Monoclonal Antibody Screening
Direct ELISA can be applied in monoclonal antibody screening when it is taken as a semiquantitative assay.
- PBS: 20 mM NaPi pH=7.5, 150 mM NaCl
- PT: 0.1% Tween 20 in PBS
- Blocking solution: 3% BSA in PT. Store at -20 C
- Developing solution: 10 mg o-phenylenediamine in 25 ml of a buffer with pH=5.5 (say, 20 ml of H2O + 5.16 ml of 0.5 M Na2HPO4 + 645 μl of 1.5 M citric acid) + 12 μl of 30% H2O2. This solution is light sensitive and should be prepared just before use.
1- Coat the plate wells with 50 μl of antigen (0.2 μg/ml in PBS). Incubate 1h at RT
2- Wash the plate (discard the well solution, add 200 μl of PT per well and wait about 2 min. Repeat this cycle another 2 times and discard the late well solution)
3- Block the plate wells with 150 μl of blocking solution. Incubate 1h at RT
4- Discard the blocking solution and add 50 μl of the hybridoma supernatants in different wells. Incubate 30 min at RT
5- Wash the plate
6- Add 50 μl of the peroxidase-coupled antimouse Igs antibody (1/5000 in blocking solution). Incubate 30 min at RT
7- Wash the plate
8- Incubate the plate wells with 100 μl of developing solution until color change is evident
9- Stop the reaction by adding 50 μl of 2.5 M H2SO4 per well
10- Measure the absorbance at 492 nm
- This is only a semiquantitative assay. If you want a quantitative assay, you have to dilute the primary antibody (step 4) at least 1/20 in blocking solution and probably much more. Anyway, direct ELISAs are adequate for quantitative assays only with pure samples. In any other case, you should use indirect ELISAs or sandwich ELISAs.
- Sodium azide is an inhibitor of peroxidase.
Learn more applications of ELISA: