ELISA screening of ScFv
Materials
1. Prepare Coating Buffer: carbonate bicarbonate Buffer
Dissolve one capsule of Sigma Aldrich C3041-100CAP in 100 ml of deionized water. Filter
sterilize. Do not store more than one month.
2. Wash Buffer: PBS+0.5% Tween (PBST)
3. Nunc immobilizer amino F96 clear plates (polysorb) catalog# 436006
4. SA-HRP antibody (BD Pharmingen. Catalog#554066)
5. TMB (KPL; catalog # KPL 52-00-00)
6. Stop solution (KPL; catalog# KPL 50-84-04)
Method
1. Coat wells with scFv eluates from high throughput purification
a. Distribute 50 µl of carbonate-bicarbonate buffer in F96 plate
b. Add 5 µl of scFv eluate in quadruplicate (2 wells for Ag, 2 wells for control)
c. Incubate for 1 hr at RT on a shaker with very gentle agitation (speed 1- 2).
2. Wash 2X with 200 µl PBST and blot after each wash
3. Antigen capture
a. Distribute 0.2µg/ml of Biotinylated antigen in 50ul of PBST per well, in duplicate
b. Distribute 0.2µg/ml of control biotinylated antigen (e.g. Bio-BSA) per well, in duplicate.
c. Incubate 1hr at RT on a shaker again with gentle agitation (speed 2-3).
4. Wash 3X with 200 µl PBST and blot after each wash
5. Antigen detection
a. Distribute SA-HRP antibody at dilution 1:1000 in PBST, 50ul per well.
b. Incubate 15mins at RT with gentle agitation (speed 2-3).
c. Aliquot TMB in a dark opaque 15 ml conical tube and prewarmed at room temperature
6. Wash 3X with 200 µl PBST and blot after each wash
7. Signal detection by colorimetry
a. Distribute 50 µl of TMB per well
b. Store the plate in the dark. Stop the reaction when the positive control is blue and the
negative control still light. It can take anywhere between 5 min to 45 min.
c. Stop the reaction with 50ul per well of stop solution
8. Read at 450nm within 30 min after stopping the reaction.
Nov 2011
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