Immunoadsorption: Principle and Procedure | Molecular Biology

In this article we will discuss about the principle, requirements and procedure for immunoadsorption.

Principle:

Antigen-antibody complexes are formed when transferred proteins on nitrocellulose paper are allowed to react with diluted antis- era. This antigen-antibody complex bound to the nitrocellulose membrane are allowed to react with alkaline phosphatase conjugated anti-rabbit-IgG. The colour develops blue to reaction with BCIP/NBT substrate which helps to identify the presence of antigen in the samples taken.

Requirements:

Reagents:

1. Tris buffered saline (TBS) pH 8.0:

Tris HCl (pH 7.5) 100 mM 1.211 g

NaCl 0.9% (W/V) 8.766 g

Distilled water to 1000.000 ml

store at 4°C for several days.

2. TBST:

TBS with Tween 20 0.10%

3. Blocking solution:

2% BSA in TBST

4. Primary antibody (experimental an­tiserum):

i. 1:1000 dilution of the antiserum in fresh blocking solution.

ii. 1:1000 dilution of preimmune se­rum.

5. Secondary antibody:

1:25,000 dilution of alkaline phosphatase conjugated anti-rabbit IgG in blocking solution.

6. AP buffer (pH 9.5):

Tris HCl: 100 mM

NaCl: 100 mM

MgCl₂ : 5 mM

7. AP substrate:

Dissolve 1 tablet of sigma fast BCIP/NBT buffered immunolocalisation substrate in 10 ml of buffer. Readymade commercial substrate also is available.

8. EDTA 20 mM in TBS.

9. Whatman 3 mm filter paper.

10. Shaker.

Procedure:

1. Wash nitrocellulose paper with protein in TBS.

2. Incubate blocking solution with agitation for 1 hour at room temperature.

3. Wash thrice in TBST and TBS for 1 hour with constant agitation.

4. Incubate the nitrocellulose paper with experimental antiserum at room tempera­ture (37°C) for 1-2 hours with constant shaking.

5. Wash for 1 hour with four changes using wash buffer.

6. Incubate for 1 hour the nitrocellulose paper with alkaline phosphatase conju­gated secondary antibody with constant shaking at room temperature (37°C).

7. Wash for 1 hour with four changes using wash buffer. The secondary antibody can be removed and stored for further use.

8. Nitrocellulose paper is equilibrated in AP buffer for 10 minutes and dried on Whatman 3 mm filter paper.

9. Immediately transfer the nitrocellulose paper to 10 ml of BCIP/NBT substrate solution.

10. Within 10-15 minutes coloured bands develop.

11. Wash nitrocellulose paper thoroughly with water and stop reaction with 20 mM EDTA, when maximum colour develop­ment takes place.

Compare with a replica blot treated in the same way except that pre-immune serum is used instead of experimental serum.