Biomedical Engineering Reference
In-Depth Information
Important!
A color change has to occur. If no change is observed,
discard the HRP.
Continue stirring at RT for 20 min and dialyze twice against 100 vol.
Soln. B each for 3 h. Transfer the dialysate into a fresh reaction
tube, add quickly 10
µ
l Soln. C, vortex, and add immediately 5 mg
antibody in Soln. D (about 10 mg
/
ml).
IncubateatRTonashakerfor2h,thenadd1
/
10 vol.ofSoln.E
and mix again. Allow the reduction of Schiff bases at 4
◦
C for 2 h,
and then dialyze against PBS overnight. Instead of dialysis, a gel
filtration on a Sepahdex G-25 column, equilibrated with PBS, is
possible. Concentrate the brownish dialysate and eluate, respec-
tively, and add BSA to a final concentration or 10 mg
/
ml,mixwith
an equal volume of glycerol, and store at 20
◦
C.
Important!
Never bring HRP solutions in contact with sodium
azide!
Instead of dialysis or gel filtration, an affinity chromatography on
Concanavalin A Sepharose (cf. Protocol 3.6.2.4) is recommended,
because on the one hand, no conjugated antibody is removed, and
on the other hand, the sugar used for elution stabilizes the enzyme-
antibody conjugate in solution.
Take the enzymatic reaction as described in Protocols 2.5.4.1
and 4.13.1.
References
Nakana PK (1980) In: Nakamura RM, Dito W, Trucker ES III (eds.) Im-
munoassays. Liss, New York, p 157
Huson L, Hay F (1989) Practical Immunology, 3rd ed. Blackwell, Oxford,
p44
4.1.7 Conjugation of Peptides to Carrier Proteins
Using Glutaraldehyde (Two-Step Procedure)
A
PBS
Solutions/Reagents
B
50% glutaraldehyde (w/v) in pure water (stabilized solution)
Dissolve 5 mg of ovalbumin, BSA, or another suited carrier protein
Universal coupling
protocol
µ
in 100
l ddH
2
O.ThepHhastobe6,becauseaggregationoccurs
above pH 7. Add Soln. B to a final concentration of 2.5% glutaralde-
hyde and stir at RT for 30 min. Filtrate the reaction mixture on
a PD-10 or Sephadex G-25 column, equilibrated with ddH
2
O, and
collect the activated protein in the void volume
4
.
4
A
stock
of
glutaraldehyde-activated
BSA
with
a
concentration
of
/
10 mg
ml may be stored frozen in aliquots. Activated BSA of high sta-
bility with about 20 moles aldehyde per mole BSA is made by a dialysis
procedure given by Zegers et al. (1990) J Immunol Meth 130:195.
