ELISA
Technique
Ma Hongbao, Ph.D.
Department of Medicine, Michigan
State University
East
Lansing, Michigan, USA
Introduction
ELISA is the abbreviation of enzyme-linked
immunosorbent assay.
ELISA is a useful and powerful method in
estimating ng/ml to pg/ml ordered materials in the solution, such as serum,
urine and culture supernatant (Savige et al. 1998).
The basic principle of an ELISA is to use an
enzyme to detect the binding of Ag and Ab. The enzyme converts a colorless
substrate (chromogen) to a colored product, indicating the presence of Ag:Ab
binding. An ELISA can be used to detect either the presence of Abs or Ags in a
sample, depending on how the test is designed.
Materials and Methods
Homogenize
tissue with 5 times of protein extract buffer ® centrifuge
10,000 rpm 20 minutes and keep the supernatant at -70oC as sample ®
passively adsorb 100 µl of coating antigen (about 25 ng/ml)
in coating buffer (PBS normally) to microtiter wells by incubation at 4˚C
overnight ® wash plates 3 times (3 minutes each time) with PBS
buffer ® add 0.1 ml supernatant sample into each well ®
over night at 4oC ® wash with PBS containing 0.5% BSA 3 times (3
minutes each time) ® add 0.1 ml diluted primary antibody 1-2 hour at
room temperature ® PBS washing 3 X 3 minutes ®
0.1 ml diluted secondary antibody 1-2 hour at room temperature ®
PBS washing 3 X 3 minutes ® dye (0.2 ml pNPP) ®
0.05 ml 3 N NaOH ® O.D. (405 nm) measurement.
1.
Extract
buffer:
50
mM Tris-HCl or 50 mM HEPES (pH 7.4)
150
mM NaCl
0.02%
sodium azide
0.1%
SDS
0.1
mg/ml
phenylmethylsulfonyl fluoride (PMSF)
0.001
mg/ml aprotinin
1%
Nonidet P-40 (NP-40) or 1% Triton X-100
(The
half-life of a 0.02 mM aqueous solution of PMSF is about 35 minutes. PMSF is
usually stored as a 10 mM or 100 mM stack solution 1.74 or 17.4 mg/ml in
isopropanol at –20oC).
2.
Homogenize
sample under ice.
3.
Centrifuge
sample at 10,000 rpm for 20 minutes at 4oC.
4.
Keep
supernatant at -70oC until usage.
5.
PBS:
Phosphate-buffered saline
(PBS), pH 7.4, 1000 ml (NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4
0.24 g, adjust to pH 7.4 with HCl). Add 0.5% BSA of 1% milk into PBS
when washing processed. It can also use Dulbecco's PBS or try others. Instead of
BSA, it can use gelatin or milk. Skim (0.5% to 1%) milk could reduce the
non-specific reaction.
6.
Primary
and secondary antibodies are normally 1:1000 to 1:2000 diluted by PBS and 0.1
ml each well.
7.
Use
alkaline phosphatase yellow (pNPP) liquid substrate as the dye for the ELISA (Derango et al. 1996). This product is supplied as a ready-to-use
buffered alkaline phosphatase substrate p-nitro-phenylphosphate (pNPP). Prior
to reaction with alkaline phosphatase, the substrate should appear as a
colorless to pale yellow solution. It will develop a yellow reaction product
when reacted with phosphatase in microwell applications. For the end-point
assays, the reaction can be stopped with 0.05 ml/well of 3 N NaOH for every 0.2
ml of substrate reaction. Following the
reaction with alkaline phosphatase, a yellow reaction product forms can be read
at 405 nm.
More Information
1.
Using
Polyclonal Antibodies:
(1) Antibody purification: Protein G column is the best for this purpose.
(2) Conjugate: Making conjugate is the most important part (e.g. horseradish peroxidase).
(3) 96-well plate: Making the solid phase using the 96-well plate.
2. Buffers and other reagents:
(1) Plate buffer: 0.1 M Sodium carbonate buffer, pH 9.5.
(2) Reaction buffer: 0.01 M Sodium phosphate buffer, pH 7.2, 0.15 M NaCl (PBS), 0.5% BSA, 0.05% thimerosal; You can also use Dulbecco's PBS or try others. Instead of BSA, you can use gelatin. Skim (0.5% to 1%) milk could reduce the non-specific reaction.
