Validation of nanodot array luminometric immunoassay: An assay for the simultaneous measurement of tumour markers

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 well 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. First… • 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

10. Standard curves

11. Intra- and inter-plate CVs: 44.5-114.1 % • LOD and recovery: • Cross-reactivity: Difficult to interpret due to high CVs and LODs

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

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