2001/10/26 NSC B

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1 NSC892323B

2 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

3 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

4 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 = ( ) / = 72.4% 125 ng/ml Log [ Log [ ( ) 250 ng/ml R L U y = x r = Log [RLU] y = x r = AFP, ng/ml Log [AFP], ng/ml 4

5 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 Type 4 Coating antibody AFP (ng/ml) 2 µg 4 µg 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 New type New type New type Type4 poly-lysine RLU 5

6 (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 labeling (signal) antibody (linear up to 500 ng/ml) y = x R 2 = r = AFP, ng/ml Log [RLU] y = x R 2 = r = Log [AFP], ng/ml 3. Determination of Antibody Affinity (Part A) OD 490 nm X of origin K D (Cat. RDI-TRK4F16-4A3) (AFP) K D M 4. Enzyme Reaction on Chips- for Indirect Chemiluminescence Assay HRP 6

7 1. Thorpe GH, Bronstein I, Kricka LJ, Edwards B, Voyta JC. Chemiluminescent enzyme immunoassay of alpha-fetoprotein based on an adamantyl dioxetane phenyl phosphate substrate. Clin Chem 1989;35: Woodhead JS, Weeks I. Immunochemiluminometric assays based on acridinium labels with a microtiter plate luminometer. Clin Chem 1991;37: Batmanghelich S, Brown RC, Woodhead JS, Weeks I, Smith K. Preparation of a chemiluminescent imidoester for the non-radioactive labelling of proteins. J Photochem Photobiol B Biol 1992;12: Kricka LJ. Strategies for improving sensitivity and reliability of immunoassays. Clin Chem 1994;40: Morgan CL, Newman DJ, Price CP. Immunosensors: Technology and opportunities in laboratory medicine. Clin Chem 1996;42: Brown RC, Weeks I, Fisher M, Harbron S, Taylorson CJ, Woodhead JS. Employment of a phenoxy-substituted acridinium ester as a long-lived chemiluminescent indicator of glucose oxidase activity and its application in an alkaline phosphatase amplification cascade immunoassay. Anal Biochem 1998;259:

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