Dilutions of
Enzyme Conjugates
I.Anti-Human-IgG Penicillinase
Conjugate :
A. Principle :
Glutaraldehyde
is an extensively used cross linking agent for the conjugation of penicillinase
with other proteins. It reacts irreversibly with e-amino group of lysine
residues of enzyme and the antibody resulting in the formation of a
crosslinkage. Single step method is widely accepted for the conjugation of
penicillinase to antibody.
B.Materials
:
a) Penicillinase
enzyme
b)
Anti-human IgG
c) 0.25 M
Sodium phosphate buffer (SPB) , pH 7.2
(Appendix-I)
d) 0.05 M
Sodium phosphate buffer (SPB) , pH 7.2
(Appendix-I)
e) 0.01 M
Sodium phosphate buffer (SPB) , pH 7.2
(Appendix-I)
f) 0.01 M
Phosphate buffer saline containing 0.05 % Tween-20 (PBS/T) (Appendix-I)
g) 3 %
Gelatin
Dissolve 3 g of gelatin in 100 ml of 0.05 M SPB (pH 7.2) and heat
indirectly until it is
dissolved completely.
h) 1 %
Glutaraldehyde
Add 0.1 ml of glutaraldehyde (25%) to 2.4 ml of distilled water.
i)
Penicillinase substrate
(Starch-iodine-penicillin 'V' reagent) (Appendix-I)
j) Pooled
positive and negative sera.
k) M.tb
ES-31/41 antigen cocktail (Appendix-II)
.
C.Methods
:
Preparation
of conjugate (Single step glutaraldehyde method) :
1.Take
0.5 ml of anti-human IgG (approximately 5 mg protein) in a plastic
tube, add 1000 units of enzyme
penicillinase, make upto 2.0 ml with SPB (0.25 M, pH 7.2) and mix.
2.Add
20 to 30 µl of 1% glutaraldehyde while stirring the mixture and keep at 250C
for 2 hr with intermittent shaking.
3. Take the mixture in a dialysis bag and dialyse
at 40 C against SPB (0.01 M, pH 7.2)
for12 hr. with 3 buffer changes
(each time 1 litre) .
4.
Centrifuge and add sodium azide (0.01%)
to the supernatant and store at 40C.Determination of optimal dilution :
1.Apply
5 µl of optimally diluted M.tb ES-31 antigen in 0.05M SPB, pH 7.2 ( 200 ng
protein/ml) on CAM square of plastic
stick.
2.Allow
to dry at room temperature for 20 to 30 min.
3.Incubate
the sticks in 3% gelatin solution in a small beaker at 370C for 2 hr.
4.Make
1:600 dilution of pooled positive serum and pooled negative serum by diluting
10 µl serum to 6 ml in PBS/T.
5.Wash
the sticks 3 times with PBS/T (For washing add PBS/T, shake gently and discard after 1 minute. Repeat this 2 times)
.
6.Add
0.5 ml of diluted pooled positive serum into each of 10 small plastic vials. Similarly add 0.5 ml diluted
pooled negative serum
into each of another set of 10
plastic vials.
7.Transfer
one antigen coated (gelatin blocked)
CAM stick to each tube and incubate at 370C for 1 hr.
8.Wash
sticks 5 times with PBS/T. (Discard PBS/T after 3 minutes during each wash) .
9.Prepare
serial (two fold) dilutions of anti
human IgG- Penicillinase conjugate in PBS/T. ( starting from 1: 500
to 1: 8000) and add 0.5 ml of
each dilution into one positive serum and one negative serum incubated tubes.
Incubate at 370C for 30 min.
10.Wash
9 times with PBS/T.
11.Change
sticks to labelled glass tubes.
12.Add
0.5 ml of starch-iodine-penicillin 'V' substrate into each tube, incubate at
370C and evaluate the results visually.
13.Note the
time of decolorization of substrate in
the tubes. Optimal dilution
of the conjugate is the
highest dilution showing
decolorization of substrate with positive serum about 4 minutes earlier
than with negative serum.
NOTE :
Some
immunoglobulin preparations give heavy precipitate after glutaraldehyde is
added. To prevent this, first add only
6 µl of glutaraldehyde followed by 2 µl each time at intervals of 1 minute.
Stop the addition if slight precipitate formation is observed.
