A simple sodium periodate method for HRP labeled antibodies

In this method, NaIO4 first oxidizes the sugar molecules on the surface of HRP to aldehyde groups, and then combines with the amino group on Ig. The yield of the obtained enzyme-labeled antibody is high. Combined with the enzyme, there is no significant loss of the activity of the enzyme and Ig, which is currently the most commonly used method.

1. Principle

In the classic sodium periodate method, dinitrofluorobenzene needs to be used to block the remaining α- and ε amino groups on HRP to avoid crosslinking between enzyme molecules. Later, Wilson et al. Used NaIO4 to oxidize HRP at low pH, thereby eliminating the dinitrofluorobenzene blocking HRP step. The aldolase formed by the oxidation of HRP by NaIO4 can be linked to the amino group of the antibody molecule to form Schiff's base, which can be further reduced with NaBH4 (or ethanolamine) to generate stable enzyme-labeled antibodies.

2. Reagents and equipment

(1) 0.1M NaIO4: Weigh 241mg sodium periodate (Guangzhou Chemical Reagent Factory, batch number 830602) and dissolve it in 10ml of distilled water.

(2) 1mM PH4.4 sodium acetate buffer: 0.2M NaAc (1.361g / 50ml) 3.7ml; 0.2M HAc (0.601ml / 50ml) 6.3ml; add distilled water to 2,000ml.

(3) 0.2M PH9.5 carbonate buffer: Na2CO3 0.32g; NaHCO3 0.586g; add distilled water to 50ml, and then use distilled water to make a 20-fold dilution, which becomes 0.01M PH9.5 carbonate buffer.

(4) NaBH4 solution (4mg / ml): Weigh 4mg of NaBH4 in 1ml of distilled water before use.

(5) For other reagents and equipment, please refer to the glutaraldehyde labeling method.

3. Marking steps

(1) Weigh 5mg HRP and dissolve it in 1ml distilled water.

(2) Add 0.2ml of newly prepared 0.1M NaIO4 solution to the upper solution, and stir at room temperature for 20 minutes in the dark.

(3) Put the above solution into a dialysis bag, dialyze against 1 mM pH 4.4 sodium acetate buffer, and overnight at 4 ℃.

(4) Add 20μl of 0.2M PH9.5 carbonate buffer to raise the pH of the above aldated RP to 9.0-9.5, then immediately add 10mg IgG (antibody, or SPA 5mg) in 1ml 0.01M carbonate In the buffer, stir gently at room temperature in the dark for 2 hours.

(5) Add 0.1ml of freshly prepared 4mg / ml NaBH4 solution, mix well, and set at 4 ℃ for 2 hours.

(6) Put the above solution into a dialysis bag, dialyze against 0.15M PH7.4 PBS, overnight at 4 ℃.

(7) Add equal volume of saturated ammonium sulfate dropwise with stirring and set at 4 ° C for 1 hour.

(8) Centrifuge at 3000 rpm for half an hour, discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate, and finally the precipitate was dissolved in a small amount of 0.15M PH7.4 PBS.

(9) Put the above solution into a dialysis bag, dialyze against 0.15M PH7.4 PBS buffered saline, remove the ammonium ion (detected with naphthalene reagent), centrifuge at 10,000 rpm for 30 minutes to remove the precipitate, the supernatant is the enzyme The conjugate is stored frozen after aliquoting.

4. Result judgment

Except for the calculation of the amount of marker IgG, which is slightly different, the rest are the same as the glutaraldehyde method.

IgG amount (mg / ml) = (OD280nm-OD403nm × 0.3) × 0.62