Antibodies Plant/Algal / Toxins / Plant toxins

SANDWICH ELISA FOR DETERMINATION OF RICIN

Materials and Reagents
1. 96-well plates
2. Suitable plate reader (depending on the detection system)
3. Plate shaker with temperature control
4. Capture antibody (#177 YO Proteins)
5. Detection antibody (conjugate) (enquire)
6. Washing solution (PBS, 0.025% Tween 20, 1% D-lactose)
7. Solution for blocking and dilution (PBS, 0.025% Tween 20, 1% D-lactose,
0.1% BSA)
8. TMB reagent
9. Sulfuric acid 10%

Protocol
1. Immobilisation of capture antibody
Dissolve capture antibody in PBS at 5 μg/ml. Add 100 μl into each well. Incubate 2
hours at 37°C. Wash twice with 300 μl washing solution.
2. Blocking
Add 250 ml blocking solution. Incubate 1 hour at 37°C. Wash with washing solution
twice 300 μl.
3. Application of control and samples.
Add 100 μl to each well: antigen (ricin) diluted from 0.5 to 100 ng/ml, sample serially
diluted. Incubate 1 hour at 37°C. Washing 3 times 300 μl.
4. Detection antibody (example based on biotin conjugate)
Add 100 μl of biotin-conjugated antibody (1 μg/ml). Incubate 1 hour at 37°C.
Washing 3 times 300 μl (not shorter than 5 min each).
5. Streptavidin-HRP conjugate
Add 100 μl of Streptavidin-HRP conjugate (diluted 10000X). Incubate 1 hour at
37°C. Washing 5 times 300 μl (not shorter than 5 min each).
6. Development
Add 100 μl of development TMB solution. Incubate 10 min at 37°C. Stop reaction
with 10 μl 10% sulfuric acid. Read OD at 450 nm