Validation of nanodot array luminometric immunoassay:

An assay for the simultaneous measurement

of tumour markers

Laura WainwrightQueen Alexandra Hospital, Portsmouth

Potential uses

•Screening of general/at risk populations•Differential diagnosis in patients displaying symptoms•Clinical 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, post- operative 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 non-malignancy•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 surrounding breast and ovarian cancer is mixed

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 examples: cytokines and tumour markers.•CVs up to 40 %: imprecision generally a problem

NALIANanodot Array Luminometric

Immunoassay

Vacuum Manifold

well

capture Ab

Agdetection Abbiotin

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

•Set 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

•Run samples, standard curve and 2 levels of control in triplicate•100 samples per marker for method comparison

First…

Standard curves

•Intra- and inter-plate CVs: 44.5-114.1 %

LOD

CEA (ng/mL) 3.9

CA 125 (U/mL) 73.7

CA 15-3 (U/mL) 235.9

CA 19-9 (U/mL) 2621.8

Free -hCG (ng/mL)

116.5

% Recovery

CEA 121-208

CA 125 8-67

CA 15-3 312-4901

CA 19-9 -868-3746

Free -hCG 73-977

•Cross-reactivity: Difficult to interpret due to high CVs and LODs

•LOD and recovery:

Scatter Plots + Spearman Rank Correlation

0

7000

14000

21000

28000

35000

0 7000 14000 21000 28000 35000

NALIA (U/mL)

DxI

(U/m

L)

CA125

0.510

0

700

1400

2100

2800

3500

0 700 1400 2100 2800 3500

NALIA (U/mL)

DxI

(U/m

L)

CA 15-3

0.499

0

300

600

900

1200

0 500 1000

NALIA (ng/mL)

DxI

(ng/

mL)

CEA

0.549

0

2000

4000

6000

0 2000 4000 6000

NALIA (U/mL)

DxI

(U

/mL

)

CA 19-9

-0.139

0

50

100

150

200

250

300

0 100 200 300

NALIA (ng/mL)

Kry

ptor

(ng/

mL)

Free -hCG

0.172

•Signed rank sum test: NALIA has a +ve bias•Bland 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

BSAbiotin

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 GabrielIan Cree

Helen SmithTORC lab members

Bernie Higgins