1. Validation of nanodot array luminometric immunoassay: An assay for the simultaneous measurement of tumour markers Laura Wainwright Queen Alexandra Hospital, Portsmouth

2. 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

3. 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

4. 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

5. 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

6. NALIA Nanodot Array Luminometric Immunoassay

7. Vacuum Manifold wel l capture Ab Ag detection Ab biotin SA-HRP

8. 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

9. 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…

10. Standard curves

11. 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 CEA121-208 CA 1258-67 CA 15-3312-4901 CA 19-9-868-3746 Free  -hCG 73-977 Cross-reactivity:Difficult to interpret due to high CVs and LODs LOD and recovery:

12. Scatter Plots + Spearman Rank Correlation CA125 0.510 CA 15-3 0.499 CEA 0.549 CA 19-9 -0.139 Free  -hCG 0.172

13. 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 BSA biotin SA-HRP

14. 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

15. 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”

16. Acknowledgements Guy Gabriel Ian Cree Helen Smith TORC lab members Bernie Higgins