Materials Required:Developing reagent: Specific antibodyalkaline 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 4 0 C.

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 l 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 l 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 l secondary antibody (antibodyalkaline 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 l 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 l 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:-

SANDWICH Elisa

SANDWICH Elisa

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

Place four 17 100mm 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 g/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 g/mL.

Prepare a standard antigen-dilution series by successive 1:3 dilutions of the homologous antigen stock in blocking buffer.
Place five 12 75mm 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 (antibodyalkaline phosphatase conjugates):

Place five 17 100mm 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 4the 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.
Developed under a Research grant from NMEICT, MHRD
by
Amrita CREATE (Center for Research in Advanced Technologies for Education),
VALUE (Virtual Amrita Laboratories Universalizing Education)
Amrita University, India 2009 - 2015
http://www.amrita.edu/create
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