Peroxidase-labeled antibody technology exchange - Database & Sql Blog Articles

On April 30, 2015, our company initiated a lively discussion on the technology of peroxidase-labeled antibodies, sharing various application methods and specific techniques for this product. Below is a summary of the meeting content, and we welcome all inquiries and visits.

Product Introduction:

Peroxidase-labeled antibodies are highly specific, sensitive, and safe immunochemicals widely used in immunology, molecular biology, and clinical medicine. The most commonly used enzyme is horseradish peroxidase (HRP), which is oxidized by sodium periodate and conjugated to an antibody IgG molecule to form an HRP-antibody complex.

All enzyme standards provided by the company are labeled using a modified sodium periodate oxidation method developed internally. A certain concentration of BSA is added to each finished product as a stabilizer. The working concentration of the product depends on the intended application.

The product should be stored at -20°C to prevent repeated freezing and thawing. It contains approximately 200 µg of HRP and 400 µg of IgG per 0.1 ml, along with 300 µg of BSA. When stored at 4°C, it can last 3–6 months, and after dilution, it can remain stable for about 30 days in PBS (pH 7.4).

Application Range:

This product is ideal for antigenic analysis, quantification, characterization, and localization of antigens and antibodies, among other applications.

HRP-Labeled Antibody Method:

There are several methods for cross-linking enzymes with antibodies, and different approaches are chosen based on the enzyme structure. For HRP conjugation, the two-step glutaraldehyde method and the sodium periodate method are commonly used. The sodium periodate method is particularly popular due to its simplicity.

Glutaraldehyde Two-Step Method:

1. Principle: Glutaraldehyde acts as a bifunctional reagent, covalently binding to both the enzyme and the amino groups of immunoglobulins to form an enzyme-glutaraldehyde-immunoglobulin conjugate.

2. Marking Steps:

(1) Dissolve 25 mg of HRP in 1.25% glutaraldehyde solution and let it react overnight at room temperature.

(2) Purify the enzyme solution using a Sephadex G-25 column, eluting with physiological saline at 1 ml/min. Collect brown fractions and concentrate if necessary with PEG.

(3) Dilute 12.5 mg of the antibody in 5 ml of physiological saline and add it dropwise to the enzyme solution under continuous stirring.

(4) Add 0.25 ml of 1 M phosphate buffer (pH 9.5) and continue stirring for 3 hours.

(5) Add 0.2 ml of 0.2 M lysine, mix, and incubate for 2 hours at room temperature.

(6) Slowly add an equal volume of saturated ammonium sulfate while stirring, then let it stand at 4°C for 1 hour.

(7) Centrifuge at 3000 rpm for 30 minutes, discard the supernatant, wash the precipitate twice with half-saturated ammonium sulfate, and finally dissolve it in 0.15 M PBS (pH 7.4).

(8) Dialyze the solution against 0.15 M PBS (pH 7.4) to remove ammonium ions, then centrifuge at 10,000 rpm for 30 minutes. Store the supernatant at 4°C.

3. Result Determination:

(1) Qualitative and potency titration: Use specific antigens or antibodies in a two-way agar diffusion or immunoelectrophoresis test. Color the precipitation arc with the enzyme substrate to assess activity. Titrate the conjugate using direct ELISA.

(2) Quantitative and molar ratio determination: Measure absorbance at 403 nm and 280 nm using a spectrophotometer. Calculate enzyme and IgG concentrations accordingly.

Enzyme amount (mg/ml) = OD403nm × 0.4

IgG amount (mg/ml) = [OD280nm – OD403nm] × 0.42 × 0.94 × 0.62

Molar ratio = (Enzyme / 40,000) ÷ (IgG / 160,000)

(3) This method is simple and reproducible, but has a low enzyme utilization rate, typically around 2–4%.

4. Reagents and Equipment:

(1) 0.1 M PBS (pH 6.8): Mix 449 ml of 0.2 M Na2HPO4 with 451 ml of 0.2 M NaH2PO4 and 1.8 g NaCl, adjust to 200 ml with distilled water.

(2) 1.25% Glutaraldehyde: Mix 50 ml of 25% glutaraldehyde with 1 ml of pH 6.8 PBS.

(3) 1 M Carbonate Buffer (pH 9.5): Combine 3 ml of 1 M sodium carbonate with 7 ml of 1 M sodium bicarbonate.

(4) 0.2 M Lysine: Dissolve 29.2 mg of lysine in 1 ml of 0.01 M carbonate buffer (pH 9.5).

(5) 0.15 M PBS (pH 7.4) and physiological saline.

(6) Saturated and semi-saturated ammonium sulfate solutions.

(7) Naphthalene reagent and PEG (MW 2000).

(8) Purified specific antibody or anti-IgG.

(9) HRP (RZ > 3.0).

(10) Sephadex G-25 chromatography column (2 cm x 50 cm).

(11) Stirrer, spectrophotometer, centrifuge.

(12) Dialysis bags, beakers, test tubes, pipettes, etc.

Shanghai Jinma Biological offers a wide range of ELISA test kits, including human, animal, plant, food, agricultural, cell, and other specialized types.

For more detailed specifications, please contact us directly to place your order.