Biomedical Engineering Reference
In-Depth Information
RT for 30 min. Remove blocking solution, rinse once with Soln. B
and add 100
µ
l/well of a dilution series (e.g., 1:500, 1:1500, 1:4500,
1:13 500, 1:40 500, 1:121 500, 1:364 500 in TBS) of antibody-enzyme
conjugate to the wells. Shake at RT for 30 min,removeconjugate
solution, knock out the plate on paper tissue, and rinse three times
with Soln. B.
Perform the enzymatic reaction with Soln. D for 10 - 30 min
(each well has to react for the same time and at the same tempera-
ture) as described above, stop with Soln. E and read O.D. at 450 nm.
Plot O.D. against conjugate dilution. The working dilution of the
conjugateshouldgiveanO.D.of1.5-2.5.
4.13.3 Isotype Determination by EIA (AP Conjugate)
This protocol describes the use of an alkaline phosphatase conju-
gate;ofcourse,aHRPconjugateworkswell,too.
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), 0.02% NaN
3
(w/v) in PBS
C 0.1% serum albumin or gelatin (w/v), in Soln. B
D1mg
/
ml p-nitrophenyl phosphate (pNP; M
r
371.15, disodi-
um salt hexahydrate), 0.5 mM MgCl
2
,0.1M diethanolamine,
pH 9.5, in ddH
2
O. Use highly pure colorless pNP and prepare
substrate solution freshly
E1N NaOH
PBS
antibody recognizing all subclasses of heavy chains of an anti-
body class of a species, e.g., anti-(mouse-
γ
-chain)-IgG (goat)
secondary isotype-specific antibody-AP conjugate, e.g., anti-
(mouse-IgG1)-IgG (goat) AP conjugate
Dilute the class-specific capture antibody to 2
µ
/
g
ml in Soln. A.
µ
Apply 100
l/well of the coating solution to a microtiter plate and
incubate at 4
◦
C overnight. Remove the liquid from the plate and
wash once with Soln. B. Block the wells with 150
µ
l Soln. C at RT for
2 h. Knock out the blocking solution and wash once with Soln. B.
Store the plate in a sealed bag at 4
◦
C.
Dilute antiserum or hybridoma supernatant in a geometric se-
ries with Soln. B. starting at 1:100 and 1:10, respectively. Pipet 100
µ
l
of each dilution in duplicates into the wells and also for positive as
well as for negative control of an appropriate dilution of a charac-
terized immunoglubulin. Incubate at RT on a shaker for 1 h.Wash
three times with at least 200
µ
µ
l of AP
conjugate dilution in Soln. B (if the conjugate is not tested and no
recommendations are given by the supplier, start with a 1:10 000
dilution). Incubate at RT for 30 min, knock out the conjugate solu-
tion, and wash three times with Soln. B. Start enzymatic reaction
by addition of 100
l/well of Soln. B. Add 100
µ
l/well of Soln. D. Stop after 10.0 min at RT with
µ
100
l/well of Soln. E. Read absorption at 405 nm in a plate reader.
