Biomedical Engineering Reference
In-Depth Information
µ
l of a suitable dilution of a secondary antibody-
HRP conjugate into each well (the dilutions range from 1:500
to 1:100 000, dependent on the quality of the conjugate, and the
amount of bound primary antibody; the dilution has to be checked
empirically). Incubate on a shaker at RT for 30 min.Washwith
Soln. B at least three times to remove traces of unbound conjugate.
Start enzyme reaction by addition of 100
Pipet 100
µ
l Soln. E, Soln. E
,or
Soln E
.
Important
: The time for enzymatic reaction has to be the same
in each well; therefore, pipet substrate solution and stop solution
exactly with the same rhythm. Enzyme reactions depend on time
and temperature!
Select the incubation time for an O.D. of the mostly colored well
between 0.8 and 2.5. These values are reached usually between 5 and
15 min. Stop color development by addition of Soln. F and Soln F
,
respectively. (If OPD or TMB are substrates, stop with Soln. F, in
the case of ABTS use Soln. F
).
Read the O.D. at 492 nm (OPD), 450 nm (TMB), and 405 nm
(ABTS), respectively, in a plate reader.
References
Avrameas S, Druet P, Masseyeff R, Feldmann G (eds.) (1983) Immunoen-
zymatic techniques. Elsevier, Amsterdam
Crowther JR (2001) ELISA - Guidebook. Humana Press, Totowa N.J.
Douillard JY, Hoffman T (1983) Meth Enzymol 92:168
Porstmann B, Porstmann T, Nugel E (1981) J Clin Chem Biochem 19:435
Porstmann T, Kiesig ST (1991) J Immunol Meth 150:5
4.13.2 Determination of Enzyme Activity by ELISA
A 5mM Na
2
CO
3
,35mM NaHCO
3
,0.02%NaN
3
(w/v), 0.001%
Solutions/Reagents
phenol red, pH 9.6, in ddH
2
O
B 0.05% Tween 20 (w/v) in TBS
C 0.1% cold fish gelatin in Soln. B
D5vol.0.1M sodium
acetate
buffer,
pH
5.0,
1
volume
of
/
ml N,N,N
,N
-tetramethylbenzidine (TMB) in ethanol
and 4 vol.of0.1%H
2
O
2
-urea adduct (w/v) in ddH
2
Oaremixed
immediately prior use
E1M sulfuric acid
TBS
1.2 mg
Dilute anti-(mouse-IgG)-IgG (in the case of monoclonal antibodies;
for testing conjugates prepared from antibodies of other origin, use
the respective anti-species specific antibody) to 5
µ
/
g
ml in Soln. A.
µ
Coatthewellsofamicrotestplatewith100
l/well of this dilution
and incubate at 4
◦
C overnight. Remove antibody solution and
wash once with TBS. Add 150
µ
l Soln. C per well and incubate at
