Flash content link: https://vlab.amrita.edu/repo/BIOTECH/IMM/InDirect_Elisa/index.swf
Materials Required:-
- Developing reagent: anti-Ig-alkaline phosphatase conjugate
- PBS containing 0.05% nan3 (PBSN)
- Deionized Water
- Blocking buffer
- Antigen solution
- Test antibody samples
- P-nitro phenyl phosphate (NPP) substrate solution
- Stop solution (0.5 M NaOH)
- 96 - well micro titer plate
- Micro titer plate reader - spectrophotometer with 405-nm filter
- Pipette and disposable pipette tips
- Plastic squirt bottles
- Vials (1.5 ml)
- Vial rack
- Plastic wrap
- Plastic container
- Incubator
- Absorbent paper
Reagent Preparation:
PBS (phosphate-buffered saline) containing 0.05% nan3 (PBSN) : Weigh 0.23 g NaH2PO4 (anhydrous; 1.9 mm),1.15 g Na2HPO4 (anhydrous; 8.1 mm), 9.00 g NaCl (154 mm)., 0.5g nan3 Add water to a volume of 900 ml. Adjust to desired ph (7.2 to 7.4) using 1 M NaOH or 1 M HCl. Add water to get a final volume of 1 liter.
Blocking buffer : Borate-buffered saline (BBS) containing 0.05% Tween 20,1 mm EDTA,0.25% bovine serum albumin (BSA),0.05% nan3.
To prepare BBS,dissolve sodium borate Na2B4O7 (5.7 g) and NaCl (7g), in 1L water,then adjust to ph 8.5 with NaOH, then add 0.372 g EDTA ( 1mm EDTA ), 0.05% tween 20,0.25% bovine serum albumin (BSA),0.05% nan3. Store at 40C.
Procedure:-
- Coat micro titer plate with antigen: Dispense 50 μl antigen solution(coating reagent) into the wells of an micro titer plate using pipette and tips.
- Wrap coated plates in plastic wrap to seal and incubate for 2 hr at 370C in an incubator.
- After incubation, uncover the micro titer plate and discard the solution into a container.
- Remove the coating solution and wash the plate twice by filling the wells with 200 µl PBS. The solutions or washes are removed by pippeting. The remaining drops are removed by patting the plate on a paper towel.
- Blocking step: Block the remaining protein-binding sites in the coated wells by adding 200 µl blocking buffer and incubate 30 min at room temperature.
- Discard the solution and perform the washing step. Gently flick microplate onto paper towels.
- Add using micropipette 50 μl of antibody solution from the vial to the wells.
- Wrap plate in plastic wrap, and incubate for 2 hr at room temperature. Discard liquid and pat bottom of plate with dry absorbent paper .
- Remove the coating solution and wash the plate twice by filling the wells with 200 µl PBS. The solutions or washes are removed by pippeting. The remaining drops are removed by patting the plate on a paper towel.
- Blocking step: Block the remaining protein-binding sites in the coated wells by adding 200 µl blocking buffer and incubate 30 min at room temperature.
- Remove the coating solution and wash the plate twice by filling the wells with 200 µl PBS. The solutions or washes are removed by pippeting. The remaining drops are removed by patting the plate on a paper towel.
- Add 50 μl secondary antibody reagent to the wells.
- Wrap the micro titer plate in plastic wrap, and incubate 2 hr at room temperature.
- Remove the coating solution and wash the plate twice by filling the wells with 200 µl PBS. The solutions or washes are removed by pippeting. The remaining drops are removed by patting the plate on a paper towel.
- Using pipette and tip, transfer 75 µl substrate solution from vial to the wells on micro titer plates.
- Wrap the micro titer plates in plastic wrap and incubate for 1 hr at room temperature.
- Using pipette and tip, transfer 25 µl stop solution (0.5 M NaOH) from vial to the wells on micro titer plates.
- Using a micro titer plate reader to measure NPP hydrolysis, use a 405-nm filter.
Advantages :
- A wide variety of labeled secondary antibodies are available commercially.
- The assay is versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
- Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
- Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
Disadvantages:
- Cross-reactivity might occur with the secondary antibody, resulting in non specific signal.
- An extra incubation step is required in the procedure
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