QUANTIFICATION OF ABSCISIC ACID USING AN IMMUNOASSAY
Developed by Michelle Hall and Dale Lynn Vogelien
Abscisic acid (ABA) is one of of the five major kinds of hormones found in plants. It has
been found in essentially all phyla of the plant kingdom and detected in every organ and living
tissue thus far examined. Like most plant hormones, ABA causes many physiological responses
in higher plants. For example, ABA has been implicated in the promotion of organ and plant
senescence, induction of bud dormancy in woody plant species, the induction of seed dormancy,
the inhibition of auxin-induced growth, the inhibition of leaf growth, the stimulation of root
growth and the emergence of lateral roots, stomatal closure during periods of stress (e.g.
freezing, salt, and water stress), and the stimulation of water uptake in roots during periods of
water stress. These processes are regulated at the cellular level through ABA’s effects on ion
movements across membranes or on synthesis of specific proteins.
Because of its effect on stomatal closure during periods of stress, ABA is often referred to
as “the stress hormone”. Stomatal closure may be hydropassive or hydroactive, and it is only the
latter mechanism of closure that involves ABA. Hydropassive closure requires no metabolic
involvement from the guard cells that form a stomata but is due directly to water loss from the
guard cells by evaporation. Hydroactive closure is metabolically dependent, and occurs when the
entire leaf is experiencing a water deficit (e.g. the rate of transpiration is greater than the rate of
water entry into leaf tissue). In general, the osmotic potential of guard cell cytoplasm becomes
less negative due to solute loss from the guard cell, resulting in a less negative water potential.
This change in water potential results in the movement of water out of the guard cells (into the
apoplast and surrounding cells) and decreased turgor, which in turn results in stomatal closure.
The initial change in osmotic potential involves a reversal of the ion fluxes that lead to stomatal
opening, specifically, K+ efflux, and this appears to be initiated by ABA.
Like most plant hormones, ABA can be detected and quantified by biological, chemical
and immunological methods. Biological assays, or bioassays, involve applying a plant extract to
a living plant system that is known to respond to the hormone of interest. Several bioassays have
been developed for ABA and include inhibition of oat coleoptile growth, inhibition of seed
germination, inhibition of giberellic acid-induced amylase synthesis in the aleurone layer of
barely seeds, induction of leaf abscission, and stomatal closure. The disadvantage of many of
these bioassays is that they are not specific for ABA (e.g. plant extracts often contain other
chemicals that can promote or inhibit the biological response being measured). Physical methods
(e.g. gas chromatography, high performance liquid chromatography) of detection are much more
reliable and sensitive than bioassays, but require several costly and time consuming preliminary
purification steps and expensive chromatography equipment. Immunoassays are another method
of detection that are gaining considerable support, particularly with the development of
commercially available kits. Immunoassays use antibodies that were raised against the plant
hormone in an animal system. These antibodies are “reactive” with the hormone, and thus can
be used in its detection. One type of immunoassay is the ELISA (enzyme-linked immunosorbent
assay). In this assay the hormone specific antibody is covalently linked to an enzyme, such as
alkaline phosphatase, and the level of the reaction catalyzed by the enzyme is used to quantify
the amount of antibody-hormone complex formed . Immunoassays are specific, sensitive (2 – 3
fold greater sensitivity than bioassays) and fairly easy to perform. The primary disadvantage of
an immunoassay is the requirement of a mircoplate reader.
In this laboratory exercise you will quantify the amount of ABA in corn shoot tissue and
monitor its level shoot tissue during the course of soil desiccation (a 7 day period of no
watering). ABA levels in shoot extracts will be quantified using a modified ELISA. The
immunoassay, available as a kit from Sigma Chemical Company, uses a monoclonal antibody
specific for ABA. A test strip, which contains 8 wells coated with unlabeled ABA-antibody, is
provided. Equal amounts of sample (which presumably contains some level of ABA) and a tracer
solution (ABA- alkaline phosphatase conjugate) are added to a well. The tracer and ABA in the
sample compete for the antibody binding sites. Unbound tracer is washed away and the substrate
for alkaline phosphatase (p-nitrophenylphosphate) is then added. The product of the reaction (pnitrophenol) is measured as absorbance at 405 by a microplate reader. The amount of product is
inversely proportional to the amount of ABA in the sample, which can be quantified by use of a
standard curve.
