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Indirect ELISA Protocol

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

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Indirect ELISA Protocol



Indirect ELISA Diagram

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


Materials

  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.



Method

  1. Dilute antigen to a final concentration of 1-20μg/mL using PBS or Bicarbonate/carbonate coating buffer. Pipette 50µl of the antigen dilution into a PVC Microtiter Plate to coat the wells. Seal the plate and incubate at 4℃ overnight or at room temp for 2 hours.
  2. Wash plate 3 times with PBS.
  3. Add 200µl blocking buffer, 5% non-fat dry milk/PBS, per well to block remaining protein binding sites. Block ACE or BSA may also be used.
  4. Cover plate with an adhesive plastic and incubate for at least 2 hours at room temperature or overnight at 4℃.
  5. Wash the plate 3 times with PBS.
  6. Add 100µl of diluted primary antibody to each well.
  7. Cover the plate with an adhesive plastic and incubate for 2 hours at room temperature.
  8. Wash the plate 4 times with PBS.
  9. Dilute 100µl of conjugated secondary antibody to the optimal concentration (according to datasheet) and then add blocking buffer immediately before use.
  10. Cover the plate with an adhesive plastic and incubate for 1-2 hours at room temperature.
  11. Wash the plate 5 times with PBS.
  12. Using a multichannel pipette, dispense 100µl (or 50µl) of the substrate solution into each well.
  13. After sufficient color development, add 50-100µl of stop solution to the wells if necessary.
  14. Record the absorbance at 450nm on a plate reader within 30 min 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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