Echelon Biosciences Inc.
675 Arapeen Drive, Suite 302
Salt Lake City, UT 84108
Telephone 866-588-0455
Fax 801-588-0497
echelon@echelon-inc.com
www.echelon-inc.com
Question and Answer Sheet
For research use only
Not intended or approved for
diagnostic or therapeutic use.
The LPA Assay Kit was co-developed using the proprietary monoclonal antibody of Lpath Inc. (San Diego, CA). Lpath, Inc. has a
number of patent applications pending or issued covering material(s) incorporated into this assay. The assay’s sole permitted use is
for non-clinical research to measure LPA in biological samples. Any other use, including drug discovery, requires a license from
Lpath (www.lpath.com).
This document reviews commonly asked questions for the Lysophosphatidic Acid (LPA) Assay kits;
catalog numbers K-2800 and K-2800B
LPA standard and % recovery of different acyl chains.
Q: What version of LPA is used for the standard in the LPA assay,
catalog number K-2805?
A: In preparing the LPA standard (K-2805) we use C18:3, Echelon catalog
number L-0183.
Product Name: D (+)-sn-1-O-linolenoyl-glyceryl-3-phosphate (Sodium Salt)
Product Number: L-0183
Formula: C21H35Na2O7P, FW: 476.45
ONa H
O
P
OH
O
0.2
0.1
0.0
DHS
Rat serum
Figure 3: Background control (No
Anti-LPA) was run with both delipidized
human sera (DHS) and Rat serum in
the K-2800 and K-2800B LPA assay
kits. Non-specific binding from
antibodies present in serum was
observed in the K-2800 kit when rat
serum was used.
C18:3
0.00
0.1
C20:0
0.5
100
1000
Note: It is assumed that these
results apply to both the K2800 and K-2800B since the
K-2800B assay utilizes the
same primary antibody and
similar assay format
10000
LOG10 [LPA], nM
Figure 2: Different mixtures of LPA in the K-2800. Mixtures were
designed based on biological activity1.
Each LPA mixture was prepared by mixing LPA at 1:1 ratio. LPA Mixture
= C14:0. C16:0. C18:0, C18:1, C18:2, C18:3, C20:0, C20:4. High
(Biological) Activity = C18:1, C18:2, C18:3. Low (Biological) Activity =
C18:0, C14:0
LPA
Spike-in
(µM)
Calculated
(µM)
% Recovery
LPA Mixture
High Activity LPA Mixture
Low Activity LPA Mixture
5
5
5
1.22
2.70
0.49
24%
54%
10%
1
10
100
1000
10000
Q: Has this assay been verified
LOG10 [LPA], nM
by mass spec analysis?
Figure: 3 A comparision of the K-2800 and K-2800B
standard curves
A: Mass spectrometry analysis,
performed by an outside lab, found
that undiluted human serum
Parameter
K-2800
K-2800B
samples run in the LPA assay gave
230 nM
15 nM
EC50
comparable LPA values. The same
0.99
0.99
R2
patient samples were anylized
160 nM
12 nM
Sensitivity
using mass spectrometry and K0.87
0.98
Z’
2800 ELISA. The samples were
run in triplicate. For more information please see “AACR poster” (available
on website) for further details of the mass spectrometry/ ELISA comparison
study.
Q: What lipids interfere with the assay?
A: We have analyzed
Structuraly Simular Lipid crossreactivity
structurally similar lipids
(Figure 4), and membrane
Glycerol Phosphate
lipids (Figure 5) in the KMG C18:1
2800 ELISA with no cross
MG C16 (migrated)
MG
C18:1
(migrated)
reactivity observed at the
LBPA C18
concentrations tested.
LBPA C12 (biotin)
LBPA C12
LPA C18:3
0.
00
LPA mixtures were prepared
based on biological activity1,2
100
1000
10000
LOG10 [LPA], nM
and assumed LPA species
Figure 1: Sensitivity and specificity of the K-2800 assay
present in serum. The percent
for different acyl chains of LPA.
recovery
of
the
different
LPA
LPA Mixture % recovery
mixtures
was
1.0
0 LPA
LPA C18:3
calculated using the
LPA Mixture
LPA C18:3 curve for
High Activity LPA
comparison.
Low Activity LPA
C20:4
0
10
Abs 450nm
.
2.
00
C18:2
1
Ba .50
se
lin
1. e
75
C18:1
1
1.
25
C18:0
Assay sensitivity and cross-reactive data with similar lipids.
Q: What is the LPA assay’s
LPA standard Curve Comparision
sensitivity?
1.00
A: The sensitivity of the LPA
ELISA (K-2800) is 0.16 μM.
