Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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