and applicable to various enzyme-labelled antibody conjugates for broad applications.