Detection of a Food Allergen by Immunoblotting

Detection of a food allergen by immunoblotting

Introduction

The lab provides an introduction to the basic steps involved in using antibodies to detect specific proteins that have been "blotted" or attached to a nitrocellulose membrane. Virtually any protein will bind very tightly to nitrocellulose when allowed to dry on the membrane. The immunoblot technique is used to demonstrate that certain proteins are present only in a particular tissue or species. The protocol differs from western blotting in that proteins are not separated by electrophoresis and then transferred to the membrane. Instead, they are simply spotted directly on the membrane, creating what is known as a "dot blot". These blots can then be processed through the standard immunodetection steps - blocking, antibody binding, enzyme-labeled secondary antibody binding (if necessary), and enzyme reaction - in about one hour instead of the more than five hours required for western blots.

Two basic formats for an immunoblot (or other immunoassay) are direct and indirect. In a direct assay, the antibody against the protein of interest (i.e. the primary antibody) is labeled for detection. A common label is an enzyme with a color-producing substrate, such as horseradish peroxidase or alkaline phosphatase. Binding of the labeled antibody is detected by color production on the membrane. In an indirect assay, an unlabeled primary antibody is allowed to bind the protein of interest, and then an enzyme-labeled antibody which will bind the first antibody (i.e. a secondary antibody; an antibody produced against antibodies) is added. The enzyme labeled- secondary antibody will bind, and color will produced, only if the primary antibody bound to target protein in the sample. While the direct assay involves fewer steps (and therefore is quicker and less expensive), the indirect method usually generates a stronger signal. Also, the indirect method is convenient if one is analyzing several different proteins, since the same labeled secondary antibody can be used with several unlabeled primary antibodies. We will be using the direct assay procedure.

Almost any protein can be detected by this method, as long as you have a specific antibody which will recognize that protein. Sigma Chemical Co. sells hundreds of different antibodies, including the secondary antibodies necessary for indirect assays. In this exercise we will perform a direct immunoassay, using an enzyme-labeled antibody against the wheat protein gliadin, which is a gluten protein thought to be responsible for allergic reactions to wheat products. The protein samples spotted on the membrane will be extracts from various flours. We will test whether the antibody against wheat gluten protein reacts with flours other than wheat. This information might be important for someone with an allergy to wheat, since proteins with similar antibody reactivities (i.e. they are crossreactive) often induce similar allergic responses in an individual.

Reagents and Supplies needed

Nitrocellulose membrane, preferably 0.45 µm pore size Antibody to wheat gliadin, labeled with horseradish peroxidase PBS with 3% casein Color reagent (4-chloro-1-naphthol dissolved in 70% ethanol) Hydrogen peroxide, 3% Micropipettor or other device to deliver about 5 µl Filter paper Transfer pipets gloves and/or forceps petri dishes Samples: Suspend 0.2 g dry material in 1 ml PBS. If using fresh (wet) material, grind 1 g in 1 ml PBS. Mix vigorously. Allow insoluble material to settle out or centrifuge briefly. The samples we are using include wheat flour, corn meal, barley flour, rye flour, spelt flour, amaranth flour, potato flour, and oat flour. Which of these do you expect to react with antibody to wheat gluten protein? Why?

Procedure

1 NOTE: Do not touch nitrocellulose with bare hands, or the proteins on your hand will bind to the membrane. Wear gloves and/or use forceps to handle the membrane, and try to handle the membrane only by the edge.

1. Place the nitrocellulose (NC) on a piece of filter paper.

2. Label the NC as illustrated below, using a ball-point pen, using abbreviations to indicate what samples will be applied to each area of the nitrocellulose. Do not actually draw circles in which to place the samples, since they may interfere with the detection step.

3. Using a capillary pipet or other micropipettor to deliver about 5 µl, place a drop of sample above its label on the NC. (Other pipets, such as pasteur pipets, will work, but will usually create larger drops that will take up more space on the membrane.)

Nitrocellulose

Sample spot B C

4. Allow the spots of sample to dry for about 10 min.

5. Place dried membrane in a petri dish. Add PBS with 3% casein to the petri dish and allow the blot to block for 5 min., occasionally swirling the dish. This step ties up any remaining protein-binding sites on the NC so that the antibody does not bind unless the antigen is present.

8. Pour off PBS/casein and add 10 ml antibody to the dish.

9. Incubate the blot in antibody for 20-30 min., swirling the dish occasionally.

10. Pour off the antibody. Rinse the blot twice with PBS by pouring about 20 ml (fill the dish about one third full), into the dish, swirling briefly, and pouring off the PBS. Repeat this rinsing step.

11. For color detection, add 20 ml PBS to the dish. Add 1 ml of color reagent and 0.2 ml of 3% hydrogen peroxide. Swirl to mix the reagents, then allow the dish to sit undisturbed.

12. A purple precipitate will appear wherever the antibody bound to the blot. Color development should be visible in a few minutes, but may take up to 15 min. to develop fully.

13. When spots are visible, rinse the blot thoroughly with distilled water or PBS, before the entire blot turns purple. Allow the blot to air dry and store protected from light.

14. Record your results.