The entire exercise will take place over several weeks. Four weeks are needed for the
sowing and growth of the seedlings. After four weeks, water will be withheld over a period of
one week. Plants experiencing increasing levels of soil desiccation will be harvested and
extracted during the first lab period. An additional lab period will be required to perform the
immunoassay and analyze data.
Plant Preparation (start 6 weeks prior to scheduled exercise; this step has been done for you)
Sowing Seeds
1. In medium size container, thoroughly moisten soil.
2. Fill 20 planting cups (enough to fill one tray) ¾ full. Make 2 depressions in each,
approximately 1 inch deep (use finger, pencil, etc.).
3. Sow one seed per depression (2 per container) and cover with soil.
4. Label pot clearly with plant sown and date of sowing.
Plant Maintenance
1. Growth chamber settings: set temperature for 25 °C and light period for 6:00 AM –
8:00 PM. Use the same photoperiod if plants are grown under a light bank.
2. Water soil until moist every 2 – 3 days, depending on soil dryness. Note: use of a
growth chamber may require a more frequent watering schedule.
3. Every 2 – 3 weeks, water with a dilute general all-purpose fertilizer.
Plant Dehydration
1. Begin to apply soil desiccation when plants are approximately 4 weeks old.
2. To have all desired desiccation treatments ready for harvest on the same day, the
following watering schedule is used:
8 days prior to the scheduled lab, water all plants moderately; on day 6, randomly
select 16 of the 20 cups to water moderately (note: the 4 unwatered cups represent
day 7 of the desiccation series and will not receive any water again); on day 3,
2
randomly select12 cups to water moderately (note: the 4 unwatered cups represent 5
days of the desiccation and will not receive any water again); on day 1 water 8 cups
sparingly (note: the 4 unwatered cups represent 2 days of dessication ); water the
remaining cups sparingly the day of lab. These plants represent day 0 of the
desiccation series.
3. On the day of the scheduled lab, 4 soil desiccation treatments should be available to
students: 0, 2, 5 and 7 days without watering (several plants at each level of soil
desiccation).
Plant Tissue Extraction (Lab Period 1)
As described in the previous section, plants experiencing varying levels of soil desiccation
are available for extraction. The entire desiccation series includes plants from which water has
been withheld 0, 2 5, and 7 days. These plants will be extracted as a class. Each group will
randomly select two treatments to extract (they need not be from the same treatment, or they can
be). Be sure to label the resulting extracts with the desiccation treatment.
Each of the desiccation treatments a group has selected are to be extracted separately. This
means that you will perform the extraction procedure twice. You MUST rinse the mortar and
pestle after each use. Also, be aware that consistency in your manipulations is important during
the extraction process. Some precautions:
•
•
•
The extraction procedure needs to be carried out in low light (e.g. keep overhead
lights off and work in natural light).
To minimize contamination of plant tissue, wear gloves during extraction
procedure.
All materials and solutions must be prepared prior to starting.
Solution Preparation (note: the Extraction Medium has been prepared for you):
Extraction Medium
80.0 ml Methanol
20.0 ml 100 mM NaHCO3 (0.17 g. NaHCO3 dissolved and brought to 20.0 ml
volume with deionized water.
1 mg BHT (butylated hydroxytoluene)
1. Prepare 100 mM NaCHO3 by dissolving 0.17 g NaHCO3 in DI water and bring to
20 ml volume.
2. Combine 20.0 ml 100 mM NaCHO3 solution with 1 mg BHT and 80.0 ml
methanol.
3. Adjust pH of final solution to pH 8 using conc. HCl. Use sparingly to avoid
altering dilution.
