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Validation of nanodot array
luminometric immunoassay:
An assay for the simultaneous
measurement of tumour markers
Laura Wainwright
Queen Alexandra Hospital, Portsmouth
Potential uses
•Screening
of general/at risk populations
•Differential
•Clinical
diagnosis in patients displaying symptoms
staging of cancer
•Estimation
of tumour volume
•Prognostic
indicator of disease progression
•Detecting
recurrence of cancer
•Monitoring
response to therapy
Tumour markers
CEA
Colorectal cancer; post-operative surveillance and during
chemotherapy
Breast cancer; detection of metastasis and during chemotherapy
in advanced disease
CA 15-3
Breast cancer; detection of recurrence and during chemotherapy
of advanced disease
CA 125
Ovarian cancer; differential diagnosis of pelvic masses, postoperative surveillance and during chemotherapy
CA 19-9
Pancreatic cancer; monitoring chemotherapy and detecting
recurrence
-hCG
Germ cell tumours and gestational trophoblastic disease;
diagnosis, staging, monitoring treatment and prognosis
Multiple markers
•Use
several markers to increase specificity and sensitivity
of detection/distinguishing malignancy from nonmalignancy
•hCG,
LDH and AFP should be used to monitor NSGCT
•EGTM
recommends measurement of CA 15-3 and CEA in
breast cancer follow-up
•Literature
mixed
surrounding breast and ovarian cancer is
Multiplex Immunoassay
•Theory:
uses less reagent, faster, needs less sample
•Dots
of immobilised Ab on a planar surface
= mini-ELISA
•Arrays
of capture Ab on 96-well plates/glass slides
•Literature
•CVs
examples: cytokines and tumour markers.
up to 40 %: imprecision generally a problem
NALIA
Nanodot Array Luminometric Immunoassay
Vacuum Manifold
wel
l
capture Ab
Ag
detection Ab
biotin
SA-HRP
Aims
•Validate
the markers currently on the array (CEA,
CA 125, CA 15-3, CA 19-9)
•Optimise
and validate -hCG onto the array
•Compare
with current routinely used assays (DxI,
Kryptor)
•Look
at how many of these markers are raised in
breast and ovarian cancer
First…
up -hCG assay as a standard ELISA
•Transfer it to NALIA
•Run all 5 assays together on NALIA
-exp with blocking, exposure time and background
subtraction
-changes to existing assay protocol
•Set
•Run
samples, standard curve and 2 levels of control
in triplicate
•100 samples per marker for method comparison
Standard curves
•Intra•LOD
and inter-plate CVs:
44.5-114.1 %
and recovery:
LOD
CEA (ng/mL)
3.9
% Recovery
CEA
121-208
CA 125 (U/mL)
73.7
CA 125
8-67
CA 15-3 (U/mL)
235.9
CA 15-3
312-4901
CA 19-9 (U/mL)
2621.8
CA 19-9
-868-3746
Free -hCG (ng/mL)
•Cross-reactivity:
116.5
Free -hCG
73-977
Difficult to interpret due to high CVs
and LODs
Scatter Plots + Spearman Rank Correlation
CEA
300
21000
14000
0
2100
1400
700
0
0
1000
0
0.499
2800
7000
0
7000 14000 21000 28000 35000
NALIA (ng/mL)
700 1400 2100 2800 3500
NALIA (U/mL)
NALIA (U/mL)
CA 19-9
6000
3500
0.510
DxI (U/mL)
DxI (U/mL)
600
500
CA 15-3
28000
900
0
CA125
35000
0.549
Free -hCG
300
-0.139
Kryptor (ng/mL)
DxI (U/mL)
DxI (ng/mL)
1200
4000
2000
0.172
250
200
150
100
50
0
0
0
2000
4000
NALIA (U/mL)
6000
0
100
200
NALIA (ng/mL)
300
•Signed
•Bland
rank sum test: NALIA has a +ve bias
and Altman plots show the same
Dotting CVs
•Dot
plates with biotinylated BSA
•Calculate
inter-well and inter-plate CVs from the raw
data to determine how spot density varies
•Within
well: 19.1 %
•Within
plate: 24.8 %
•Occurs
randomly over the plate
well
BSA
biotin
SA-HRP
So…
•Not
ready for routine use
•CEA,
then CA 125 were the best of the five
Drawbacks of NALIA
•Main
-
problem: very high assay CVs
dotting inconsistencies
buffer flow variations over the plate when in manifold
differing viscosities of serum samples
uneven well-emptying during incubations
manual process for conversion of image data to numerical format
•Very
low S/N ratio
•Data
acquisition process not practical for routine use
•Very
time consuming and labour-intensive
Future
•Much
additional work needs to be performed
- sort out previously mentioned problems
- reagent stability
- effect of lot number change
•Need
more research into the use of multiple markers
•Requesting tests just because they are there will not improve
patient care
•Temptation
to use array-based assays as a cancer “screen”
Acknowledgements
Guy Gabriel
Ian Cree
Helen Smith
TORC lab members
Bernie Higgins