|
Technical
Files
Home Page
ELISA Reagents
Indirect ELISA, alkaline phosphatase conjugate
Content List
|
Lot Number |
Item |
500
wells |
1000
wells |
5000
wells |
Storage |
|
|
Detecting conjugate, alkaline phosphatase (Bottles A and B) |
0.25 ml (A)
0.25 ml (B) |
0.50 ml (A)
0.50 ml (B) |
2x1.25 ml (A)
2x1.25 ml (B) |
4 oC |
|
|
96-well ELISA plates |
5 |
10 |
50 |
Room temperature |
|
|
Instructions |
1 |
1 |
1 |
|
ELISA Reagents
Indirect ELISA, alkaline phosphatase conjugate
Safety and Storage
Always wash hands thoroughly after using this product. Prevent direct skin
and eye contact with, or ingestion of, product components. Obtain medical
attention in case of accidental ingestion of reagent components.
All reagent components should be stored at the recommended temperature to
assure their full shelf life. Do not store prepared working solution from day to
day.
Please contact AC Diagnostics, Inc. if you have any questions about safety and storage
of this product.
Preparing For The Test
Check all the components in the package of ELISA Reagents.
Prepare all of buffer solutions according to the attached buffer formulations.
Make sure all laboratory equipments and facilities required are ready for the
test.
Prepare a humid box for incubation steps.
Make a copy of the attached recording sheet and create a loading diagram by
recording the locations of your samples, controls, and other reagents needed.
Preparing Samples
Select symptomatic and/or infective tissues for the test. Leaf tissue is
often used in ELISA testing. However, plant tissues such as stem, sprout, seed,
tuber, root and others can also be tested. Remove any soil from the tissue to be
tested. Because contamination with soil can interfere with ELISA tests.
AC Diagnostics’s SB3 buffer should be used as extraction buffer for most of plant
sample. Grind sample with a mortar and pestle, or other grinding devices. If you
are using a mortar and pestle, wash and rinse it thoroughly between samples.
If you extract plant sap, dilute the sap into SB3 sample extraction buffer at
a ratio of 1:100 (sap volume: buffer volume). Or you can grind plant tissue in
SB3 extraction buffer at a 1:100 ratio (tissue weight: Extraction Buffer
volume).
If you have any questions about sampling, sample preparation, or the
appropriate extraction buffer for your samples, please contact AC Diagnostics, Inc.
Sample Dispensing and Incubation
About 100 µl of diluted sample extract is needed per test well. Always have an
additional amount
to assure easy dispensing. A convenient way to prepare this diluted sample is to
measure 10 µl
of undiluted sap into a small test tube, then add 1 ml of extraction buffer.
Dispense the prepared samples into wells of ELISA plate following your
loading diagram on your recording sheet. Add 100 µl of positive control into
positive control well and 100 µl of negative control or extraction buffer into
negative control well.
Put the plate inside the humid box and incubate for 2 hours at room
temperature (21-24 ° C)
Preparing Enzyme Conjugate
Always make enzyme conjugate solution within 10 minutes before use. Prepare
the enzyme conjugate, using AC Diagnostics’s ECB1 buffer and a cleaning container.
The volume of ECB1 buffer required depends on the number of test wells used,
with 100 µl needed per test well. To estimate the volume needed, prepare 1 ml
for each 8-well strip used, or 10 ml for each 96-well plate.
The volume of enzyme conjugate required for each test is calculated based on
the volume of ECB1 buffer used and on the dilutions given on the bottles. Use a
new, sterile pipette tip and change the tip for each pipetting of each bottle to
prevent contamination.
First dispense appropriate volume of ECB1 buffer into a cleaning container,
then add enzyme conjugate bottles A and B according to the dilutions given on
the label.
For example, if the dilutions given on bottles A and B are both 1:200 and you
are preparing 2 ml of enzyme conjugate, you should first dispense 2 ml of ECB1
buffer. Then add 10 µl from bottle A and 10 µl from bottle B to the ECB1
buffer.
After adding the conjugates from bottles A and B, mix the conjugate solution
thoroughly. If you prepare the conjugate in a test tube, invert it several
times. If you prepare the conjugate in a beaker, stir the conjugate solution
with a glass bar. It is important to mix the enzyme conjugate well for a
consistent test result.