3
Tissue Extraction
1. Gently brush off dirt or particles that may be on plant leaves to be extracted.
2. Work in low light (e.g. with overhead lights off). Cut a plant at the base of the stem.
Using scissors, cut plant leaves into small pieces into tared weigh boat. START
CUTTING AT THE TIP AND CUT “BACK” TOWARDS THE BASE OF THE
SHOOT. USE ONLY LEAF TISSUE – AVOID THE “STALK”. Approximately 5 g
of tissue is desired, so don’t cut any more than you need. If less than 5 g is obtained,
the volume of the extraction medium added will be adjusted accordingly (see step 4
below). Record actual fresh weight used for each sample in Table 3 (pg 79).
3. Transfer tissue in a mortar and carefully add liquid nitrogen (fill the mortar). When the
liquid nitrogen has boiled off, grind tissue to a coarse powder consistency. Warning:
Liquid nitrogen is so cold that it can cause burns. Wear gloves at all times. Mortar and
pestle will become extremely cold following the addition of liquid nitrogen.
4. While the tissue is still frozen, transfer to a 40.0 ml centrifuge tube (use a flat end
spatula) and combine with extraction medium. Use 15.0 ml of cold extraction medium
for every 5 g. fresh weight of tissue. Adjust volume accordingly for the amount of
tissue you were able to obtain. If the ground tissue has already begun to thaw, add half
the extraction medium to the mortar and use a transfer pipette to transfer the
homogenate to the centrifuge tube. Wash the mortar with the remaining extraction
medium and add to the centrifuge tube. The goal here is to recover as much of the
ground tissue as possible. Record the actual volume of Extraction Medium used for
each sample in Table 3.
5. Extract for 48 hours at 4 °C.
6. Create weight balanced tubes and centrifuge the tissue samples at 10,000 x g for 30
minutes at 4°C.
7. Transfer supernatant to a clean tube screw-capped tube (label tube contents with
initials, and sample ID).
8. Extract the pellet again with 5 ml cold extraction medium at 4 °C for 24 hours.
Centrifuge again at the same conditions. Combine the second supernatant with the first
one and record the total extract volume.
9. Concentrate samples using the SpeedVac vacuum concentrator. Use moderate heat
(e.g. 45 °C) for 1 hour. Record volume of each sample after concentration (Table 3)
10. Store all extracts (concentrated and unconcentrated) at -20 ° C until next lab period.
4
11. In addition to the desiccation treatments listed, prepare as a control one additional
sample that receives 100 µl of a 10 mM ABA stock and process this sample as
described in steps 5 – 10. This is will serve as a positive control.
The Immunoassay for ABA (Lab Period 2)
In order for samples to be analyzed using the immunoassay, standards with known
concentrations must also be run to determine the relationship between ABA
concentration and absorbance. When running the immunoassay, whether for standards or
samples, timing is very important. With the exception of the substrate solution, which has
an 8-hour shelf life (and is prepared just prior to use), all solutions should be prepared
prior to beginning the immunoassay. Because the volumes of solutions involved in an
immunoassay are very small, solutions will be measured and transferred using
micropipettes. It is important to use a new pipette tip for every delivery in order to
prevent contamination. Practice pipetting techniques before actually performing the
assay, as incorrect pipetting can result in significant error. The smallest air bubble in a
pipette tip can alter the volume of a solution significantly.
Materials
Solutions
Tracer Solution
TBS Buffer
Substrate solution
Wash solution
Stop reagent
ABA Stock Solution (10 umole/ml)
Absolute methanol
Desiccation samples
Equipment
1 and 10 ml pipettes
pipette aids
15 ml tube with cap
sharpie
8 glass 7 ml test tubes with screw caps
test tube rack/holder
10, 100 and 1000 µl micropipettes and sterile pipette tips
waste container (for used tips)
4 testwells (equivalent to ½ of a test strip: two groups will share 1 strip)
37 °C incubator
refrigerator at 0 – 4 °C
5
Preparation of ABA Standard Solutions for Standard Curve
1. Dilute 1.0 ml of the 10.0 µmol/ml ABA stock with 9.0 ml absolute methanol. Label
this solution as Standard Stock (SS) ABA. SS ABA concentration is 1.0 µmol/ml.
