Direct ELISA Procedure

COATING Make 1-10 µg/ml solution of antigen (peptide or protein) in Coating buffer (do not store diluted antigen). Coat High Binding ELISA Strip Plates by dispensing 100 µl of antigen solution per well. Cover the plates with Adhesive Films. Incubate at RT for 2-6 h or overnight at 4oC with gentle orbital shaking. Aspirate solution and wash plates 2X with Wash Buffer.

BLOCKING Prepare Blocking Buffer by diluting from the stock. Dispense 250 µl per well and Incubate plates for 2-6 h at RT or overnight at 4°C with gentle mixing. Aspirate blocking buffer solution and tap over paper towels to remove all solution. The plates can be used directly for the assay or kept at 4°C in sealed bag for future use.

ANTIBODY ASSAY Dilute preimmune and immune sera in Antibody/Conjugate Diluent (Serial dilution’s of 1:100, 1:1K, 1:10K, 1:100K). Dispense 100 µl of each antibody and preimmune dilution to antigen coated wells. Cover the plates with Adhesive Films. Incubate at RT for 30 min with gentle orbital shaking. Aspirate solution and wash plates 5X with Wash Buffer (200-300 µl wash buffer should be used for each washing; an improper washing will lead to high background). Dilute Anti-Rabbit IgG-HRP Conjugate (1:5K-1:20K) in Antibody Conjugate diluent. Dispense 100 µl of conjugate per well (An improper dilution of conjugate may give too low color or high background). Cover the plates with Adhesive Films. Incubate at RT for 30 min with gentle orbital shaking. Aspirate plates and wash plates 5X with Wash Buffer. Tap over paper towels to remove all solution. Dispense 100 µl/per well of ready-to-use TMB Substrate solution. Cover the plates with Adhesive Films. Incubate at RT for up to 15 min with gentle orbital shaking. A blue color will develop in positive wells. Dispense 100 µl diluted solution per well. The blue color will turn into yellow. Read absorbance at 450 nm.

NOTE: ALL reagents must be dispensed using fresh tips and Reagent reservoir. A specially designed Microtubes may be suitable for this purpose.