Exercise Your Options – Student Protocol
Laboratory – Exercise Your Options
Student Version
Procedures – note: buffer recipes follow
1. Prepare buffers.
 Each ELISA will use approximately 10ml plate-coating buffer, 150 ml phosphatebuffered saline (PBS), 20ml assay-blocking buffer, and 10ml substrate buffer. Except for
the assay-blocking buffer, these can be prepared well ahead of time and stored, long-term,
at 4oC. Assay-blocking buffer starts to grow microbes, so it should be prepared less than
a week before use, stored at 4oC, and tossed out when cloudy.
2. Each group should obtain one 96-well plate and enough plate coating buffer to coat the entire
plate at 100l/well.
3. Using the first antibody ordered (primary, or capturing, antibody), follow the manufacturer’s
instructions to make the proper dilution in the 10 ml of plate coating buffer.
4. Add 100l/well of this first antibody/plate-coating buffer solution. Cover and incubate
overnight at 4oC. Alternatively, incubate 37oC for 30 minutes.
5. Remove the plate-coating buffer by flicking into the sink and lightly tapping the plate on a
paper towel.
6. Wash the plate three times with PBS. Fill each well and flick PBS into sink. After the third
wash, lightly tap the plate on a paper towel.
7. Add 50l/well of the insect cell supernatant to each of the wells. Notice there are 96 wells in
the ELISA plate and 96 wells to the supernatant plate. Add to the ELISA wells supernatant
from the corresponding wells of the supernatant plate. Notice the supernatant plate has one
empty row. You will use this row on your ELISA plate for positive and negative controls.
BE SURE TO CHANGE TIPS AFTER EACH SAMPLE IS ADDED! Add appropriate
positive and negative controls (50µl each) to the wells in row H.
8. Incubate one hour at room temperature. This is an important incubation; if possible, longer is
better. Overnight at 4oC is also possible.
9. Remove the supernatant by flicking into the sink.
10. Wash the plate three times with PBS. Fill each well and flick PBS into sink. After the third
wash, lightly tap the plate on a paper towel.
11. Using the second antibody ordered (secondary, or detecting, antibody), follow the
manufacturer’s instructions to make the proper dilution in enough assay-blocking buffer to
add 100l/well.
12. Add 100l/well of this second antibody/assay-blocking buffer solution. Incubate 30 minutes
at room temperature.
13. Remove the antibody solution by flicking into the sink.
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Exercise Your Options – Student Protocol
14. Wash the plate three times with PBS. Fill each well and flick PBS into sink. After the third
wash, lightly tap the plate on a paper towel.
15. Using the third antibody ordered (conjugated antibody), follow the manufacturer’s
instructions to make the proper dilution in enough assay-blocking buffer to add 100l/well.
16. Add 100l/well of this conjugated antibody/assay-blocking buffer solution. Incubate 30
minutes at room temperature.
17. Remove the antibody solution by flicking into the sink.
18. Wash the plate three times with PBS. Fill each well and flick PBS into sink. IT IS VERY
IMPORTANT TO WASH WELL AFTER THIS STEP, SO REPEAT WASHES
AGAIN AND DRY PLATE ON PAPER TOWEL BEFORE DEVELOPING.
19. Prepare development solution by mixing 10 ml substrate buffer and one OPD tablet. Let
tablet dissolve and add 10l 30% H2O2. DO NOT PREPARE THIS SOLUTION UNTIL
JUST PRIOR TO USE—OPD WILL TURN COLOR IN AIR OVER TIME!!!
20. Add 100l per well of this development solution.
21. Observe for a color change. OPD should turn yellow within 15 minutes. If color develops
sooner, stop the reaction before the negative control starts to turn yellow.
22. Stop the reaction with 100l 2M H2SO4 (or HCl). If no color change is noticed within 15
minutes, all wells are considered negative.
23. Read plate in ELISA plate reader at 490nm. If you are not using the plate reader, score wells
as positive (+++, +) or negative (-). Wells will be obviously positive or negative with the
naked eye.
How to interpret results:
 The plate reader is measuring the absorbance, or optical density, of each well (**note: if
you have stopped the ELISA with acid, the OPD turns from yellow to orange).
 The darker the color, the higher the absorbance (O.D.).
 The higher the O.D., the more necrotin in the supernatant.
 Generally, an O.D. less than 0.100 is considered negative, but you should use your
negative and positive controls to assess each well.
Wrap-up Questions:




What is the connection between O.D. and necrotin?
Do you have any positive wells?
What would you do next?
If you do not have any positive wells (not even the positive control), what could have
gone wrong?
 If you have ALL positive wells, what could have gone wrong?
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Exercise Your Options – Student Protocol
ELISA Buffer Recipes
PBS
It is recommended that you prepare 10x PBS and dilute to 1x prior to use.
To prepare 10x PBS:
1. Prepare 1 liter of buffer A
2 g KCl
2 g KH2PO4
80 g NaCl
21.6 g Na2HPO4 • 7H2O
 Prepare 1 liter of buffer B
1 g CaCl2
1 g MgCl2 • 6H2O
Mix 450 ml buffer A with 50 ml buffer B.
This gives you 500 ml of 10x PBS.
To make 1xPBS, dilute 10x 1:10.
Use 1x PBS in all steps of the ELISA
Plate-Coating Buffer
0.1M NaHCO3, pH 9.6
NaHCO3 (MW 84): 8.4 g/L
Protein is diluted in this buffer. This buffer increases the ability of protein to stick to the
plate. The protein can either be an antigen or an antibody.
Assay-Blocking Buffer
0.5% bovine serum albumin (BSA) : 5 g/L
0.1% Tween-20 (not essential) : 1 ml/L
dissolve in1x PBS
This buffer prevents non-specific interactions from giving false positive results. It is also
used to make antibody dilutions.
Substrate Buffer
7.14 g/L citric acid
17.96 g/L dibasic sodium phosphate
pH 5.0
This buffer is used to make the developing buffer.
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Exercise Your Options – Student Protocol
Developing Solution
Per 10 ml: does one entire 96 well plate
10 ml Substrate buffer
10 l 30% H2O2
3-5 mg tablet o-phenylenediamene (OPD)
This solution must be made fresh, immediately prior to use.
Stop Solution
2M H2SO4 or other acid
Wash Solution
1x PBS
This solution is used to wash the plate after each step of the ELISA.
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