The
substrate is blue in color due to the formation of starch-iodine complex. The
enzyme penicillinase converts penicillin 'V' to penicilloic acid, which in turn
removes iodine from starch-iodine
complex making the substrate colorless.
II.Preparation of Bm mf ES Ag-peroxidase conjugate by periodate method :
A.Principle :
The
periodate method was introduced by Nakane and Kawaoi in 1974 and modified by
Wilson and Nakane. This gives excellent enzyme antibody (IgG) ratios. It involves the oxidation of peroxidase
with m-periodate and generates some proportion of conjugate in polymeric form.
However, this does not usually present a problem, and the conjugation procedure
is uncomplicated and gives reliable results. For this reason it is a
recommended procedure.
B.Materials:
a.Sodium
carbonate bicarbonate buffer-1 pH 9.5
(Appendix-I)
b.Sodium
periodate 0.2 M (4.3 mg in 1 ml D.D.W.)
c.Sodium
acetate 1.0 mM pH 4.4 (Appendix-I)
d.Sodium
carbonate bicarbonate buffer-2, pH 9.5 (0.2M)
(Appendix-I)
e.Borate
buffer, 0.1M pH 7.4 (Appendix-I)
f.Sodium
borohydrite solution (4 mg in 1 ml of D.D W)
g.Bm
mf ES antigen
h.HRPO
[Sigma]
C. Method :
1.Dialyse
Bm mf ES Ag overnight against sodium carbonate bicarbonate buffer 1 pH 9.5.
2.Dissolve
2 mg of HRPO in 0.5 ml D.D.W.
3.Prepare fresh
0.2 M solution of sodium
m-Periodate (NaIO4) &
then add 0.1ml
to
HRPO solution in a glass bottle-the mixture immediately turns green.
4.Stir
the bottle gently for 20 min on shaker at Room
Tempature (250C)
5.Dialyse
against 1.0 mM sodium acetate buffer pH 4.4 at 40C for 5 hrs with 4-5 changes
6.Raise the
pH 9.0-9.5 by
addition of approximately 10 ml of 0.2M sodium carbonate
bicarbonate buffer 2 pH 9.5. Immediately add 500 µg of Bm mf ES Ag.
7.Stir
the mixture gently on shaker for 2 hrs at room temperature (250 C) .
8.Add
100 ml of freshly prepared sodium borohydride solution. Keep the mixture for 2
hr at 40C.
9.Dialyse
the conjugate overnight against
0.1 M borate buffer pH 7.4 at 40C with 2-3 changes.
10.Add
BSA to the conjugate to a final conc.
of 5 mg/ml, distribute into aliquates
in vials and add one granule
of thymol & store at -200C.
Determination of optimal dilution :
1.Add
and incubate 16 wells in a microtitre plate with Rabbit anti BM mf
'S' antibodies (5ng/100 ml in corbonate
buffer, pH 9.6) and equal number of
wells with only carbonate buffer
(control wells) at 370 C for 3 hrs.
2.Wash
once with PBS/T (for washing add PBS/T in each well shake gently and discard after 1 minute) .
3.Remove
the contents and add 100 µl of 1 % BSA in 0.06M Carbonate buffer pH 9.6 and
incubate at 370C for 2 hrs.
4.Wash
twice with PBS/T (as described above) .
5.Add
100 ml of two fold serial
dilutions (1:1,000 to
1:16,000) of BM mf ES antigen
-HRPO conjugate diluted
in PBS / T ( each
dilution to be
added in dupicate in both antibody coated and control wells ) and incubate at 370C.
6.Wash
5 times with PBS/T (as described above) .
7.Add
100 ml OPD substrate and incubate
for 10 min. at room temperature
(25OC) in dark.
8.Stop the
reaction by adding
50 ml of 5N HCI
to each well and read the
OD using ELISA reader.
9.The
conjugate in highest dilution which
gives maximum difference between
antibody coated and control wells can
be considered as optimal dilution.
Note :
The absorbance of control well should be
< 0.1 and that of the antibody coated well should be > 1
References :
1)
Avrameas S., Coupling of
enzymes to protein with glutaraldehyde. Use of conjugates for the
detection of antigen and antibodies, Immunochem.,6(1969) 43.
2)
Catty D., Antibodies Volume II, A practical approach 106-108.