0.75
K-2800B
However, the Universal LPA
K-2800
0.50
ELISA (K-2800B) has a much
higher sensitivity of 12 nM
0.25
(Figure 3; Table 2).
1.
00
C16:0
2
Different LPA chain lengths
and saturation were run in the
K-2800 LPA assay (See
figure 1). A decrease in chain
length and saturation resulted
in decreased potency in the
assay.
0.
75
C14:0
0 LPA
O.D.450
0.3
0.
50
LPA Acyl Chain Comparison
O.D.450
0.4
O
3
0.0
10
K-2800B
K-2800
0.5
0.
25
O
Non-specific binding
0.6
Q: I am running mouse samples, why
do they contain such high levels of
LPA?
A: The K-2800 assay kit utilizes an
anti-mouse-HRP conjugated secondary
antibody. This antibody has been
known to cause high background in
mouse and rat serum samples due to
endogenous antibodies present in the
serum. The K-2800B kit does not use
this secondary antibody and therefore
does not have elevated non-specific
binding from these sample sources
(Figure 3).
O.D.450
NaO
Primary and/or secondary antibody questions.
Q: What is the isotype of the primary antibody?
A: Mouse IgG
Ab450
Assay platform:
Q: Why do higher levels of LPA cause a decrease in the assay signal?
A: The LPA assays are competitive assays, in which the LPA bound to the
plate, competes for binding to the antibody with the LPA in solution. As the
LPA in solution increases the amount of anti-LPA antibody that binds the
plate is reduced, lowering the observed signal.
Figure 4: Structuraly simular lipids to LPA were ran in the LPA ELISA
(K-2800) at 2 M concentration.
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Common membrane lipids crossreactivity
References:
2.0
baseline
OD 450 nm
1.5
1.
Jalink K, Hengeveld T, Mulder S, Postma FR, Simon MF, Chap H, van der
Marel GA, van Boom JH, van Blitterswijk WJ, Moolenaar WH. Biochem
Lysophosphatidic acid-induced Ca2+ mobilization in human A431 cells:
structure-activity analysis. Biochem J. 1995 Apr 15;307 ( Pt 2):609-16
2.
Tokumura A, Sinomiya J, Kishimoto S, Tanaka T, Kogure K, Sugiura
T, Satouchi K, Waku K, Fukuzawa K.Human platelets respond
differentially to lysophosphatidic acids having a highly unsaturated fatty
acyl group and alkyl ether-linked lysophosphatidic acids. Biochem J. 2002
Aug 1;365(Pt 3):617-28.
1.0
10 M
0.5
2 M
pl
e
D
ilu
en
t
SP
C
Sa
m
P
SP
PS
S1
PE
PG
PC
PA
G
A
LP
C
D
LP
A
0.0
Lipids
Figure 5: Cell membrane lipids were tested for cross reactivity in the K-2800 assay kit. All
lipids were prepared in DHS at 10 µM and 2 µM. Run in triplicate. LPA =
Lysobisphosphatidic Acid C18:3, DAG = Diacylglycerol, LPC = Lysophosphotidylcholine,
PA = Phosphatidic Acid, PC = Phosphatidylcholine; C16, PE = Phosphatidylethanolamine;
C16, PG = Phosphatidylglycerol, PS = Phosphatidylserine; C16, S1P = Sphingosine 1
Phosphate, SP = Sphingosine, SPC = Sphingosylphosphorylcholine, Sample = Random
Human Sera, tested in 1X & 10X dilution, Diluent = Delipidized Human Sera (DHS), use as
baseline
Sample Preparation for other sources of LPA
Q: What other types of samples have been verified in the LPA assays and
what is the proper dilution?
A: The K-2800 and K-2800B were developed for the determination of LPA
in human serum and plasma. However, due to the interest in other sources of
LPA we are currently working to broaden the use of these kits (see table). An
example of human, goat and rat serum is shown below (Figure 5).
Sample Source
Cat #
K-2800/K-2800B
K-2800B
K-2800B
Dilution factor
of sample
Neat
*
*
Human Sera/Plasma
Tissue
Cell lysate
Animal sera/plasma
Mouse/Rat
Diluted in
DHS
DHS
DHS
K-2800B
*
DHS
* To be determined; work in progress.
Performance of different serum sources
1.75
1.50
K-2800B
uM LPA
1.25
K-2800
1.00
0.75
0.50
0.25
oa
t
G
um
H
H
um
an
(#
62
)
an
R
at
(#
sp
36
ik
)
ed
0.
5
M
G
oa
ts
R
pi
at
ke
d
0.
5
M
0.00
Figure 5. An example of Human, Rat, and Goat serum
with and without 5 M LPA spike. Goat and rat serum
anylized in both LPA assays.
.
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