Basic HTML Version
Molecular Pathogens
MP2011, Vol.2, No.3
http://mp.sophiapublisher.com
- 25 -
(nutrient broth) at 28
℃
for 48 h. The bacterial cells
were collected by low-speed centrifugation (4,200 xg)
and washed once in sterile 0.85% NaCl. The bacterial
suspension was adjusted spectrophotometrically to
A
600
0.2 (about 10
8
CFU/mL) and heated to 85
℃
for
15 min. The killed cell suspension was used as antigen
for antiserum production.
3.4.2 Antiserum production
Antiserum against
Xac
(BP210) was prepared using
two New Zealand white female rabbits. 1 mL of
antigen mixed with Freund’s complete adjuvant (1:1
v/v) was immunized to each rabbit by subcutaneous
injection on the first day. Then, 1 mL of antigen
(mixed with Freund’s incomplete adjuvant (1:1 v/v))
was injected subcutaneously 7 day later. After that,
1 mL of bacterial antigen without adjuvant was
injected intravenous on day 14 and day 21. One week
after the last injection, the blood was drawn from the
central ear artery then kept in refrigerator overnight.
The antiserum was clarified by centrifugation at 3,000 xg
for 15 min at 4
℃
and stored at
-
80
℃
for future use.
This antiserum was used for indirect ELISA assay for
the antibody titer, the sensitivity and the cross reaction
of the antiserum. The pre-immune serum was bleed
before immunization to use as negative control in the
ELISA assay. Absorbance values of at least double of
the control were considered positive (OEPP/EPPO, 2005).
3.4.3 Indirect ELISA assay
The indirect enzyme-linked immunosorbent assay
(ELISA) was carried out as described by Alvarez et al.
(1991). The bacterial concentrations were adjusted
from 10
3
~10
8
CFU/mL in 0.05 mol/L carbonate
coating buffer (0.015 mol/L Na
2
CO
3
, 0.035 mol/L
NaHCO
3
, 0.003 mol/L NaN
3
; pH 9.6). Half volume of
each diluted bacterial suspension were treated at 85
℃
for 15 min to kill the cells and used as dead cells
antigen. 100 μL of each diluted bacterial suspensions
(both live and dead cells antigen) were coated in
microtiter plate well at 4
℃
overnight. The plate was
washed three times with phosphate buffer
saline+0.05% Tween20 (PBST) and 200 μL of 2%
skim milk were added and incubated for 1 h at 37
℃
.
Then, the plates were washed as mentioned earlier and
100 µL of diluted antiserum were added to the wells.
The ELISA plates were incubated at 37
℃
for 1 h then,
three times washing were performed. Goat anti-rabbit
gamma globulin conjugated to alkaline phosphatase
(whole molecule, enzymatic activity 1 mg/mL)
obtained from Sigma Chem. Co St Louis, Mo
(Production#F0382) was diluted to 1:10,000 and
100 µL was added in each well, followed by 1 hour
incubation at 37
℃
. The wells were repeatedly washed
and 100 μL of enzyme substrate (1 mg/mL p-
Nitrophenyl-phosphate (PNPP) in 10 mmol/L
Diethanolamine; Thermo Scientific#34047) were
added in each well. The reaction were stopped by
adding equal volume of 0.75 mol/L NaOH and the
O.D. at 405 nm were measured after 15~30 min
incubation in the dark at room temperature. The live
and dead cells of
E. coli
(10
6
CFU/mL) and
pre-immune serum were used as negative control.
3.4.4 Antibody titer and sensitivity tests
The antiserum was diluted to different dilution
(1:1,000, 1:2,000, 1:4,000, 1:8,000 and 1:1,600 v/v) in
conjugate buffer (2% polyvinyl pyrrolidone (PVP)+2%
ovul albumin in PBST) and tested with both live and
dead cells of bacterial antigen (10
4
~10
8
CFU/mL) by
indirect ELISA assay to test the titer of the antiserum.
For sensitivity testing, the 1:2,000 (v/v) diluted
antiserum was used to test both live and dead cells of
bacterial antigen (10
3
~10
8
CFU/mL
Xac
BP210) by
indirect ELISA assay as described above. All
experiments were done in duplicated and repeated at
least 3 different times.
3.4.5 Cross-reaction test
The antiserum was diluted to 1:2,000 (v/v) and used to
test the cross reactivity with other
Xanthomonas
bacteria at the concentration of 10
6
CFU/mL by
indirect ELISA assay as described above. The percent
of cross-reaction was calculated by; {[A405 of each
Xanthomonas
species-A405 of negative control] /
[A405 of positive control-A405 negative control]} × 100.
3.5 Detached leaf assay
3.5.1
Xac
inoculation
Full expanded young leaves of lime were washed in
running tap water and surface sterilized in 1% sodium
hypochlorite for 1~4 min. The leaves were aseptically
rinsed thoroughly with sterile distilled water. Each
leaf was divided into 4 parts for treatment separation,