Virus antigen was purified using cesium chloride gradient centrifugation and dialysis against 10mM HEPES buffer, pH 7.4, 10% glycerol and 1mM EDTA. Nine male BALB/c mice of approximately six weeks were immunized intraperitoneally (i.p.) as follows20: injection of 50μg adenovirus hexon protein (pure antigen) diluted in Freund's adjuvant. The booster dose was performed with i.p. injection of approximately 50μg antigen in Freund's incomplete adjuvant. The third dose was performed i.p. injection of approximately 50μg antigen in Freund's incomplete adjuvant. The fourth dose was administered with injection with 50μg antigen in Freund's incomplete adjuvant. Finally, spleen cells were used for the fusion.

Production/purification of polyclonal antibodies in rabbit was performed as previously described.20–22

Selection of positive mice, i.e., those that produced adenovirus antigen-specific antibodies, was based on the result of indirect ELISA test. The plate was coated with purified “adenovirus hexon” diluted in 0.05M carbonate/bicarbonate buffer, pH 9.6 (50μL/well), at a concentration of approximately 10μg/well. The plate was incubated overnight at 4°C and subsequently washed once with 0.05% PBST, and blocking solution (1% tryptone, 1% BSA, 3% sucrose (150μL/well)) in PBS for 1h at 37°C was then added. The plate was washed once with 0.05% PBST. Next, 50μL of sera were added to the ELISA plate, which was then incubated at 37°C for 1h. The negative control was PBS. The plate was washed 3 times with 0.05% PBST and then incubated with secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Immunopure Pierce), diluted 1/10,000 in 0.05% PBST. Peroxidase activity was revealed by the addition of ELISA substrate TMB BD OptEIA. The reaction was stopped by adding 50μL/well of 3N H2SO4 and the plate read on a TP Reader Thermo Plate at a wavelength of 450nm.20

Fusion was performed according to the protocol proposed by Dr. Leticia Barboza's group (Bacteriologia – Instituto Butantan). Myeloma cells from the Sp2/0-Ag14 line (ATCC CRL 1581-Köhler and Milstein, 1976) were purchased from the Rio de Janeiro Cell Bank (BCRJ). Cells were previously thawed, grown in pre-fusion medium (RPMI 1640 medium (Invitrogen) plus 10% NCTC (Invitrogen) and 10% FBS) and kept in an incubator at 37°C in 5% CO2.

For the preparation of hybridoma conditioned medium (feeder), mouse macrophages of the BALB/c strain were obtained by the intraperitoneal lavage procedure. Macrophages were obtained by introducing 5mL of RPMI 1640 medium (4°C) into the peritoneum. The medium was withdrawn with the macrophages and then centrifuged at 1200rpm for 5min. Finally, the cells were resuspended in RPMI 1640 medium with 10% NCTC and 10% FBS and incubated at 37°C in 5% CO2.

Initially, two 25-cm2 and three 75-cm2 flasks of SP20 cells were used in the assay. The cells were then counted in a Neubauer chamber and placed in a 5% CO2 incubator at 37°C, in approximately 5mL of pure DMEM. The BALB/c mice received the fourth booster dose of 50μg of purified “adenovirus” diluted in PBS three days prior to the fusion assay. The immunized animals were sacrificed and the spleen and lymph nodes removed. The spleen and lymph nodes were placed on a screen on top of a beaker and DMEM was injected with a needle and syringe (they swelled and burst easily and the cells fell through the screen). The tissue was macerated on the screen with the plunger of a glass syringe. The macerates were placed in a Falcon tube and centrifuged at 1200rpm for 5min. The supernatant was discarded and the splenocytes and lymph node cells were resuspended in 5mL of medium. The cells were then counted in a Neubauer chamber. SP2 cells and splenocytes (in 1:2 ratio) were mixed in a Falcon tube. Next, 10mL of pure DMEM were added and the tube was centrifuged at 1200rpm for 5min. The supernatant was discarded with the aid of a Pasteur pipette and the pellet was gently broken with 0.5mL of pure DMEM. Subsequently, 1mL of PEG was added with 50μL of DMSO for 1min, dropwise with slow stirring, in a beaker with water at 37°C, after which time the cells were allowed to remain another minute in the beaker. Subsequently, 2mL of pure DMEM were added dropwise with gentle shaking; an additional 20mL of pure DMEM were then added, and the cells were allowed to stand for 4min in water at 37°C. Thereafter, the tube with the cells was centrifuged at 1200rpm for 5min at room temperature. For lymph node cells, the pellet was resuspended in 10mL of complete medium: DMEM+3% HAT+15% FBS and plated (100μL) in a 96-well plate. For splenocytes, the pellet was resuspended in 30mL of complete medium: DMEM+3% HAT+15% FBS and plated (100μL) in three 96-well plates. Finally, the cells were incubated at 37°C, 10% CO2. The medium was changed every three days. After one week, 3% HAT was substituted with 2% HAT for another three weeks.21

