HRP Antibody Conjugation Technology
Introduction
Antibody-HRP (Horseradish Peroxidase) conjugation technology involves chemically linking an antibody to the HRP enzyme to form a stable complex. This technique is widely used in immunoassays (e.g., ELISA, Western Blot, immunohistochemistry) to detect target molecules via HRP-catalyzed chromogenic or chemiluminescent reactions using substrates like TMB or DAB.
Principle
The sugar groups of HRP are oxidized into aldehyde groups by sodium periodate. After adding the antibody, the aldehyde groups on HRP combine with the amino groups of the antibody to form Schiff's base, which is stabilized by sodium borohydride.
Protocol
1.Reagent Preparation
（1）10 mg/ml Horseradish Peroxidase(HRP): Weigh 100 mg of HRP and dissolve it in 10 mL of deionized water.
（2）0.06 mol/L Sodium periodate(NaIO4): Weigh 128 mg of NaIO4 and dissolve it in 10 mL of deionized water.
（3）0.16 mol/L Ethylene glycol solution: Dissolve 0.09 mL ethylene glycol in 10 mL deionized water.
（4）Saturated Ammonium sulfate((NH4)2SO4): weighed 7.8 g of ammonium sulfate and dissolved it in 10 mL of deionized water at 80℃. Then, it was filtered through a 0.22 μM filter and the pH value was adjusted to 7.2.
（5）5 mg/mLSodium borohydride(NaBH4): Weigh 50 mg of NaBH4 and dissolve it in 10 mL of deionized water.
（6）0.1 M carbonate buffer(pH 9.6)：Weigh 10.6 g of anhydrous sodium carbonate and 8.4 g of sodium bicarbonate. Add it to approximately 900 mL of distilled water in a beaker. Adjust the pH of the buffer to 9.6 by adding the required quantity of diluted hydrochloric acid or sodium hydroxide. Then, add the required volume of distilled water to achieve a total volume of 1,000 mL.
2. Operation steps
（1）Antibody Pretreatment
Dissolve 2 mg monoclonal antibody in 0.1 M carbonate buffer (pH 9.6) and dialyze to remove interfering substances (e.g., Tris, sodium azide). Adjust antibody concentration to 2 mg/mL.
（2）Activation of HRP
Take 10 mL of 10 mg/mL HRP, add 10 mL of freshly prepared 0.06 mol/L NaIO4 solution, mix and incubate at 4℃ for 30 minutes (solution turns dark green) . Terminate activation by adding 10 mL of 0.16 mol/L ethylene glycol and stir in the dark at room temperature for 30 minutes.
（3）Conjugation Reaction
Add 300 μL of activated HRP for each 1 mg of antibody. Mix the antibody with the activated HRP thoroughly.
（4）Purification and Termination Reaction
Transfer the mixed solution to a dialysis bag, place it in 5 L of 1×CBS buffer solution, and stir slowly. Let it stand for overnight for dialysis. Remove the mixed solution from the dialysis bag, add 40 uL of freshly prepared 5 mg/ml NaBH4 solution, mix well, and store at 4℃ in the dark for 2 hours.
（5） Precipitated Conjugation-Antibody
Add an equal volume of saturated ammonium sulfate solution, mix well, and then incubate at 4℃ for 30 minutes. Centrifuge at 13000 rpm for 5 minutes and remove the supernatant.
（6）Dissociation Conjugated Antibody
Add 1 mL of 1× PBS buffer and dissolve the precipitate.
Quality control
1.Conjugation Ratio: Analyze via SDS-PAGE to confirm molecular weight shifts (HRP: ~44 kDa; antibody: ~150 kDa). Optimal ratio: 1:1–2:1 (HRP) . Calculate Conjugation Ratio through Image J software.
2.Activity Assays: The activity of the conjugated antibody was verified by direct ELISA.
Notes
1.Interfering Substances: Avoid amine-containing buffers (e.g., Tris) to prevent reduced conjugation efficiency .
2.Oxidation Control: Excess NaIO₄ or prolonged reaction time may inactivate HRP. Strictly control timing and protect from light.
3.Purification Optimization: Increase HRP-to-antibody ratio if separation of conjugated products is challenging.
