Minitube Immunoblot

Background

One of the major lines of defense against foreign substances entering the body of mammals is called the humoral, or body fluid immune response. Humoral immunity is based on the production of special cell markers called immunoglobulins (antibodies) which detach from the cells that produce them and circulate in the bloodstream, lymph, and other body fluids. Each immunoglobulin is specific in its ability to bind to a foreign substance called an antigen at key portions of the antigen molecule called antigenic determinants or epitopes. Once immunoglobulins have become bound to antigens, they can facilitate several reactions, including precipitation of the antigen out of the fluid, agglutination or clumping of antigens together to facilitate their removal from the body, detoxification of poison substances, and delivery of nonspecific immune chemicals such as complement which lyses cells and enhances phagocytosis by wandering macrophages and neutrophils. The study of antibody-antigen reactions is called serology, and knowledge of serological reactions has led to the development of many techniques for the identification of antigen-carrying cells, ranging from the well-known blood type agglutination reactions to complement fixation tests used to diagnose the presence of endotoxins released by gram-negative bacteria, and fluorescent antibody techniques used for the identification of human pathogens such as Treponema pallidum, the etiologic agent of syphilis.

In this lab, we will utilize a technique for the identification of a plant pathogen which utilizes two antibodies, a primary antibody specific for antigens expressed on the surface of the tobacco mosaic virus (TMV), and a secondary antibody produced against the primary. The secondary is called a conjugated antibody, since it carries an enzyme called alkaline phosphatase which has been chemically attached to it. The primary antibody is produced by injecting laboratory rabbits with TMV, which, while not pathogenic to the animals, elicits the production of antibodies which circulate in the serum. Blood is removed from the rabbits and the serum fraction containing the antibodies purified. Some of the primary antibodies are then injected into the bloodstream of goats, which stimulates the production of the secondary immunoglobulin. Secondary antibodies are then purified and chemically bound to alkaline phosphatase enzyme.

Fluid taken from a plant suspected to be infected with TMV is placed on a nitrocellulose membrane and allowed to dry. The TMV antigen, if present, will adhere to the membrane. The primary antibody will then bind to the antigen. Excess unbound antigen and antibody are washed away, then the membrane is exposed to the conjugated secondary antibody. The secondary will bind to the primary, exposing the alkaline phosphatase enzyme. Excess secondary antibody is then washed from the membrane. Finally, a substrate for the enzyme called nitroblue tetrazolium (NBT) is added. If the antigen-primary-secondary complex is present on the membrane, the enzyme will react with NBT and cause a blue color to appear.

Materials

Whatman filter paper
goat-anti-rabbit IgG AP conjugate (Sigma # A0418)
1X TBS (Tris-Buffered Saline pH7.4)
Sterile diH₂0
2% nonfat dry milk in PBS-Tween
sterile plastic petri dishes
Sigma FastTM BCIP/NBT tablets (Sigma # B5655
10 X 75 mm disposable culture tubes
0.2 mm nitrocellulose sheets cut into 2 X 80 mm strips
TMV-specific polyclonal antibodies (ATCC # PVAS-135 or freshly prepared rabbit anti-TMV)
PBS-Tween (PBS, 0.05% Tween-20)
1000 ml and 10 ml pipettors
vinyl or latex gloves

Procedure

Remember to always wear gloves when preparing and handling samples.

1. Label two culture tubes C (control) and T (test).

2. Place approximately 0.1 g fresh cigarette tobacco in a 1.5 ml eppendorf microcentrifuge tube. Add 1000 ml sterile 1 X TBS and grind with a mini pestle or clean glass rod until the tobacco is finely macerated.

3. Place approximately 0.1 g fresh tobacco leaf in a separate tube and repeat step 2.

4. Place the two samples in a microcentrifuge and spin for 1minute to pellet the leaf matter.

5. Soak two nitrocellulose strips in diH₂O in the bottom of a plastic petri dish until they are completely wetted. Remove and place on a paper towel. Gently blot with a chemwipe to remove excess moisture.

6. Remove the strips from water and place them on a sheet of filter paper. Dot 2 ml anti-rabbit 2^o antibody on one strip near the bottom (positive control) and 2 ml sterile diH₂O approximately 1 mm above the first dot (negative control). Allow the membrane to air dry (see diagram).

7. On the second strip, dot 2 ml cigarette supernatant, then 2 ml of the ground tobacco leaf supernatant approximately 1 mm above the cigarette dot. Allow the strip to air dry.

Note: Air dried samples can be sealed in a dry plastic Ziplock bag and stored at room temperature for several days prior to use.

8. Soak dried nitrocellulose strips in 1X TBS for 20 seconds, then transfer them to the appropriate tubes.

9. Add 1000 ml of the blocking buffer to each tube and incubate for 20 minutes at room temperature.

10. Prepare 2000 ml 1:5000 dilution of the primary antibody.

11. Discard blocking buffer, rinse once with diH₂O, then add 1000 ml of the primary antibodies to each tube. Incubate for one-half hour at room temperature.

12. Prepare 2000 ml 1:1000 dilution of the secondary (anti-rabbit goat) antibody.

13. Discard primary antibody, rinse once with diH2O, then add 1000 ml of the secondary antibody to each tube. Incubate at room temperature for one-half hour.

14. Discard secondary antibody, then rinse three times 10 minutes each, with PBS-Tween.

15. Prepare NBT-BCIP solution by adding one Sigma Fast^TM BCIP/NBT tablet to 10 ml diH₂O and mixing with a vortex until the tablet is dissolved.

16. Discard the wash, then add 400 ml of the BCIP/NBT solution to each tube. Immediately place the tubes into the dark and incubate until a blue color appears. Stop the reaction by immersing the nitrocellulose strips in diH₂O. Allow the samples to air dry.