|
Table
I ELISA Development with PBS-4% FBS
Plate
|
No
primary Antibody
|
SQE
#3
|
SQE
#9
|
SQE
#16
|
SQE
#18
|
Pre-immune mouse serum
|
Post-immune
mouse serum
|
|
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
|
Immulon II
U-Bottom
|
0.21*
(0.01)**
|
0.07
(0.00)
|
1.10
(0.47)
|
3.23
(0.06)
|
1.50
(0.18)
|
3.23
(0.12)
|
2.82
(0.66)
|
4.04
(0.05)
|
0.68
(0.16)
|
2.87
(0.15)
|
0.39
(0.11)
|
0.20
(0.01)
|
1.68
(0.59)
|
2.41
(0.01)
|
|
Immulon
II Flat Bottom
|
0.09
(0.01)
|
0.08
(0.02)
|
1.10
(0.35)
|
2.28
(0.13)
|
1.26
(0.09)
|
2.22
(0.03)
|
3.19
(0.12)
|
3.17
(0.06)
|
2.85
(0.12)
|
3.03
(0.02)
|
0.03
(0.06)
|
0.19
(0.01)
|
1.79
(0.32)
|
1.01
(0.11)
|
|
Costar
Tissue Cult. U-Bottom
|
0.10
(0.03)
|
0.07
(0.01)
|
0.15
(0.01)
|
1.32
(0.02)
|
0.17
(0.06)
|
1.20
(0.11)
|
0.12
(0.10)
|
2.58
(0.09)
|
0.17
(0.04)
|
2.02
(0.27)
|
0.08
(0.01)
|
0.16
(0.05)
|
0.23
(0.08)
|
1.09
(0.02)
|
|
Costar
Tissue Cult. Flat Bottom
|
0.07
(0.01)
|
0.07
(0.01)
|
0.14
(0.01)
|
1.13
(0.08)
|
0.15
(0.04)
|
2.07
(0.08)
|
0.14
(0.10)
|
2.11
(0.09)
|
0.18
(0.02)
|
1.62
(0.08)
|
0.10
(0.01)
|
0.22
(0.10)
|
0.23
(0.01)
|
1.16
(0.02)
|
|
Immulon
4HBX
|
0.09
(0.01)
|
0.76
(0.00)
|
0.33
(0.05)
|
1.96
(0.13)
|
0.70
(0.03)
|
2.99
(0.13)
|
0.55
(0.19)
|
3.12
(0.05)
|
0.36
(0.08)
|
2.78
(0.09)
|
0.20
(0.06)
|
0.32
(0.12)
|
0.43
(0.17)
|
1.49
(0.13)
|
|
F96
Maxisorp
|
0.23
(0.16)
|
0.14
(0.01)
|
0.36
(0.10)
|
2.11
(0.11)
|
0.29
(0.08)
|
2.02
(0.11)
|
0.32
(0.10)
|
3.02
(0.05)
|
0.30
(0.01)
|
1.72
(0.06)
|
0.20
(0.13)
|
0.20
(0.01)
|
0.87
(0.62)
|
1.70
(0.09)
|
|
Millipore
IP ELISA Washer
|
0.13
(0.08)
|
0.11
(0.03)
|
0.18
(0.02)
|
1.23
(0.08)
|
0.27
(0.14)
|
1.13
(0.20)
|
0.17
(0.05)
|
2.77
(0.07)
|
0.19
(0.05)
|
2.72
(0.06)
|
0.14
(0.02)
|
0.24
(0.03)
|
1.45
(0.60)
|
1.99
(0.06)
|
|
Millipore
IP Hand Washed
|
0.07
(0.02)
|
0.07
(0.01)
|
0.14
(0.08)
|
1.03
(0.06)
|
0.01
(0.04)
|
1.19
(0.10)
|
0.08
(0.02)
|
2.75
(0.19)
|
0.19
(0.09)
|
2.78
(0.07)
|
0.07
(0.00)
|
0.24
(0.01)
|
0.23
(0.04)
|
0.72
(0.10)