(3) Washing buffer: 0.05% Tween-20, 0.01 M Sodium phosphate buffer, pH 7.2 or 0.05% Tween-20, 0.15 M NaCl.
(4) Developing buffer: 0.05 M Sodium acetate buffer, pH 5.5.
(5) TMB stock solution: Tetramethylbenzidine 1 mg/ml in DMSO.
3. Making Conjugate
(1)
Nakane's
method.
(2)
Glutaraldehyde
method.
(3)
Maleimide
method.
4. Steps.
(1)
2
mg Horseradish peroxidase (HRP) in 1 ml water: A.
(2)
21.4
mg NaIO4 (never to be NaIO3!) in 1 ml water: B.
(3)
100
micro-l of B into A: Color will change to the dark green!
(4)
Wait
for 10 min at room temperature.
(5)
Put
into the dialysis tube (such as Molecular cut off 20,000).
(6)
F.
Put the tube into 5 mM NaAcetate buffer, pH 4.0 in a 2 to 3 l flask.
(7)
Dialysis
overnight: Color will change to the gold.
(8)
Raise
the pH of the HRP solution to pH = 9.0 by the addition of 0.2 M NaCarbonate
buffer, pH 9.5 (try an aliquot of 0.05 ml).
(9)
Mix
with the antibody solution (8 mg of IgG in 1 ml), which has been pre-dialyzed
to 0.01 M NaCarbonate buffer, pH 9.0 overnight.
(10)
Incubate
the mixture for 2 hr at room temperature.
(11)
Put
freshly prepared 0.1 ml, 0.1 M NaHBr4 in water to the solution.
(12)
Incubate
at 4 degree for 2 hr.
(13)
Put
the mixture into a dialysis tube and dialyze against PBS overnight.
(14)
Now
the conjugate solution is ready for use. Add thimerosal to a final
concentration of 0.02% for preservation. Add glycerol to a final concentration
of 10% (optional). If you stock the conjugate solution for a long period such
as years, stock it at -80 degree. But, in this case, don't repeat freeze-thaw.
You can stock the solution at 4 degree at least 6 months.
5. Preparation of ELISA Plate. This will take 2 hr to overnight.
Overnight is preferable.
(1)
Dilute
antibody (IgG) by Plate buffer: 5 to 10 micro-g/ml.
(2)
Put
the diluted antibody solution, 0.1 ml to the wells of 96-well ELISA plate.
(3)
Incubate
for 2 hr at room temperature or overnight at 4 degree.
(4)
Discard
the solution and wash the plate three times by washing buffer. Put 200 micro-l
into wells using micro-pipette or just put the Washing buffer using some
devices.
(5)
Discard
the Washing buffer by tapping against paper towel.
(6)
Put
0.15 to 0.2 ml of reaction buffer. Now, the plate is ready for use. You can
stock the plate at least for 6 months. Take care not to dry up the plate.
6. Using Monoclonal Antibodies
Antibody purification:
Antibody purification step is the only special part comparing with materials
and methods in using polyclonal antibody. For most monoclonals, except for IgM,
Protein G column will be good for the practical use. If you failed by this method,
confirm your procedure again before proceeding to the other methods such as
DEAE column. When your monoclonal antibody is IgM, try Protamine column
combined with molecular sieving column. Others are the same as above mentioned
in "Using polyclonal antibodies.
Try skim milk (any kind of
powdered milk such as powdered milk for babies) instead of BSA. It's really
cheap! Try 1% to 3%. It will decrease the background!! Thing is stability. It
will form precipitate if you keep it for a few months. If you are running many
plates, it is good alternative.
References
Derango R, Page J. The quantitation of coupled
bead antibody by enzyme-linked immunosorbent assay. J Immunoassay. 1996;17:145-153.
Savige
JA, Paspaliaris B, Silvestrini R, Davies D, Nikoloutsopoulos T, Sturgess A,
Neil J, Pollock W, Dunster K, Hendle M. A review of
immunofluorescent patterns associated with antineutrophil cytoplasmic
antibodies (ANCA) and their differentiation from other antibodies. J Clin Pathol
1998;51:568-575.