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 methods of application and specific techniques for this product. Below is a summary of the meeting content. We welcome you to inquire and explore further.

Product Introduction:

Peroxidase-labeled antibodies are highly specific, sensitive, and safe immunochemical reagents 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 IgG antibody 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 specific application method. It should be stored at -20°C to avoid repeated freeze-thaw cycles.

This product contains approximately 200 µg of HRP per 0.1 ml, 400 µg of IgG, and 300 µg of BSA per branch. When stored at 4°C, it can last about 3 to 6 months. After dilution with PBS (pH 7.4), it can be kept for up to 30 days at 4°C.

Application Range:

Antigenic analysis, quantification, characterization, and localization of various antigens and antibodies, as well as related analyses.

HRP-Labeled Antibody Method:

There are multiple methods for cross-linking enzymes with antibodies, depending on the enzyme structure. The two-step glutaraldehyde method and the sodium periodate method are commonly used for HRP conjugation. The sodium periodate method is particularly popular due to its simplicity and reproducibility.

Glutaraldehyde Two-Step Method:

1. Principle: Glutaraldehyde acts as a bifunctional reagent that covalently binds to both the enzyme and the amino groups on the immunoglobulin, forming an enzyme-glutaraldehyde-immunoglobulin conjugate.

2. Labeling Steps:

• (1) Dissolve 25 mg of HRP in 1.25% glutaraldehyde solution and let it stand overnight at room temperature.
• (2) Purify the enzyme solution using a Sephadex G-25 column with physiological saline as the eluent, collecting brown fractions. If the volume exceeds 5 ml, concentrate to 5 ml using PEG.
• (3) Dilute 12.5 mg of the target antibody to 5 ml with physiological saline and add it dropwise to the enzyme solution while 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 at room temperature for 2 hours.
• (6) Slowly add an equal volume of saturated ammonium sulfate, let it stand at 4°C for 1 hour, then centrifuge at 3000 rpm for 30 minutes.
• (7) Wash the precipitate twice with half-saturated ammonium sulfate, then dissolve it in 0.15 M phosphate buffer (pH 7.4).
• (8) Dialyze the solution against 0.15 M PB (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 a specific antigen or antibody and an enzyme-labeled antibody in a two-way agar diffusion or immunoelectrophoresis test. Color the precipitation arc with the enzyme substrate to assess activity, and determine the optimal working concentration via direct ELISA.
• (2) Quantitative and molar ratio determination: Measure absorbance at 403 nm and 280 nm using a spectrophotometer.

Enzyme Amount (mg/ml) = OD403nm × 0.4

IgG Amount (mg/ml) = (OD280nm – OD403nm) × 0.42 × 0.94 × 0.62

Molar Ratio = (Enzyme Amount / 40,000) ÷ (IgG Amount / 160,000) = Enzyme Amount × 4 / IgG Amount

4. Disadvantages: This method has low enzyme utilization, typically only 2–4% of the enzyme binds to the protein.

5. Reagents and Equipment:

• (1) 0.1 M pH 6.8 PBS: 449 ml of 0.2 M Naâ‚‚HPOâ‚„ + 451 ml of 0.2 M NaHâ‚‚POâ‚„ + 1.8 g NaCl + distilled water to 200 ml.
• (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): 3 ml of 1 M Naâ‚‚CO₃ + 7 ml of 1 M NaHCO₃.
• (4) 0.2 M lysine: Dissolve 29.2 mg in 1 ml of 0.01 M carbonate buffer (pH 9.5).
• (5) 0.15 M PBS and physiological saline.
• (6) Saturated and semi-saturated ammonium sulfate solutions.
• (7) Naphthalene reagent, PEG (MW 2000).
• (8) Purified specific antibody or anti-IgG.
• (9) HRP (RZ > 3.0).
• (10) Sephadex G-25 column (2 cm x 50 cm).
• (11) Stirrer, spectrophotometer, centrifuge.
• (12) Dialysis bags, beakers, test tubes, pipettes.

Shanghai Jinma Biological Products Include:

• 1. Human ELISA kits
• 2. Animal ELISA kits
• 3. Plant ELISA kits
• 4. Food ELISA kits
• 5. Agricultural product ELISA kits
• 6. Cell ELISA kits
• 7. Other ELISA kits

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