Prepare enzyme conjugate just before use. Keep the prepared enzyme conjugate
at safe place and use it after washing the plate.
Washing Plate
Wash the plate when the incubation is complete. Use a quick flipping motion
to empty the wells into a sink or waste container without mixing the contents.
Wash the plate by filling the wells with PBST, then quickly emptying them
again. Repeat 6 to 8 times.
To remove drops of PBST from the wells after washing, hold the frame upside
down and tap firmly on a folded paper towel.
Enzyme Conjugate Incubation
Dispense 100 µl of prepared enzyme conjugate per well.
Incubate the plate in the humid box for 2 hours at room temperature (21-24 °C).
Preparing Substrate Solution
Concentration of PNP in substrate is 1 mg/ml. Each PNP tablet will make 5 ml
of PNP solution, which is enough for five 8-well strips.
Do not touch the PNP tablets or expose the PNP solution to strong light.
Light or contamination could cause background color in negative wells.
Prepare PNP substrate about 10-15 minutes before the end of the above
incubation step. Measure 5 ml of PNP buffer for each tablet, then add the PNP
tablets to the buffer. Mix by vortexing or stirring to let the PNP tablet full
dissolving in the buffer.
Washing Plate
Wash the plate 6 to 8 times with PBST as above. Then fill the wells with PBST
and keep soaking the wells for 1-2 minute. Empty the wells and tap on a folded
paper towel.
Incubation With Substrate
Dispense 100 µl of PNP substrate solution per well.
Incubate the plate for 30 to 60 minutes in a humid box at room temperature
(21-24 °C).
To stop reaction, add 50 µl of 3M sodium hydroxide to each well. This step
is optional. The plate can be interpreted visually or with a plate reader
without adding the stop solution.
Evaluating Results
Test results can be examined by eye, or measured on a plate reader at 405 nm.
Development of yellow color in test wells indicate positive results. Wells in
which there is no significant color development indicate negative results. Test
results are valid only if positive control wells give a positive result and
negative control wells remain clear.
Results may be interpreted after more than 60 minutes of incubation as long as
negative
control wells remain virtually clear.
Buffer Formulations
SB3 Buffer
Sodium carbonate
(anhydrous)
1.60 g
Sodium
bicarbonate
2.92 g
Polyvinylpyrrolidone (PVP) MW
24-40,000 10.0 g
Sodium azide
0.2 g
Dissolve in distilled water to 1000 ml. Adjust pH to 9.6. Store at 4° C.
PBST Buffer
Sodium phosphate, dibasic
(anhydrous)
1.15 g
Potassium phosphate, monobasic
(anhydrous)
0.2 g
Sodium
chloride
8.0 g
Potassium
chloride
0.2 g
Tween-20
0.5 g
Dissolve in distilled water to 1000 ml. Adjust pH to 7.3.
ECB1 Buffer
Bovine serum albumin (BSA)
2.0 g
Polyvinylpyrrolidone (PVP) MW
24-40,000
10.0 g
Sodium azide
0.2 g
Dissolve in 1000 ml 1X PBST. Adjust pH to 7.3. Store at 4° C.
PNP Buffer
Diethanolamine
97.0 ml
Magnesium
chloride
0.1 g
Sodium azide
0.2 g
Dissolve in 800 ml distilled water. Adjust pH to 9.8 with hydrochloric
acid.
Adjust final volume to 1000 ml with distilled water. Store at 4° C.
RECORDING SHEET OF ELISA
TEST:
DATE:
BY:
TIMING: Coating
Sample
EC
Substrate
KEY POINTS:
Coating
Antibody:
ul, Coating Buffer:
ml,
Enzyme Conjugate:
ul, ECB1:
ml
PNP Substrate:
ml
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
A |
|
|
|
|
|
|
|
|
|
|
|
|
|
B |
|
|
|
|
|
|
|
|
|
|
|
|
|
C |
|
|
|
|
|
|
|
|
|
|
|
|
|
D |
|
|
|
|
|
|
|
|
|
|
|
|
|
E |
|
|
|
|
|
|
|
|
|
|
|
|
|
F |
|
|
|
|
|
|
|
|
|
|
|
|
|
G |
|
|
|
|
|
|
|
|
|
|
|
|
|
H |
|
|
|
|
|
|
|
|
|
|
|
|
RESULTS/CONCLUSIONS:
1.
2.
3.
|