|
*
Absorbance at 405 nm
 ** Std.
Deviation
Abbreviations used: ELISA – enzyme-linked immunosorbent assay; FBS – fetal
bovine serium; SQE – squalene; ISP – isopropanol
Table
II ELISA Development with Gelatin
Plate
|
No
primary Antibody
|
SQE
#3
|
SQE
#9
|
SQE
#16
|
SQE
#18
|
Pre-immune mouse serum
|
Post-immune
mouse serum
|
|
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
ISP
|
SQE
|
|
Immulon II
U-Bottom
|
0.16*
(0.11)**
|
0.28
(0.07)
|
0.49
(0.16)
|
1.08
(0.05)
|
0.30
(0.11)
|
1.05
(0.14)
|
1.02
(0.12)
|
2.24
(0.17)
|
1.55
(0.72)
|
3.14
(0.25)
|
0.37
(0.06)
|
0.33
(0.25)
|
1.37
(1.09)
|
1.26
(0.11)
|
|
Immulon
II Flat Bottom
|
0.21
(0.15)
|
0.12
(0.01)
|
1.38
(0.23)
|
0.64
(0.02)
|
1.54
(0.13)
|
0.59
(0.07)
|
2.69
(0.16)
|
1.10
(0.09)
|
2.65
(0.21)
|
1.74
(0.07)
|
0.74
(0.60)
|
0.21
(0.01)
|
2.32
(0.40)
|
0.93
(0.10)
|
|
Costar
Tissue Cult. U-Bottom
|
0.50
(0.32)
|
0.30
(0.12)
|
1.48
(0.55)
|
0.89
(0.08)
|
2.41
(0.24)
|
0.59
(0.16)
|
2.95
(0.35)
|
2.82
(0.39)
|
2.08
(0.30)
|
2.57
(0.14)
|
1.21
(0.35)
|
0.28
(0.20)
|
1.67
(0.35)
|
1.56
(0.65)
|
|
Costar
Tissue Cult. Flat Bottom
|
0.10
(0.02)
|
0.24
(0.14)
|
0.15
(0.03)
|
0.61
(0.12)
|
0.14
(0.01)
|
0.65
(0.11)
|
2.03
(0.15)
|
3.11
(0.04)
|
0.95
(0.04)
|
2.38
(0.15)
|
0.11
(0.00)
|
0.18
(0.02)
|
0.32
(0.04)
|
0.84
(0.11)
|
|
Immulon
4HBX
|
0.08
(0.00)
|
0.16
(0.00)
|
0.22
(0.08)
|
0.55
(0.06)
|
0.20
(0.07)
|
0.61
(0.05)
|
0.03
(0.10)
|
1.18
(0.11)
|
0.25
(0.05)
|
1.38
(0.22)
|
0.25
(0.06)
|
0.27
(0.02)
|
0.31
(0.13)
|
0.74
(0.03)
|
|
F96
Maxisorp
|
0.10
(0.01)
|
0.19
(0.08)
|
0.36
(0.14)
|
0.80
(0.12)
|
0.52
(0.21)
|
0.61
(0.10)
|
0.60
(0.50)
|
1.63
(0.33)
|
0.47
(0.02)
|
1.86
(0.18)
|
0.81
(0.61)
|
0.29
(0.03)
|
0.54
(0.35)
|
0.87
(0.05)
|
|
Millipore
IP ELISA Washer
|
0.28
(0.21)
|
0.43
(0.05)
|
0.33
(0.08)
|
0.53
(0.14)
|
0.29
(0.07)
|
0.32
(0.02)
|
0.32
(0.05)
|
1.29
(0.26)
|
0.25
(0.05)
|
1.15
(0.30)
|
0.58
(0.15)
|
0.83
(0.09)
|
1.37
(0.23)
|
1.89
(0.11)
|
|
Millipore
IP Hand Washed
|
0.24
(0.14)
|
0.33
(0.03)
|
0.24
(0.15)
|
0.30
(0.07)
|
0.20
(0.09)
|
0.23
(0.01)
|
0.15
(0.01)
|
0.45
(0.06)
|
0.16
(0.02)
|
0.45
(0.08)
|
0.03
(0.02)
|
0.40
(0.12)
|
0.95
(0.03)
|
1.52
(0.09)
|
*
Absorbance at 405 nm
** Std.
Deviation
Abbreviations
used:
ELISA – enzyme-linked immunosorbent assay; SQE – squalene; ISP –
isopropanol
Table
III Binding Specificities of
Monoclonal Antibodies to Squalene1
|
Clone
No.
