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HRP acridinium ester ELISA validation pattern AFP PMMA Keywordstumor marker, immunoassay, microchip, chemiluminescence Cancer is the first leading cause of death in Taiwan. Our ability to decrease the number of death caused by cancer depends, in part, on early detection and treatment. Serum tumor markers have been used for many years in the management of cancer patients. However, their poor sensitivity and specificity precluded their use in screening and in many instances in predicting the patients prognosis. New technology in immunoassay may allow the improved sensitivity and specificity required to permit rational use of tumor markers in clinical oncology. Many forms of immunoassays, ranging form test tubes to microwells are used in clinical setting for qualitative or quantitative assays. A recent trend has been to fashion immunoassay tests into disposable devices processed on a dedicated analyzer. Micromachining and microfabrication provides a route to total system integration for the multi-step immunoassay. The low internal volume of microchip analyzers leads to many advantages including low reagent consumption, low sample volume as well as waste production, faster response time and simple to operate point-of-care diagnostics. This study is designed for the development, optimization and validation of a microchip device to measure multi-biomarkers for earlier cancer detection and risk assessment. The main themes of this project will include technological development and refinement, as well as the 2
laboratory validation of immunobiochemical reaction on chips. By using the techniques of Micro-Electro-Mechanical Systems (MEMS) and the development of solvent programming system, the feasibility of enzyme analysis on the chip will be investigated by photometric and chemiluminescent detection scheme. We studied direct chemiluminescence immunoassay using acridinium-labeled antibody as the detection antibody and indirect chemiluminescence immunoassay using horseradish peroxidase (HRP) as the tracer in the enzymeimmunoassay on surface-modified chips. The solid phase enzyme chemiluminescence immunometric assay (ECMA) on beads has been successfully established in the laboratory. The results of the first year study showed that PMMA activated with polypeptide was the best substrate for the chip. The antibody affinity determined by K D was 8 x 10-9 M. The total reaction time reduced from 48 hours to 4 hours on chips. The acridinium ester-labeled ECMA assay for AFP on chips had better linearity and sensitivity, compared to the bead system. In conclusion, the ECMA reaction on chip for the determination of serum AFP showed the great access to the (immunoassay) (risk assessment) (screening) - (differential diagnosis primary vs. metastatic) (prognosis) (monitoring clinical course) [1] 1960 Radioimmunoassay, RIA ng Enzyme immunoassay, EIA (Chemiluminescent immunoassay, CIA) [2-12] [13] ( ) RIA microfabrication microchip analyzer µl ρl microchip immunoassays µchip AFP 10 ρg/ml (Light-addressable potentiometric sensor) 25 ρg / (capillary electrophoresis, CE) 2 µg / µchip-ce 10 ng/ml / µchip (IgG) 3
chemiluminescence acridinium ester (lucigenin) (signal antibody) horseradish peroxidase (HRP) luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) H 2 O 2 camera luminometer (photomultiplier tube 1. AFP-ECMA on Beads Labeled Recovery = (326547 + 289595) / 850510 = 72.4% 125 ng/ml Log [ Log [ ( ) 250 ng/ml 200000 5.5 R L U 150000 100000 50000 y = 523.36x + 24992 r = 0.985 Log [RLU] 5 4.5 4 3.5 y = 0.3846x + 4.1584 r = 0.968 0 0 100 200 300 AFP, ng/ml 3 0 0.5 1 1.5 2 2.5 3 Log [AFP], ng/ml 4
2. Coating Anti-AFP Antibody on Chips 2001/10/26 1). ( PMMA ) coating chips with 2 nd set antibody 5 AFP antigen PMMA 2). poly-l-lysine 24 (a) Direct coating of labeled (signal) antibody on chips (PMMA, PDMS) poly-lysine PMMA signal antibodypdma poly-lysine signal antibody (b) Coating chips with 2 nd set (capture) antibody type 2 type 4 capture antibody coating Type 2 Coating antibody AFP (ng/ml) 2 µg 4 µg 0 55787 40076 12.5 26600 39326 62.5 50205 68464 Type 4 Coating antibody AFP (ng/ml) 2 µg 4 µg 0 11785 24348 12.5 38346 29618 62.5 56286 47677 2 µg capture antibody coated on type 4chip had the best linear reaction. (c). Direct coating of labeled anti-afp antibody (signal antibody) on new chip Poly-L-lysine (+) Poly-L-lysine (-) P (+) Ab coating New type 1 1262274 42559 30 New type 2 1071016 71794 15 New type 3 1355225 33599 40 New type 4 1397764 127213 11 Type4 poly-lysine RLU 5
(d). Coating secondary set antibody (capture antibody) on chips and run an ELISA assay on chips using different concentration of antigens (AFP) coating secondary set (capture) antibody antigen RLU 400000 350000 300000 250000 200000 150000 100000 50000 0 labeling (signal) antibody (linear up to 500 ng/ml) y = 765.24x + 39905 R 2 = 0.9258 r = 0.962 0 100 200 300 400 500 AFP, ng/ml Log [RLU] 6 5.5 5 4.5 4 3.5 3 y = 0.6105x + 3.9144 R 2 = 0.9622 r = 0.981 0 0.5 1 1.5 2 2.5 3 Log [AFP], ng/ml 3. Determination of Antibody Affinity (Part A) OD 490 nm 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 0.0005 0.001 0.0015 0.002 X of origin K D (Cat. RDI-TRK4F16-4A3) (AFP) K D 8 10-9 M 4. Enzyme Reaction on Chips- for Indirect Chemiluminescence Assay HRP 6
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