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Direct ELISA Protocol

Available ELISA Protocols: Direct ELISA | Indirect ELISA | Sandwich ELISA


Direct ELISA Protocol

Direct ELISA Diagram

Sandwich ELISA diagram courtesy of Univ. of Florida. Click to enlarge.


  1. Bicarbonate/carbonate coating buffer (100mM)
    Dilute the antigen or antibody in coating buffer to immobilize them to the wells:
    3.03g Na2CO3, 6.0g NaHCO3, 1000ml distilled water, pH 9.6.
  2. PBS:
    1.16g Na2HPO4, 0.1g KCl, 0.1g K3PO4, 4.0g NaCl (500ml distilled water) pH 7.4.
  3. Blocking solution:
    Common blocking agents: 1% BSA , serum, non-fat dry milk, casein, gelatin in PBS.
  4. Wash solution:
    PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).
  5. Antibody dilution buffer:
    Dilute primary and secondary antibody in 1x blocking solution to reduce non-specific binding.


  1. Dilute the antigen to a final concentration of 10µg/ml in PBS or other carbonate buffer. Pipette 100µl of the antigen dilution into a PVC Microtiter Plate to coat the wells. Dilute the plate down as necessary. Seal the plate and incubate at 4℃ overnight or at room temp for 2 hours.
  2. Wash plate 3 times with PBS.
  1. Add 200µl blocking buffer, 5% non-fat dry milk/PBS, per well to block remaining protein binding sites. BlockACE or BSA may also be used.
  2. Cover plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4℃.
  3. Wash the plate twice with PBS.
  4. Dilute 100µl of the antibody to the optimal concentration (according to manufacturer datasheet) and then add blocking buffer immediately before use.
  5. Cover the plate with an adhesive plastic and incubate for 2h at room temperature.
  6. Wash the plate 5 times with PBS.
  7. Using a multichannel pipette, dispense 100µl (or 50µl) of the substrate solution per well.
  8. After sufficient color development, add 50-100µl of stop solution to the wells if necessary.

Record the absorbance at 450 nm on a plate reader within 30min of stopping the reaction.

**This is a suggested protocol and should be adjusted by the user accordingly**

Available ELISA Protocols: Direct ELISA | Indirect ELISA | Sandwich ELISA
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