2. Prepare standard solutions by diluting SS ABA with TBS buffer as outlined in Table 1.
Each solution should be prepared in a small glass screw capped tube. Labe each tube
as indicated in column 1.
Table 1 ABA Standard Solutions
Solution Code
ABA Solution
TBS Buffer
pmol/ml ABA Dilution Factor
NSB
4.99 ml
1000
1:1000
5 µl of SS
B
100
1:10
500 µl of NSB 4.50 ml
C
2.00 ml
20
1:5
500 µl of B
D
2.00
ml
4
1:5
500 µl of C
E
2.00 ml
0.8
1:5
500 µl of D
F
2.00 ml
0.16
1:5
500 µl of E
G
2.00 ml
0.032
1:5
500 µl of F
B0 _
--------------0
----100 µl of TBS
NOTE: NSB will allow you to estimate the level of nonspecific binding of the Tracer
during the assay; B0 should allow for 100% binding of Tracer.
The Immunoassay
1. Two lab groups will receive 1 test strip to share. Each test strip consists of 8 microwells that have been pre coated with ABA-antibodies. Each group will use 4 wells in
the test strip.
2. As a class, the following solutions must be run through the immunoassay
Std. NSB- in duplicate
Std. B - in duplicate
Std. C - in duplicate
Std. D - in duplicate
Std. E - in duplicate
Std. F - in duplicate
Std. G - in duplicate
Std. B0 - in duplicate
DAY 0 A & B – each in duplicate
DAY 2 A & B – each in duplicate
DAY 5 A & B – each in duplicate
DAY 7 A & B – each in duplicate
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3. Your instructor will assign solutions to each lab group to run through the assay.
Regardless of individual assignments, all groups will follow the same steps.
4. Using a 100 µl micropipette, add 100 µl of a standard solution or sample solution to a
test well. Use a clean tip to deliver each solution. Use Table 2 as a guide. Load all
standards/samples quickly and move immediately to the next step. NOTE: record
immediately any deviations from the recommended loading format shown in Table 2.
5. To each well, add 100 µl of Tracer solution. Use a clean tip for each delivery. Be
careful not to touch the pipette tip to the well bottom. When all wells have been loaded
with Tracer, mix the solutions by gently tapping the strip. Take care to not spill or
splash contents from wells in the process. Tap repeatedly to mix well.
Table 2 Recommended Well Assignments
Well
row/columns
A/1&2
B/1&2
C/1&2
D/1&2
E/1&2
F/1&2
G/1&2
H/1&2
Volume of Solution
100 µl NSB
100 µl B
100 µl C
100 µl D
100 µl E
100 µl F
100 µl G
100 µl B0
Well
row/column
A/3
B/3
C/3
D/3
E/3
F/3
G/3
H/3
Volume of Solution
100 µl DAY 0 – A
100 µl DAY 0 – A
100 µl DAY 0 – B
100 µl DAY 0 – B
100 µl DAY 2 – A
100 µl DAY 2 – A
100 µl DAY 2 – B
100 µl DAY 2 – B
Well
row/column
A/4
B/4
C/4
D/4
E/4
F/4
G/4
H/4
Volume of Solution
100 µl DAY 5 – A
100 µl DAY 5 – A
100 µl DAY 5 – B
100 µl DAY 5 – B
100 µl DAY 7 – A
100 µl DAY 7 – A
100 µl DAY 7 – B
100 µl DAY 7 – B
Volume of Diluted Tracer
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
Volume of Diluted Tracer
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
Volume of Diluted Tracer
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
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Well
row/column
A/5
B/5
C/5
D/5
E/5
F/5
G/5
H/5
Volume of Solution
100 µl of positive control
100 µl of positive control
100 µl of 1:10 of + control
100 µl of 1: 10 of + control
100 µl of 1:100 of + control
100 µl of 1:100 of + control
100 µl of unknown
100 µl of unknown
Volume of Diluted Tracer
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
100 µl
6. Snap all test strips into the strip holder (see instructor) and cover strips with the plate
sealer (a sheet of plastic cut to fit the plate holder with one sticky face). Incubate for 3
hours at 4 °C. You will resume with step 7 in lab.
