Immuno Assays

ELISA

  • An ELISA is used to react each of the crude venoms or fractions with monoclonal antibodies produced against important venom proteins.

  • The procedure used to insure the production of a monoclonal antibody is a series of three limiting dilutions.

  • Each time the limiting dilutions are carried out, only one cell is in the last well to ensure monoclonal production.

  • In ELISA, 50 µL of each venom sample are added to individual wells of a 96-well microtiter plate and incubated at 37˚C for 30 min.

  • The plate is rinsed three times with 0.01 M phosphate-buffered saline (PBS), 0.15 M NaCl, pH 7.4. Approximately 500 µL of powdered milk (5% in PBS) are added to each well and incubated at 37˚C for 30 min.

  • The plate is again rinsed with PBS three times. Tissue culture fluid (50 µL) from each monoclonal antibody is added to the wells and the plate is again incubated.

  • Once again the plate is rinsed with PBS three times.

  • Fifty microliters of goat anti-mouse IgG conjugate with alkaline phosphatase are added to each well at a rate of 1:4 with PBS.

  • The plate is again incubated at 37˚C for 30 min.

  • After incubation, the microtiter plate is rinsed three times with Tween 80 (125 µl Tween 80, 250 ml PBS) and three times with Milli-Q water.

  • Fifty microliters of the color development reagent (i.e., 1 tablet of P-nitrophenyl reagent in 5 mL of 10 % Diethanolamine, pH 9.8) are added to each well and incubated for 30 min.

  • Following this, the absorbance is measured at 405 nm using a Beckman Coulter AD340 reader.

Limiting Dilution

  • Two hundred microliters of the hybridomas supernatant that tested positive by ELISA are transferred to the second row of a 96-well plate.

  • From row 3-well 1, a two-fold dilution is made of the supernatant well 2 (100 µl of supernatant from well 1 and 100 µl of complete media placed in well 2).

  • Two-fold serial dilutions are made until well 11 is reached. After 72 hr, cells are screened in wells 11 and 12. Wells 1-10 are saved for back-ups.

  • The positive cells are plated out again in a 96-well plate as described above.

  • A week later, the cells in the last two rows are screened. This procedure is repeated.

Western Blot

  • Four hundred microliters of a 5 mg/ml of crude sample are electrophoresed by Native Page gradient 8-25 (Rael et al., 1984).

  • The gel is placed in 10 ml of 0.07% acetic acid (transfer buffer) and placed at 37˚C for 15 min.

  • A transfer membrane (Immobilon™-PSQ) is soaked in methanol followed by water and placed over the gel.

  • The gel/membrane is placed between damp paper towels with the gel on top of the membrane.

  • A four-liter flask containing water is placed on top of the damp paper towels.

  • After 15 hr, the gel/membrane is soaked in distilled water to separate.

  • The membrane is blocked for 2 hr in 5% powder milk solution on a Junior orbitor shaker at 80 rpm.

  • The gel is developed by silver stain.

  • The membrane is rinsed with PBS four times and placed in 6 ml of tissue culture medium containing antibodies against a sample or fraction.

  • The membrane is incubated for 2 hr in the shaker.

  • The membrane is rinsed four times with PBS and 8 ml of the goat antimouse IgG with horseradish peroxidase enzyme (diluted in PBS 1:3000) are added to the membrane and incubated for 2 hr.

  • The blot is rinsed as before and developed using color reagents A and B.

  • Reagent A consists of 10 ml of TBS (0.02 M Tris-HCl buffer pH 8.0 with a 0.5 M NaCl) and six microliters of hydrogen peroxide.

  • Reagent B consists of 6 mg of 4-chloro-naphtol and 2 ml of methanol.

  • The mixture of reagents A and B is sufficient for one blot.