Double-Blotting Protocol - Creative Diagnostics

Introduction of Double-Blotting Protocol

The development of a test for anti-doping control of erythropoietin(EPO), a hormone used in endurance sport to stimulate red blood cell production, has been a long and exacting task. The method is based on differentiation of natural and recombinant(used in case of doping) hormones in urine by their isoelectric profiles. For this, urine is first submitted to ultra-filtration to concentrate EPO in retentates that are then subjected to isoelectric focusing. Immunoblotting of EPO is then performed using primary monoclonal anti-human EPO antibodies and secondary biotinylated goat anti-mouse IgG antibodies. The major drawback of the ultrafiltration step is the resulting very high protein content of the retentates that are then subjected to the next step of isoelectric focusing. This is particularly true for urine samples taken at the end of a competition, due to proteinuria induced by physical exercise. This results in retentates with huge total protein contents(about 50 g/L for samples taken at rest and up to 200 g/L for samples taken after a physical exercise) for an EPO concentration generally no more than 4 μg/L. Such a situation is a real challenge for the classical immunoblotting procedures. Indeed, a strong nonspecific binding of secondary antibodies to some proteins presentin the retentates was observed, completely masking the detection of EPO. All attempts to prevent or reduce this nonspecific binding were ineffective when working directly on the blotting membrane. The problem was solved by isolating the primary antibody from the interfering proteins on a second membrane that was then probed by the secondary antibody without any risk of nonspecific binding. For this, after it has been probed by the primary antibody, the membrane with the blotted proteins is assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules are thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remain bound to the first one. The second membrane can then be probed by the secondary antibodies without the risk of nonspecific binding.

Materials of Double-Blotting Protocol

Since double blotting (DB) takes place after probing of the blotting membrane (B membrane) with a primary antibody and before probing with the secondary one, the reagents and materials for these steps (blotting membrane, blocking and washing buffers, primary and secondary antibody solutions, possibly amplification system, development reagents) will not be indicated here, being specific for the application in which DB is introduced Only the materials used for the DB step itself will be detailed.

Semidry transfer unit.
Roller.
Immobilon P polyvinylidene fluoride membranes (PVDF), 0.45μm pore size.
Durapore hydrophilic PVDF membranes, 0.65μm pore size
Filter paper sheets: Electrode paper Novablot.
0.7 % (v/v) acetic acid solution.
Phosphate buffered saline (PBS) pH 7.4.
Sealing film, Parafilm.

Methods of Double-Blotting Protocol

Proceed to the usual blotting, blocking, primary antibody probing and washing steps of your B membrane according to your application. DB is performed after the last wash of your B membrane.
Cut two stacks of nine filter paper sheets, a Durapore (intermediate membrane) and an Immobilon P(DB membrane) membrane to the dimensions of the blotting membrane.
Condition them in 0.7 % acetic acid: just immerse the Durapore in the acidic solution for at least 10 min, pre-wet the Immobilon P membrane in methanol for 3s, rinse in water for 2 min before equilibration in acidic solution for 10 min. The stacks of filter paper are moistened in acetic acid solution by capillary action.
During the same time, perform a rinsing of the B membrane in two changes of PBS.
Layer the B membrane of your application onto a first stack of filter paper with the blotted proteins facing up and cover it with the intermediate and DB membranes successively. Quickly put the second stack of filter paper onto the DB membrane to prevent the membranes from drying.
Position this sandwich on the anode plate of the semidry electrophoretic blotting instrument so that the B and the DB membranes will face the anode and the cathode, respectively.
Place a sealing film onto the sandwich and carefully press out the air bubbles with the roller. Remove the film.
Place the cathode plate on the sandwich and connect the blotting instrument to the power supply.
Apply a constant intensity of 0.8 mA per cm² for 10 min.

Figure 1. Experimental set-up for DB.

Disconnect the blotting instrument and disassemble the membranes.
Keep the B membrane in PBS at 4 °C.
Rinse the DB membrane quickly in two changes of PBS.
Proceed to the blocking of the DB membrane according to your usual procedure.
Proceed to the usual steps of your application from the probing with the secondary antibody to the final development, on the DB membrane.

Notes of Double-Blotting Protocol

Reagents for detection of erythropoietin. The method is illustrated by its application to immunodetection of erythropoietin. The specific reagents used in the case of EPO are:
(a) Primary antibodies: monoclonal mouse anti-human EPO.
(b) Secondary antibodies: biotinylated goat anti-mouse IgG(H + L).
(c) Amplifying systems: Streptavidin:biotinylated peroxidase complexes.
(d) Development system: Chemiluminescence.
(e) Blocking buffer: 5 % (w/v) nonfat milk in PBS buffer.
DB has been developed using PVDF as blotting membrane and has not been tested with other types of membranes.
Be aware that the DB membrane is very sensitive to drying when not saturated. This, while being not visually perceptible during the handling of the membrane, will produce a high background in the final image. It is thus extremely important to quickly perform the steps in which the membrane is handled without any liquid contact.
The acidic pH of this step induces dissociation of the primary antibody molecules from their corresponding antigen. The released antibodies, being positively charged due to the acidic pH, migrate towards the cathode, passing through the intermediate membrane and thus are transferred onto the DB membrane. Since the acidity does not affect hydrophobic interactions with PVDF, the antigen and the unrelated proteins (interfering proteins and proteins used for blocking the B membrane) are retained on the B membrane. It should be noted that the result is actually an "image" of the probed antigen since it is only the antibody on the second membrane and not the antigen that gives rise to the final signal. However, this image is quite representative of the probed antigen.
The interposition of the intermediate membrane between the B and the DB was very useful in our application (EPO analysis) which uses nonfat milk as a blocking agent, some "holes" in the final image obtained with the DB membrane being sometimes observed when this intermediate membrane was omitted. Though the explanation for this is purely hypothetical (local releases of clumps of casein precipitated by the acidic pH), the interpolated microporous Durapore membrane worked as a barrier definitely remedying this problem. It is possible that this membrane is not necessary in other applications but its use is strongly advised in case of "holes" in the final image.
The use of an electric field speeds up the transfer of the primary antibody from the blotting to the DB membrane. However, a simple contact between the membranes (passive transfer) without applying an electric field for a prolonged time (30 min) has been tested in dot blot experiments and proved to be usable too.
Storage of the B membrane (on which the blotted proteins are retained) in PBS may be useful. If some problem is observed on the final image, e.g., high background, it is possible to reuse the stored B membrane to perform a second DB. In this case, the B membrane is re-incubated in the primary antibody of the application and DB is then performed as described above. If desired, it is possible to probe the stored B membrane with a different primary antibody prior to performing a second DB. This enables obtaining an image of another antigen from the same sample.