7. Prior to the end of the incubation period, prepare Substrate solution by dissolving 1
substrate table in 5 ml Substrate diluent. Evenly aliquot among groups.
8. Following the incubation period, remove the plate sealer carefully (e.g. do not allow
well contents to contaminate it), for it will be needed for the next incubation.
9. Remove your test strip from the strip holder and wash wells as follows: add 200 µl of
Wash solution to each well using a different pipette tip for each delivery: dump wash
into sink or waste container. Repeat wash two more times. Carefully shake out
remaining drops of solution by tapping upside down on a paper towel (hold test wells
firmly!)
10. Add 200 µl Substrate solution to each well (again, use a clean pipette tip for each
delivery).
11. Place your strip in the strip holder and reseal with plate sealer. Place in a humid box
and incubate for a minimum of 1 hour at 37 ° C.
12. After approximately 1 hour, scan test wells using a microplate reader set at 405 nm.
Your instructor will provide instructions for use of the microplate reader. All strips are
read at the same time. Note: the reading for standard B0 should be greater than 0.750
O.D. units. If this is the case proceed to the next step. If not, return plate to 37 °C
incubator for and additional 15 – 30 minutes.
13. Add 50 µl of Stop reagent to each test well. Wait 5 minutes.
14. Read test wells at 405 nm in the plate reader. Record optical densities in Table 4 and 5.
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Data Calculations, Generation of Standard Curve, and Sample Analysis (Lab Period 3)
Data Calculations
1. Determine the log10 equivalent of the ABA concentration for standards B1 – G1.
2. Calculate the percent binding for standards B1 – G1 as follows:
% Binding (%B) = (Standard or Sample O.D. – NSB O.D.) x 100
(B0 O.D. – NSB O.D.)
•
•
•
NSB O.D. represents the level of nonspecific binding of the tracer to
wells. It must be subtracted from all values.
B0 O.D. represents the O.D. level for 100% binding of tracer.
% Binding is nothing more than the % of total possible binding that
was observed for a given concentration of ABA after correcting for
nonspecific binding.
3. Record these values in Tables 4 and 5.
4. Calculate Logit %Binding as follows:
Logit % Binding = ln
% Binding
100 – %Binding
5. Record these values in Tables 4 and 5.
Preparing the Standard Curve for the ABA Immunoassay
To quantify ABA levels in the various plant extracts, a standard curve must first be
generated. To construct the standard curve for this study, a series of solutions with known
ABA concentrations were prepared and analyzed. Typically, the concentrations of ABA
standard solutions are plotted on the x-axis, and the corresponding measurements of %
Binding (based on O.D. values of the known solutions) are plotted on the y-axis. However,
the resulting relationship is curve-linear. Such a graph is difficult to use as a standard
curve. A linear relationship can be obtained by transforming the data and graphing it as
Logit % binding (y-axis) vs. Log10 ABA Concentration (x-axis).
1. Use Excel to construct a standard curve by plotting the Logit % Binding (y-axis) vs.
Log10 ABA Standard Concentration (x –axis). Determine the equation for the resulting
trend line and the R2 value (also using Excel). Note: to establish a trend line, the trend
line equation, and R2 value, right-click on a series point within the graph; select “Add
trend line”; Under “Types” subheading, select “linear”; Under “Options”, select (;)
“display equation on chart” and “display R-squared value on chart”. This will give the
equation of the trend line produced, which can be used to determine the concentration
of ABA in unknown samples based on % binding.
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Sample Calculations and Analysis
1. Use the trend line equation for the standard curve to determine the log10 ABA
concentration in samples.
2. Convert values to antilog equivalents.
3. Calculate the average the ABA concentration for each day in the desiccation series.
Record values in Table 5.
4. Use Excel to prepare a graph that shows ABA concentration over the course of soil
dehydration. Note: this can be a bar graph or a line graph (connect the data points; do
not use a trend line).
