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SANDWICH Elisa
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Materials Required:-

 

  • Developing reagent: Specific antibody–alkaline phosphatase conjugate
  • PBS containing 0.05% NaN3 (PBSN)
  • Deionized Water
  • Blocking buffer
  • Capture Antibody solution
  • Test antigen samples
  • p-nitrophenyl phosphate (NPP) substrate solution
  • Stop solution (0.5 M NaOH)
  • 96 - well microtiter plate
  • Microtiter 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

 

Buffers and Reagents:

 

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.

 

Samples and standard solutions :

 

Standards of known amounts are required for positive controls, and estimation of levels within the samples(via comparison to a titration of known amounts). All standards should be diluted in a buffer  as near as, or identical to that of sample solution. Standards are best obtained from a reliable commercial source having its mass been caliberated.

 

Procedure:-

 

  1. Coat microtiter plate with capture-antibody solution: dispense 50 μl capture-antibody solution into the  wells of a microtiter plate using pipette and tips. Tap or shake the plate to ensure that the antigen solution is evenly distributed over the bottom of each well.
  2. Wrap coated plates in plastic wrap to seal and incubate for 2 hr at 370C in an incubator.
  3. After incubation, uncover the microtiter plate  and discard the solution into a container.
  4. Washing step: Rinse coated plate by filling wells with deionized water dispensed from a plastic squirt bottle .Flick the water into the container and rinse with water two more times.
  5. Remove residual liquid by gently flicking it face down onto several paper towels laying on the bench top.
  6. Blocking step: Fill each well with blocking buffer dispensed as a stream from a squirt bottle and incubate 30 min at room temperature.
  7. Discard the solution and perform the washing step. Gently flick microplate onto paper towels.
  8. Add using micropipette  50 μl of test antigen solution and standard antigen solutions  from the vial  to  the wells.
  9. Wrap plate in plastic wrap, and incubate for 2 hr at room temperature.
  10. Discard liquid and pat bottom of plate with dry  absorbent paper.
  11. Perform the washing step: Rinse coated plate by filling wells with deionized water dispensed from a plastic squirt bottle. Flick the water into the container and rinse with water two more times.
  12. Blocking step: Fill each well with blocking buffer dispensed as a stream from a squirt bottle and incubate 30 min at room temperature .
  13. Perform the washing step: Rinse coated plate by filling wells with deionized water dispensed from a plastic squirt bottle. Flick the water into the container and rinse with water two more times.
  14. Add50 μl secondary antibody (antibody–alkaline phosphatase conjugates) reagent to the wells.
  15. Wrap the microtiter plate in plastic wrap, and incubate 2 hr at room temperature.
  16. Washing step: Rinse coated plate by filling wells with deionized water dispensed from a plastic squirt bottle .Flick the water into the container and rinse with water two more times.
  17. Remove residual liquid by gently flicking it face down onto paper towels laying on the bench top.
  18. Using pipette and tip,  transfer 75 µl substrate solution from vial to the wells on microtiter plates.
  19.  Wrap the microtiter plates in  plastic wrap and incubate  for 1 hr at room temperature.
  20. Using pipette and tip,  transfer 25 µl stop solution (0.5 M NaOH) from vial to the wells on microtiter plates.
  21. Using a microtiter plate reader to measure NPP hydrolysis, use a 405-nm filter.

 

Analysis of Data:-

 

 Prepare coating-reagent dilutions by diluting specific antibody or immunoglobulin fraction in PBSN.

 

  • Place four 17 × 100–mm test tubes in a rack and add 6 ml PBSN to the last three tubes.
  • In tube  prepare a 12-ml solution of coating reagent at 10 μg/ml in PBSN.
  • Transfer 6 ml of tube 1 solution to tube 2. Mix by pipetting up and down five times.
  • Repeat this transfer and mix for tubes 3 and 4; the tubes now contain the coating reagent at 10, 5, 2.5, and 1.25 μg/mL.

 

Prepare a standard antigen-dilution series by successive 1:3 dilutions of the homologous antigen stock in blocking buffer.

 

  • Place five 12 × 75–mm test tubes in a rack and add 3 ml blocking buffer to the last four tubes.
  • In tube 1, prepare a 4-ml solution of secondary reagent at 200 ng/ml in PBSN. Transfer 1 ml of tube 1 solution to tube 2. Pipette  up and down five times.
  • Repeat this transfer and mix for tubes 3 to 5; the tubes now contain the secondary reactant at 200, 50, 12.5, 3.125, and 0.78 ng/ml.

 

Prepare dilutions of test antigen solutions in blocking buffer :

 

It may be necessary to assay one or two serial dilutions of the initial antigen test solution to ensure that at least one of the dilutions can be accurately measured. For most assay systems, test solutions containing 1 to 100 ng/ml of antigen can be accurately measured.

 

Prepare developing-reagent dilutions (antibody–alkaline phosphatase conjugates):

 

  • Place five 17 × 100–mm test tubes in a rack and add 3 ml blocking buffer to the last four tubes.
  • In tube 1, prepare a 6-ml solution of developing reagent at 500 ng/ml in blocking buffer.
  • Transfer 3 ml of tube 1 solution into tube 2 and mix.
  • Repeat this transfer and mixing for tubes 3 and 4—the tubes now contain the developing reagent at 500, 250, 125, 62.5, and 31.25 ng/ml.
     

Prepare a standard curve constructed from the data produced by serial dilutions of the standard antigen.Plot antigen concentration on the x axis which is a log scale, and absorbance on the y axis which is a linear scale. Interpolate the concentration of antigen in the test solutions from the standard curve.

 

NOTE: While standard curves are necessary to accurately measure the amount of antigen. In test samples, they are unnecessary for qualitative “yes/no” answers.
 

 

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