|
SQE
|
DMPC
|
DMPG
|
Lipid
A
|
Hexadecane
|
Pristane
|
Stearylamine
|
Cholesterol
|
SQA
|
Arachidic
acid
|
Behenic
acid
|
Palmitic
acid
|
Stearic
acid
|
Lignoceric
acid
|
Lauric
acid
|
Myristic
acid
|
|
1
|
+
|
+
+ +
|
+
|
+
+ +
|
+
|
-
|
+
|
+
|
-
|
-
|
+
+ +
|
+
+ +
|
+
+ +
|
+
|
-
|
+
+
|
|
2
|
+
|
+
+
|
+
|
+
+ +
|
-
|
-
|
+
|
+
|
+++
|
-
|
+
+
|
+
+
|
+
+ +
|
+
|
-
|
+
+
|
|
3
|
+
|
+
+
|
+
+
|
+
+ +
|
+
|
-
|
+
|
+
+ +
|
+++
|
-
|
+
+ +
|
+
+ +
|
+
+ +
|
+
|
-
|
+
|
|
4
|
+
|
+
+ +
|
-
|
-
|
-
|
-
|
+
|
+
+ +
|
+
|
-
|
+
|
+
|
+
+ +
|
-
|
-
|
-
|
|
5
|
+
|
+
+ +
|
+
|
+
+ +
|
-
|
-
|
+
|
+
+ +
|
-
|
-
|
-
|
+
+
|
+
+ +
|
+
|
-
|
-
|
|
6
|
+
|
+
+ +
|
-
|
+
+ +
|
-
|
-
|
+
|
+
+ +
|
-
|
-
|
+
|
+
+
|
+
+ +
|
+
|
-
|
-
|
|
7
|
+
|
+
+ +
|
+
|
-
|
-
|
+
|
+
+
|
+
+ +
|
++
|
-
|
-
|
+
+ +
|
+
+ +
|
+
+
|
-
|
-
|
|
8
|
+
|
-
|
-
|
+
|
-
|
-
|
-
|
+
|
+++
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
|
-
|
+
|
|
9
|
+
|
+
+
|
+
|
+ + +
|
+
|
-
|
+
|
+
|
++
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+
|
+
|
+
|
|
10
|
+
|
+
+
|
+
|
+
+ +
|
+
|
-
|
+
|
+
|
+
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
|
-
|
+
+
|
|
11
|
+
|
+
|
-
|
+
+ +
|
+
|
-
|
+
|
+
|
-
|
+
+ +
|
+
+ +
|
+
|
+
+ +
|
+
|
-
|
+
|
|
14
|
+
+
|
+
|
-
|
+
+ +
|
-
|
-
|
+
|
+
|
+++
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
-
|
+
+ +
|
|
16
|
+
+ +
|
+
+ +
|
+
|
+
+ +
|
+
+
|
+
|
+
|
+
|
+++
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+
|
-
|
+
+ +
|
|
18
|
+
+
|
+
+ +
|
+
|
+
+ +
|
+
+
|
+
|
+
+
|
+
|
++
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
-
|
+
+ +
|
|
19
|
+
|
++
+
|
+
|
+
+ +
|
+
|
+
|
+
+
|
+
|
+++
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+ +
|
+
+
|
-
|
+
+ +
|
1
-
<3X Background
+
3X-6X Background
++
7X-10X Background
+++
>10X Background
Abbreviations
used:
SQE – squalene; SQA – squalane; DMPC – dimyristoyl
phosphotidylcholine; DMPG – dimyristoyl phosphotidylglycerol | 
