Grid-Immunoblotting Protocol

Overview

Introduction of Grid-Immunoblotting Protocol

Grid-immunoblotting is a technique developed by Reese et al. that could alleviate many problems often encountered in allergy diagnosis and research. This method is particularly valuable for it requires only about 150–200 μL of the serum sample compared to ten times more serum required for enzyme-linked immunosorbant assay (ELISA). Furthermore, this technique permits simultaneous testing of up to 20 different antibodies against 20 different allergens. In clinical or laboratory settings, the amount of sera drawn from allergic subjects is often limited. This limitation really becomes a barrier when blood is drawn from allergic children or patients with complicated preexisting conditions who are unable to give sufficient amounts of blood.

Figure 1. Grid-Immunoblotting. (Reese G, et al.; 2001.)

The grid-immunoblotting procedure consists of three basic steps. First, the protein is immobilized on a carrier nitrocellulose membrane. This membrane is placed onto a multichannel manifold (Surf-blot apparatus) and various proteins are applied to the channels. After a 1-h incubation period, the membrane is washed and blocked. Second, the blot is incubated with the primary antibody. Finally, specific binding is detected using a detection system.

Materials of Grid-Immunoblotting Protocol

Protein Immobilization and Blocking

12.5 ×12.5 cm reinforced nitrocellulose membrane (pore size 0.45μm).
Cyanogen bromide (CNBr) used to activate the nitrocellulose membrane and improve protein binding.
Tris-buffered saline (TBS): 100 mM Tris–HCl, 100 mM NaCl, 2.5 mM MgCl₂ , pH 7.4.
Surf-blot apparatus (Idea Scientifi c, Minneapolis, MN, USA).
ELISA carbonate/bicarbonate coating buffer: 60 mM Na₂CO₃, 140 mM NaHCO₃, pH 9.6.
Allergen solutions (200 μL/channel).
0.01 % Pyronin Y solution (200 μL).
Washing buffer (TBST): TBS supplemented with 0.05 % Tween-20.
Blocking solution: 200 mL of TBST containing 1 % (w/v) nonfat dry milk powder.

Blot Incubation with Primary Antibody

Antibodies specific to the allergens.
Detection antibody.

Detection of Specific Binding with a Detection System

Alkaline phosphatase (AP) buffer: 100 mM Tris–HCl, 100 mM NaCl, 5 mM MgCl₂ , pH 9.5.
Substrate/chromogen mixture for alkaline phosphatase at 37 °C (450 μM 5-bromo-4-chloro-indonyl-phosphate disodium salt (BCIP) and 400μM nitroblue tetrazolium chloride (NBT) solubilized in AP buffer).

Methods of Grid-Immunoblotting Protocol

Protein Immobilization and Blocking

Activate reinforced nitrocellulose membranes with CNBr according to the technique used by Demeulemester and colleagues (can store this membrane at 4 °C for extended periods of time).
Soak the membranes for 1 min in Tris-buffered saline.
Place the soaked membranes onto the Surf-blot apparatus and assemble the apparatus according to the manufacturer's instructions.
Dilute the allergens in standard ELISA carbonate/bicarbonate coating buffer with a concentration ranging from 100 μg to 1,000 μg/mL.
Pipette 200 μL of allergen solution into each channel of the assembled Surf-blot apparatus except for the last channel. Coat the last channel with 200 μL of a 0.01 % Pyronin Y solution instead of the allergen in order to avoid confusion about the orientation of the membrane. This will result in a permanently stained pink lane on the blot.
Incubate the Surf-blot apparatus for 1 h at room temperature (RT) with end-over-end rotation .
Perform three washing steps (5 min each) with 2 mL per channel of washing TBST.
Disassemble the apparatus using a regular 1 mL pipette and block the unoccupied binding sites on the membrane with 200 mL of TBST containing 1 % (w/v) nonfat dry milk for 1 h at RT.
Rinse the membranes twice for 10 min in 200 mL of the washing buffer TBST and air-dry until usage.

Blot Incubation with Primary Antibody

For detection of specific antibody reactivity, soak the membrane in TBST for 1 min.
Arrange the membranes in such a way that the antigen-coated lanes on the membrane are perpendicular to the incubation channels of the manifold, thus permitting each antibody to interact with each of the 20 antigens.
Remove the excess buffer and pipette 150–200 μL of diluted (1:100) antibody-containing solution into the lanes.
Incubate the blot for 1 h with end-over-end rotation.
Perform another washing procedure (3×) with TBST and incubate the membrane in 100 mL of TBST containing 1 % nonfat dry milk powder and detection antibody for 1 h.

Detection of Specific Binding with a Detection System

Use calorimetric detection method to make the antibody binding visible.

Calorimetric Detection

Wash the membranes with 100 mL of TBST and 50 mL TBS- AP buffer for 5 min each.
Incubate the blots in substrate/chromogen mixture for alkaline phosphatase at 37 °C till spots appear.
Stop color development by washing with TBST.

Notes of Grid-Immunoblotting Protocol

For the detection of mouse antibodies, use 1:20,000 diluted alkaline phosphatase-conjugated goat anti-mouse IgG + IgM antibody. For the detection of IgE antibodies, incubate the blot with 1:1,000 diluted alkaline phosphatase-conjugated monoclonal anti-human-IgE.
Aspirate any TBS remaining in the channels before the membrane is coated with allergen.
Make sure that the antigen evenly coats the membrane and that no bubbles are trapped in the channels.
As an alternative to colorimetric detection, one can use a chemiluminescence substrate for alkaline phosphatase. The following materials ( see (a)–(c)) and methods ( see (d)–(g)) need to be used for this procedure.
(a) Washing assay buffer (100 mM diethanolamine/HCl, 1.0 mM MgCl₂ , pH 10).
(b) 1:20 diluted nitroblock chemiluminescence enhancer.
(c) 250 μM CSPD (disodium 3-(4-methoxy- spiro{dioxetane-3, 2'-(5' chloro) tricyclo [3.3.1.1. ^3,7 ] decan}-4-yl)phenyl phosphate; Tropix).
(d) Wash the blots with freshly prepared assay buffer (100 mM diethanolamine/HCl, 1 mM MgCl₂ , pH 10).
(e) Incubate the blots in the 1:20 diluted Nitroblock chemiluminescense enhancer for 5 min.
(f) Incubate the blots in 250 μM CSPD (disodium 3-(4-methoxy-spiro{dioxetane-3,2'(5' chloro) tricyclo[3.3.1.1. ^3,7 ] decan}-4-yl)phenyl phosphate; Tropix) for 5 min and drain any excessive liquid.
(g) Expose the blots to autoradiography film for 15, 30, 60, and 120 s sealing the blots between transparencies.

References

John M. Walker. Western blotting: methods and protocols. Humana Press. 2015, Electronic ISSN:1940-6029.
Reese G, et al.; Grid-immunoblotting: a fast and simple technique to test multiple allergens with small amounts of antibody for cross-reactivity. J Chromatogr B Biomed Sci Appl. 2001, 756(1-2):151-6.

Return to Resources

Reagent Solutions