Magnetically Promoted Rapid Immunoreactions Using Functionalized Fluorescent Magnetic Beads: A Proof of Principle

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1 Magnetically Promoted Rapid Immunoreactions Using Functionalized Fluorescent Magnetic Beads: A Proof of Principle S. Sakamoto, K. Omagari, Y. Kita, Y. Mochizuki, Y. Naito, S. Kawata, S. Matsuda, O. Itano, H. Jinno, H. Takeuchi, Y. Yamaguchi, Y. Kitagawa, and H. Handa April Copyright 2014 by the American Association for Clinical Chemistry

2 Background Over the last 2 decades, a plethora of new diagnostic methods have been proposed in the academic literature, but few have made it to end users. A hurdle is that for a new diagnostic method to take hold, vastly different facets of the technology, including sample handling, assay chemistry, and device hardware, must be considered from the outset to function together seamlessly in the final implementation. For example, to develop a faster test, improved chemical reagents alone are insufficient; rather, the reagents must also work with equipment easily accessible to the end users. See Editorial by Sia SK. On the Path from Materials Chemistry to Clinical Use. Clinical Chemistry 2014; 60:

3 Introduction Immunoassays are commonly used in the measurement of biomarkers in both the research and clinical arenas. Although ELISA is commonly used, it is a time-consuming and relatively complex approach. Immunoreaction using magnetic particles has attracted attention as a means to improve assay efficiency. Reports of immunoassays with magnetic particles have mainly involved magnetically assisted separation of antibody-coated magnetic particles from reaction medium to facilitate assay processes.

4 Questions Is it possible to construct a rapid and reliable immunoreaction system with functionalized magnetic particles? Is it possible to utilize magnetic force for acceleration of an immunoreaction? Is it possible to detect an immunoreaction without use of enzymes?

5 Materials and Methods Fluorescent Ferrite beads (FF beads) Dispersion Magnetic collection Visible light Ultraviolet Can be well-dispersed Can collect magnetically Show high fluorescence

6 Question FF beads are functional materials that possess dual functions (strong magnetic force and highly fluorescence intensity). Can FF beads be used effectively for achieving a rapid immunoreaction?

7 Materials and Methods Immunoassay with magnetic collection of FF beads Immunohistochemistry with magnetic collection of FF beads

8 Results A B Figure 1. Detection of BNP by sandwich immunoassay using FF beads coated with anti-bnp antibody. Results of BNP detection with magnet (A) and without magnet (B). Red, blue, green, and orange lines represent 200, 20, 2.0, and 0 pg/ml of BNP, respectively.

9 Results C D E Figure 1. Detection of BNP by sandwich immunoassay using FF beads coated with anti-bnp antibody. Results of BNP detection at a single incubation time: 1 min (C); 2 min (D); 3 min (E). Blue and red markers represent BNP detection with magnet and without magnet, respectively. All data are expressed as mean (SD) of 3 measurements.

10 Results A B Figure 2. Detection of PSA by sandwich immunoassay using FF beads coated with anti-psa antibody. Results of PSA detection with magnet (A) and without magnet (B). Red, blue, green, red broken, blue broken, green broken, and orange lines represent 6.3, 2.0, 0.64, 0.20, 0.064, 0.020, and 0 ng/ml of PSA, respectively.

11 Results C D E F Figure 2. Detection of PSA by sandwich immunoassay using FF beads coated with anti-psa antibody. Results of PSA detection at a single incubation time: 1 min (C), 3 min (D), 10 min (E), 30 min (F). Blue and red markers represent PSA detection with magnet and without magnet, respectively. All data are expressed as means (SD) of 3 measurements.

12 Results Figure 3. Comparison of PSA concentrations measured by the conventional immunoassay (CLEIA) and by the sandwich immunoassay with FF beads. All data are expressed as means (SD) of 3 measurements.

13 Results A B C Figure 4. Immunohistochemical staining of A431 cells (human epidermoid cancer cell, high EGFR expression level) (A); H69 cells (small-cell lung cancer cell, low EGFR expression level) (B); and needle-core biopsy samples of breast cancer using anti-egfr antibody-coated FF beads (C). Left, HE staining; middle, paraffin-embedded staining; right, immunohistochemical staining with magnetic collection of FF beads.

14 Results The results of the sandwich immunoassay Magnetic collection of FF beads in the immunoreaction step enabled the detection of disease markers within 5 min after sample addition. The results of the immunohistochemical staining A 10-min magnetic collection of FF beads in the incubation step enabled the ultra-rapid and reliable discrimination of carcinoma cells.

15 Question for Discussion Why does the magnetic collection of FF beads enable a rapid immunoassay and a rapid immunohistochemistry?

16 Discussion The results clearly indicate that the magnetic force enhances and accelerates specific interactions between antigens and antibodies, and the application of magnetic collection of FF beads in a particular space to the immunoreaction can dramatically improve both the total assay time and the number of steps required. The successful rapid and analytically sensitive detection of target molecules by magnetic collection of FF beads in the immunoreaction steps can be explained by the magnetic concentration of both antigens and antibodies locally on the plate or samples, followed by direct florescent measurement without the need for signal amplification.

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