Assignment/Questions
1. Provide Table 4. For standard D1 (4 pmole/ml), show stepwise all calculations leading to %
binding and Logit % Binding.
2. Provide the standard curve generated from the immunoassay (use Excel). Note that the
immunoassay is a competition enzyme-linked immunosorbent assay, or ELISA. The kind of
immunoassay used should be reflected in the graph title.
3. Provide Table 5. The ABA concentration in the last column is the concentration of ABA in
the concentrated sample. In the event that all samples were not extracted uniformly or did
not concentrate uniformly in the CentriVap, these values must be adjusted to reflect the
amount of ABA (in pmoles or nanomoles or micromoles) per g of tissue extracted. Provide
ABA concentration for each treatment on a mole ABA per g fresh weight extracted.
Step 1 (correct for vacuum concentration of extract): divide pmole/ml by the concentration
factor achieved by vacuum concentration (do this for each sample).
Step 2(determine the amount of ABA in the initial extract): multiply the above values by the
volume of Extraction Medium used for a given sample in the initial extraction process
Step 3 (express this amount on a g fresh weight): divide the above value by the sample g
fresh weight used for extraction.
4. Provide a graph that shows the relationship between the level of soil desiccation and ABA
concentration per g fresh weight (use Excel).
5. Write the narrative that would accompany this graph in a formal lab report.
6. What was the concentration of ABA in the Unknown sample provided by the instructor?
7. What was the observed concentration of ABA in the + control? How does this compare to
the expected concentration? (Note: you will have to calculate the expected concentration;
refer to step 11 under Tissue Extraction).
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Table 3 Sample Extraction Data
Sample
Extraction
Sample Fresh Medium
Weight (g) Volume (ml)
Volume of Extract Following
Concentration with the CentriVap
Day 0 – A
Day 0 – B
Day 2 – A
Day 2 – B
Day 5 – A
Day 5 – B
Day 7 – A
Day 7 – B
+ control
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Table 4 ABA Standard Curve Data and Calculations
Standard
NSB
B1
C1
D1
E1
F1
G1
B0
ABA conc.
(pmol/ml)
1000
100
20
4
0.8
0.16
0.032
0
Log
(Conc.)
O.D.
Reading
%
Binding*
Logit %
Binding*
Table 5 ABA Levels in Corn Shoot Tissue Experiencing Increasing Levels of Soil
Desiccation Over a One Week Desiccation Period
Sample
(days from
watering)
0–A
O.D.
Reading
Avg. O.D.
Reading
%
Binding
0–B
2–A
2–B
5–A
5–B
7–A
7–B
+ control
(undiluted)
1:10 dilution of +
control
1:100 dilution of +
control
unknown
72
Logit %
Binding
Log
(conc.)
ABA
conc.
(pmol/ml)
References
Blackman, P.G., and W.J. Davies, 1985. Root to Shoot Communication in Maize Plants of the
Effects of Soil Drying. Journal of Experimental Botany 36: 39 –48.
Harris, Daniel C. (1998) Quantitative Chemical Analysis . New York: W.H. Freeman and Co.
Plant Growth Reguarory Immunoassay Detection Kits (PRG-1), Sigma Chemical Col, St. Louis,
Missouri, 63178, USA (Jan. 19, 2000)
Salisbury, F.B. and C.W. Ross (1992) Plant Physiology, the 4th Edition, Wadsworth Publishing
Co., California.
Taiz, L. and E. Zeiger (1991) Plant Physiology (1st edition), Benjamin/Cummings Publishing
Company, Inc., California.
Xin, Z., X. Zhou and P. Pilet (1996) Level Changes of Jasmonic, Abscisic, and Indole-3yl-acetic
Acids in Maize Under Desiccation Stress. Journal of Plant Physiology 151: 120 –124.
Zhang, J. and W.J. Davies (1987) Increased Synthesis of ABA in Partially Dehydrated Root Tips
and ABA Transport from Roots to Leaves. Journal of Experimental botany 38: 2015-2023.
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