(a) Dilute the antigen to an appropriate final concentration
in PBS; for example, 2 μg/ml is a frequently used concentration for purified recombinant protein. Use the
amount of the antigen per microplate well recommended
by the supplier, or optimized by yourself.
(b) Coat plate wells by pipetting 50 μl of the prepared antigen solution, and incubate overnight at 4
C (see Note
14).
(c) Remove the coating solution and wash plates four times
by filling the wells with 300 μl PBST (see Note 15).
(d) Add 300 μl blocking buffer (2% BSA/PBS) to each well
and incubate 90 min at 37
C.
(e) Wash the plate twice with 300 μl PBST at RT (20–25
C).
(f) Dilute sera samples (collected after animal immunization)
in the blocking solution (2% BSA/PBS). Serum dilution
depends on the antibody concentration in your samples
and should be optimized; for example, you can begin
with 50-, 100-, and 200-fold dilutions.
(g) Add 50 μl of the diluted tested sera to each well and
incubate overnight at 4
C.
(h) Wash plates five times with 300 μl 0.1% Tween/PBS
(chicken sera samples) or 0.05% Tween/PBS (mouse
sera samples) at RT (20–25
C).
(i) Add 50 μl of HRP- or AP-conjugated (anti-host) secondary antibody, diluted in 2% BSA/PBS. Use dilution
recommended by the manufacturer.
(j) Incubate for 1 h at 37
C.
(k) Wash the plate five times with PBST at RT (20–25
C).
(l) Add 50 μl/well of substrate solution: TMB for HRP or
pNPP for AP. Incubate at RT (20–25
C) in the dark for
30 min.
(m) Stop the HRP and AP reactions by adding 50 μl of 0.5 M
H 2 SO 4 and 3 M NaOH, respectively.
(n) Read the optical density (using a microplate reader) at
450 nm for HRP-based procedure and 405 for AP-based
procedure (see Note 16).
4 Notes
1. PTAI-6-8 or PTAI-10-14 with chain lengths of 6–8 or 10–14
isoprene units, respectively, are derived from the natural mixtures of polyprenols with indicated chain length, whereas
PTAI-7 and PTAI-11 are derived from single polyprenols
400
Monika Rak et al.
in PBS; for example, 2 μg/ml is a frequently used concentration for purified recombinant protein. Use the
amount of the antigen per microplate well recommended
by the supplier, or optimized by yourself.
(b) Coat plate wells by pipetting 50 μl of the prepared antigen solution, and incubate overnight at 4
C (see Note
14).
(c) Remove the coating solution and wash plates four times
by filling the wells with 300 μl PBST (see Note 15).
(d) Add 300 μl blocking buffer (2% BSA/PBS) to each well
and incubate 90 min at 37
C.
(e) Wash the plate twice with 300 μl PBST at RT (20–25
C).
(f) Dilute sera samples (collected after animal immunization)
in the blocking solution (2% BSA/PBS). Serum dilution
depends on the antibody concentration in your samples
and should be optimized; for example, you can begin
with 50-, 100-, and 200-fold dilutions.
(g) Add 50 μl of the diluted tested sera to each well and
incubate overnight at 4
C.
(h) Wash plates five times with 300 μl 0.1% Tween/PBS
(chicken sera samples) or 0.05% Tween/PBS (mouse
sera samples) at RT (20–25
C).
(i) Add 50 μl of HRP- or AP-conjugated (anti-host) secondary antibody, diluted in 2% BSA/PBS. Use dilution
recommended by the manufacturer.
(j) Incubate for 1 h at 37
C.
(k) Wash the plate five times with PBST at RT (20–25
C).
(l) Add 50 μl/well of substrate solution: TMB for HRP or
pNPP for AP. Incubate at RT (20–25
C) in the dark for
30 min.
(m) Stop the HRP and AP reactions by adding 50 μl of 0.5 M
H 2 SO 4 and 3 M NaOH, respectively.
(n) Read the optical density (using a microplate reader) at
450 nm for HRP-based procedure and 405 for AP-based
procedure (see Note 16).
4 Notes
1. PTAI-6-8 or PTAI-10-14 with chain lengths of 6–8 or 10–14
isoprene units, respectively, are derived from the natural mixtures of polyprenols with indicated chain length, whereas
PTAI-7 and PTAI-11 are derived from single polyprenols
400
Monika Rak et al.