The plate was sensitized with adenovirus (serotypes 2, 3, 5, and 41) diluted with 0.05M carbonate/bicarbonate buffer, pH 9.6 (50μL/well), at a concentration of approximately 0.5μg/mL. In addition, all hybridoma supernatants were tested against parainfluenza virus (negative control) respiratory tract cell antigens at the same concentration, with overnight incubation at 4°C. Subsequently, the plate was washed once with 0.05% PBST and then incubated with blocking solution (1% tryptone, 1% BSA, 3% sucrose) in PBS (150μL/well) for 2h at 37°C. Wells were washed once with 0.05% PBST and then 100μL of purified monoclonal antibodies (5μg/mL) were added, followed by incubation at 37°C for 1h. The plate was incubated with anti-adenovirus monoclonal antibody (positive control) at 1/3000 dilution and washed six times with 0.05% PBST, and the secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) (Immunopure Pierce), diluted 1/10,000 in PBS, was added, followed by incubation for 40min at 37°C. The reaction was stopped by adding 50μL/well of 3N H2SO4 and TMB BD OptEIA was added as substrate. The plate was read on a plate spectrophotometer (TP Reader Thermo Plate) at 450nm.21

ELISA for isotype was performed as previously described.21

The antibody(s) were resuspended (at a final concentration of 1mg/mL) in 0.2M NaCl–borate buffer pH 9.0, and then dialyzed against 2mM borate–NaCl buffer, pH 9.0 for 2h. The amount of antibody required to stabilize gold was determined: 100μL of colloidal gold (20nm, 1 OD or 0.01%, BBI) was added in aliquots containing 10μL of dilutions of dialyzed monoclonal antibody (in 2mM borate). After 10min, 11μL of 10% NaCl was added to each tube. The amount of antibody sufficient to stabilize the gold was the dilution in which the solution did not change color, i.e., insufficient amount of antibody changes the coloration of the solution from red to blue. Considering a dialyzed solution of antibodies at the approximate concentration of 1mg/mL, the volumes of 1–4μL were tested. Subsequently, the pH of the gold to be conjugated to pH 9.0 was adjusted with 0.1M K2O3 solution.

The colloidal gold was added to the stirring antibody, allowed to react for 30min at room temperature (10mL of gold colloidal in the amount of antibody determined in step 3). In order to stabilize the reaction, a 10% BSA solution (10% BSA in 0.01M borate buffer, pH 9.0) was mixed in sufficient volume to reach a final concentration of 1% and incubated for 30min. The solution was centrifuged at 15,000×g for 20min at 4°C and the supernatant discarded. The pellet was resuspended in 2mL of a 2% BSA in 0.01M borate buffer and centrifuged at 12,000×g for 20min at 4°C. Finally, the pellet was resuspended in 1mL of buffer (3% BSA, 3% sucrose, 0.01M sodium borate and 0.05% sodium azide) and stored at 4°C.23

Each paper type was treated separately before assembling the tape, as shown below: the sample pad(s) were immersed in buffer (20mM sodium borate buffer, 0.1% Triton X-100 and 0.1% sodium azide, pH 8.5) and stored in a vacuum chamber at room temperature for 2h or held at 56°C for 2h. Conjugate pad(s) (solid phases with conjugate) were soaked with 20μL of the pAb-gold solution. The fibers were then kept in an oven at 37°C for 2h. Nitrocellulose membranes (HiFlow Plus nitrocellulose 180 – Merck) were incubated with 0.1M Tris–HCl, pH 7.5, with 0.4% glycerol for 15min on a shaker. After immersion, the membranes were washed 1× with 10mM Na2HPO4 wash buffer pH 7.5 and then placed in the oven at 37°C for 18–20h for drying. Subsequently, 1.5μL 6D-1G monoclonal antibody (concentration 1.2mg/mL) diluted in 0.01M PBS pH 7.2 (test line) were applied. Then, 1.2mg/mL rabbit anti-IgG was applied after 0.5cm of the test line (control line). The membranes were then kept in a vacuum chamber for 2h. The strips were set for drying at room temperature overnight or in the vacuum chamber for 2h. The absorbing pad(s) (solid absorption phases) need no treatment. The diluent (0.01 PBS containing 0.1% Triton X-100 and 0.5% BSA) improved the migration rate of the sample (1:1 diluent/sample). For assembling the tests, the pretreated papers were overlapped according to Fig. 1.23

Fig. 1.

Prototype of immunochromatographic test for detection of adenovirus. Test line (positive) and control line (validation).

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