FIGURES
Figure 1|
H3N2
H3N2
B virus
B virus
HANA H1N1
BSA
PBS
H5N3
Parainfluenza Virus
HANA H1N1
SDS
HANA H3N2
HANA H3N2
SDS
Adenovirus
H5N3
H1N1
H1N1
SDS
HANA B
HANA B
SDS
Hu NP A
Hu NP A Tw20
H7N3
H5N1
H7N3
SDS
Hu NPB
Hu NPB
H5N1
SDS
Av NPA
Av NPA Tw20
SDS
SDS
SDS
Tw20
MAb 2A12
Figure 2|
H5N3
H5N3
H7N3
H7N3
Virus
B Virus
B
H5N1
H5N1
H3N2
H3N2
H1N1
H1N1
130
130
130
100
100
70
70
70
55
55
55
A.L.
A.L.
MDCK cells
cells
MDCK
Adenovirus
Adenovirus
Kb
Kb
MAb 33G2
Figure 3|
100
70
70
55
55
40
40
c
Kb
170
130
100
70
55
40
H5N3
100
Adenovirus
Kb
H1N1
25
25
BSA
H5N3
Avvirus
NPA
B
H1N1
NPB
Hu NPA
NPA
b
AvNPA
BSA
BSA
Av NPA
AvNPA
MAb 4A11
NPB
HuNPA
B virus
NPB
NPB
HuNPA
Hu NPA
Kb
NPB
BSA
NPB
NPB
NPB
NPB
NPB
Virus
B
virus
B
B
Virus
BVirus
Virus
B
H1N1
H1N1
H1N1
H1N1
H1N1
a
Kb
Kb
Kb
Kb
Kb
225
225
225
150
150
150
150
150
100
100
100
100
100
75
75
75
75
75
50
50
50
50
50
35
35
35
35
25
35
25
MAb 7G11
MAb 45A1
Figure 4|
H3N2 deg
H3N2 sub
H3N2
170
130
100
70
55
40
35
Adenovirus
H7N3
H5N3
H5N1
B sub
H1N1
Kb
kD
kD
100
75
50
MAb 1H11
Supplementary Figure 1 |
H3N2 Deg
H3N2 Deg Kit
H3N2
H3N2
H1N1
Deg Deg
Kit
H1N1
H1N1
H1N1
H7N3
Deg Deg
Kit
H7N3
H7N3
H7N3
SUPPLEMENTARY FIGURES
Table 1| Immunization regimen.
Table 2 | Reactivity of MAbs.
Table 3 | Troubleshooting table.
Step
13 and 34
Problem
Hybridomas
Possible reason
Contamination can
contamination
during
the
collection
Solution
occur Avoid notching or cutting the
feeder
and
cell gut
spleen
dissection
Avoid touching with scissors or
tweezers
the
fur
or
any
external mouse part
62
Shape and
Various
spotting size
solution
different
or
spotting Optimize
array
printing
surfactant condition by trying different
concentrations can affect spotting buffers at different pH
spots
size,
shape
and and
surface binding properties.
62
“Comet-tails”
High
shape in the
proteins
printed array
buffer can increase the risk
concentration
in
the
by
adding
different
concentration of surfactants.
of Print
the
antigen
under
printing different concentrations
of comet tails caused by
excess of unbound material
in the spot
74
High background
High background caused by Decrease secondary
high
antibodies antibodies concentration
concentration,
or
prolongation of secondary
Decrease time of secondary
antibodies incubation
antibody incubation.
Do a third washing step after
removing the gene frames
BOX 1
Myeloma cell culture
1. Grow myeloma cells (ATCC P3x63Ag8.653) in complete IMDM/20%FCS at 37°C
and 5% CO2.
2. Subculture myeloma cell line every 2 days by splitting in a ratio 1:2 or 1:3.
CRITICAL STEP Before adding the cells, it is best to put the fresh medium into a
sterile flask and place the flask/medium into the CO2 incubator to warm the medium
and to allow the CO2 to "dissolve" in the medium. The cells should be kept in logphase growth.
Cell splitting procedure
3.
Once cells are semiconfluent, collect medium (containing floating cells) from
the flask and store on ice in 50-ml tubes.
4.
Wash adherent cells remained in the flasks ones with ice-cold Ca++Mg++ freePBS/1mM EDTA, rinse and throw.
5.
Detach adherent cells from the flask bottom by adding 10 ml of ice-cold
Ca++Mg++ free-PBS/1mM EDTA solution.
6.
Incubate flasks 4-5 min on ice giving to the flask every now and then sharp
raps with the palm of your hand, collect the supernatant and add it to the falcon.
7.
Centrifuge the cells for 10 minutes x 800g at 4°C.
8.
Discard the supernatant and resuspend the pellet beating gently with fingertips
on bottom tube and adding fresh incomplete IMDM medium.
9.
Split the cells in new flasks containing complete IMDM/20%FCS.
Freezing procedure
Collect the cells as described in “cell splitting procedure” from point 1 to 6 and
resuspend the pellet with 3 or 4 ml of ice-cold 90% FCS/10 % DMSO. Spilt the cell
suspension in cold criovials. Use a freezing container to allow a slow freezing of cells
at -80°C for 1 night and then move samples in liquid nitrogen.