Antibody Labeling with HRP

Antibody labeling involves the covalent attachment of a marker (enzyme, fluorescein, biotin, etc.) to an antibody, which reacts specifically with the substance to be detected to form a multiplex, and the test result is observed or determined by direct microscopic examination with an instrument. Some substances that are both easy to measure and highly sensitive, such as horseradish peroxidase (HRP), biotin, etc., are labeled to specific antibody molecules, and the nature and content of the antigen or antibody in the reaction system can be displayed or quantified by the enhanced amplification effect of these markers.

HRP Antibody Labeling Protocol

The most classic method for labeling HRP with antibodies is the NaIO₄ oxidation method. First, HRP sugar groups are oxidized into aldehyde groups. The aldehyde groups react with the -NH₂ of the antibody to form a Schiff's base reaction, and the antibody molecules form a stable structure with the HRP molecules. When labeling, the molar ratio of antibody molecules to HRP molecules is generally 1:4. At this time, the mass of the two is about 1:1. To prevent the coupling of the aldehyde group of the sugar in HRP to its own protein amino group, the amino group was blocked with dinitrofluorobenzene prior to oxidation with sodium periodate. At the end of the oxidation reaction, Schiff's base is stabilized with NaBH₄.

Materials required

The labeling step takes 2 mg of the antibody as an example.

Antibody/protein treatment

• Dialyze the antibody/protein to be labeled in 50mM carbonate buffer (0.015M Na₂CO₃, 0.035M NaHCO₃, pH9.6), change the solution twice during the period, and adjust the antibody/protein concentration to 2mg/ml.
• Or dilute the high-concentration antibody to 2mg/ml with 0.5M carbonate buffer (0.15M Na₂CO₃, 0.35M NaHCO₃, pH9.6), if the original antibody/protein solution contains free amino reagents such as Tris and NH4+, Must be removed by dialysis.

HRP enzyme oxidation

• Weigh 2 mg of HRP dry powder and dissolve in 100 μL ddH₂O.
• Weigh 21 mg of NaIO₄ and dissolve it in 1 mL of ddH₂O. Draw 100 μL of NaIO4 solution and 100 μL of HRP solution and mix slowly, and let the mixture stand at 4°C for 30 minutes, until the solution turns green.

(Note: All subsequent operations are carried out under dark conditions.)

• Add 2 µL of ethylene glycol slowly to the oxidized HRP solution, and let it stand in the dark for 30 minutes at room temperature. At this time, the solution turns brown.

Antibody labeling

• Add the oxidized HRP solution directly to the treated antibody/protein solution, and react at room temperature for 2 hours.
• Weigh 0.4 mg of NaBH₄, dissolve it in 20 µL of ddH₂O, add all of it to the reaction solution in the previous step, let it stand at 4°C for 2 hours, and shake it every 30 minutes.
• Dialyze with PBS (pH 7.2) overnight, add 30% to 50% glycerol by volume to the marking solution, mix well, and store at -20°C.

HRP antibody labeling is a powerful technique that enhances the sensitivity and specificity of various immunoassays. The technique involves covalently attaching the HRP enzyme to an antibody molecule, allowing for the detection and quantification of specific proteins in biological samples. To optimize labeling efficiency, researchers should consider various factors such as the antibody concentration, pH of the labeling buffer, ratio of HRP to antibody, and reaction time. Creative Diagnostics offers a wide range of high-quality HRP-conjugated antibodies and labeling kits that simplify HRP antibody labeling. If you have any requirements for HRP antibody labeling, please contact us for more information.