Biomedical Engineering Reference
In-Depth Information
4.1.9 Alkaline Phosphatase-Immunoglobulin Conjugate
(Glutaraldehyde Protocol)
The advantage of the use of alkaline phosphatase (AP) lies in the
high purity of commercially available enzyme. But it is difficult to
get such highly specific activities as for HRP, because there is no
simple procedure for separation of unbound antibodies from the
conjugate.
A 1% glutaraldehyde (w/v) in ddH
2
O
B 0mM Tr i s
·
HCl, pH 8.0, 1 mM MgCl
2
,0.02%NaN
3
(w/v)
PBS
Mix 5 mg of affinity-purified immunoglobulin in 1 ml PBS with
10 mg AP
5
in 1 ml PBS. The molar ratio should be 1 mol IgG: 2 mol
AP (M
r
IgG 150 kD,M
r
AP 140 kD). Dialyze the mixture twice at
4
◦
C against 100 ml PBS for 6 - 9 h each.
Add 0.15 ml Soln. A to the dialysate. Agitate at RT for 2 h and
continue at 4
◦
C overnight. Dialyze three times against 100 ml PBS
for 3 h each. Bring the dialysate up to 2 mg BSA and fill up to 10 ml
with Soln. B. Aliquote and store at −20
◦
C.
A separation of conjugated AP from unbound enzyme in EIA
is not necessary, because free AP is removed during the washing
steps.
4.1.9.1 Enzymatic Reaction of Alkaline Phosphatase
from Calf Intestine
This protocol gives soluble colored reaction products used for en-
zyme immunoassays in test tubes. A procedure yielding insoluble
reaction products is given in Protocol 2.5.4.2.
A .7mM p-nitrophenyl phosphate (pNP; M
r
371.15, disodium
Solutions/Reagents
salt hexahydrate), 0.5 mM MgCl
2
,0.1M diethanolamine, pH
9.5, in ddH
2
O. Use highly pure colorless pNP
B .1M EDTA in 2 N NaOH
Wa r m 0 . 5 ml of Soln. A up to 37
◦
C and add 5
µ
l conjugate and
µ
conjugate dilution in PBS, resp. Stop with 100
l Soln. B after 5
to 10 min. If the assay is done in parallels, each probe has to be
stopped after exactly the same time. Read the O.D. at 405 nm.
4.1.10 Labeling of Immunoglobulins with Fluorescent Dyes
Solutions/Reagents
A .2M carbonate buffer, 0.5 M NaCl (2.12 g anhydrous Na
2
CO
3
,
2.92 g NaCl in 100 ml ddH
2
O),adjusttopH9.2withHCl
PBS
5
If AP is supplied as ammonium sulfate suspension, spin an aliquot con-
taining 10 mg with 12 000
g at 4
◦
C for 15 min.Discardthesupernatant
completely, add 1 ml PBS, followed by 1 ml of antibody solution in PBS.
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