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 2014www.clinchem.org/content/60/4/610.full© Copyright 2014 by the American Association for Clinical Chemistry

Background Over the last 2 decades, a plethora of new diagnostic
methods have been proposed in the academic literature, butfew 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 seamlesslyin the final implementation.
 For example, to develop a faster test, improved chemical
reagents alone are insufficient; rather, the reagents mustalso work with equipment easily accessible to the end users.
See Editorial by Sia SK. On the Path from Materials Chemistry to Clinical Use. ClinicalChemistry 2014; 60: 573-574.

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction
 Immunoassays are commonly used in the measurement ofbiomarkers 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 havemainly involved magnetically assisted separation ofantibody-coated magnetic particles from reaction medium tofacilitate assay processes.
© Copyright 2009 by the American Association for Clinical Chemistry

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

© Copyright 2009 by the American Association for Clinical ChemistryMaterials and Methods
 Fluorescent Ferrite beads (FF beads)
Dispersion

Magnetic collection

Visible light

Ultraviolet• Can be well-dispersed• Can collect magnetically• Show high fluorescence© Copyright 2009 by the American Association for Clinical Chemistry

Question
 FF beads are functional materials that possessdual functions (strong magnetic force and highly
fluorescence intensity). Can FF beads be usedeffectively for achieving a rapid immunoreaction?

© Copyright 2009 by the American Association for Clinical ChemistryMaterials and Methods
 Immunoassay with magnetic collection of FF beads

 Immunohistochemistry with magnetic collection of FFbeads© Copyright 2009 by the American Association for Clinical Chemistry

Results
AB

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.

© Copyright 2009 by the American Association for Clinical Chemistry

ResultsCDE

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.
© Copyright 2009 by the American Association for Clinical Chemistry

ResultsAB

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.
© Copyright 2009 by the American Association for Clinical Chemistry

ResultsCDEF

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 andred markers represent PSA detection with magnet and without magnet, respectively. All data are expressed
as means (SD) of 3 measurements.
© Copyright 2009 by the American Association for Clinical Chemistry

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.

© Copyright 2009 by the American Association for Clinical Chemistry

ResultsABCFigure 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); andneedle-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.
© Copyright 2009 by the American Association for Clinical Chemistry

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.

© Copyright 2009 by the American Association for Clinical Chemistry

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

© Copyright 2009 by the American Association for Clinical Chemistry

Discussion The results clearly indicate that the magnetic force
enhances and accelerates specific interactions between
antigens and antibodies, and the application ofmagnetic collection of FF beads in a particular space tothe immunoreaction can dramatically improve both thetotal 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 explainedby the magnetic concentration of both antigens andantibodies locally on the plate or samples, followed bydirect florescent measurement without the need forsignal amplification.
© Copyright 2009 by the American Association for Clinical ChemistryThank you for participating in this month’s
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