1. Magnetically Promoted Rapid Immunoreactions Using Functionalized Fluorescent Magnetic Beads Satoshi Sakamoto and Co Department of Biological Information Graduate School of Biosciences and Biotechnology Tokyo Institute of Technology, Yokohama, Japan Imtiaz Ahmed 98-arid-230 1

2. Contents: Introduction Material and Methods Results Discussion Conclusion 2

3. Introduction  Accurate Detection & Monitoring of a Disease-related Biomarkers is important in understanding pathophysiology  Devised a Rapid Immunoreaction System that containing Ferrites (magnetic iron oxide) Fluorescent 3

4.  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 immuno reaction?  Is it possible to detect an immuno reaction without use of enzymes? 4

5.  Biomarkers provides information Disease Progression and Prognosis Diagnosis of Disease  Immunoassays are commonly used in the measurement of biomarkers in both the research and clinical arenas 5

6. Types of ELISA 6 http://www.abnova.com/

7.  ELISA time-consuming and relatively complex approach  Platforms shorten assay time and reduce the number of steps involved  Immuno reaction using magnetic particles has attracted attention as a means to improve assay efficiency 7

8.  Reports of immunoassays with magnetic particles have mainly involved magnetically assisted separation of antibody- coated magnetic particles from reaction medium to facilitate assay processes  Here they describe a magnetically prompted immunoreaction system with unique polymer-coated magnetic beads containing highly analytically sensitive fluorophores 8

9.  Immunoassay  Immuno-histochemical Staining require no signal amplification step for the detection of biomarkers Analytically sensitive and rapid detection of disease related biomarkers 9

10. Material and Methods  Brain natri-uretic peptide (BNP) and antiBNP antibodies  Prostate specific antigen (PSA) and anti PSA antibodies  immunohistochemical staining Needle core biopsy samples were obtained from breast cancer patients (2006–2009) Samples from esophageal cancer patients undergoing surgery (2001–2006)  Magnetic plate with 96 magnets  Transmission electron microscope (TEM) 10

11.  PREPARATION OF FLUORESCENT FERRITE BEADS  PREPARATION OF ANTI BNP ANTIBODY–COATED FF BEADS  PREPARATION OF ANTI PSA ANTIBODY–COATED FF BEADS 11

12.  MAGNETICALLY INDUCED SANDWICH IMMUNOASSAY WITH FF BEADS FOR DETECTION OF BNP 96 well microplates coated with antiBNP antibody BNP Anti BNP antibodies Antibody coated FF beads magnet plate for incubation times of 0.5–3 min fluorescence measurements 12

13.  MAGNETICALLY INDUCED SANDWICH IMMUNOASSAY WITH FF BEADS FOR DETECTION OF PSA 96 well microplates coated with anti PSA antibody Prostate specific antigen (PSA) Anti PSA antibody coated FF beads magnet plate for incubation times of 0.5–3 min fluorescence measurement 13

14.  IMMUNOHISTOCHEMICAL STAINING OF PARAFFIN EMBEDDED SAMPLES  RAPID IMMUNOHISTOCHEMICAL STAINING OF TISSUES USING FF BEADS 14

15. Results  PRODUCTION OF UNIQUE 140nm–DIAMETER FF BEADS Fig:1 15

16. 16 Fig. 1. FF beads.(A), Schematic of the structure of FF beads. (B), FF beads dispersed in PBS (left) or collected by magnet (right) in visible light (above) and under ultraviolet irradiation (below). GMA, glycidyl methacrylate.

17.  MAGNETICALLY PROMPTED RAPID SANDWICH IMMUNOASSAY USING FF BEADS Fig:2  Detection antibody–coated FF beads and samples were added on a capture antibody–coated microplate, a magnet was attached under the plate for 1–2 min to concentrate the FF beads onto the immobilized capture antibody, unbound FF beads were washed out (similar to standard immunoassays), and the fluorescence of the remaining FF beads held on the plate through the antigen–antibody reaction was measured directly 17

18. Fig. 2. Schematic of a standard immunoassay scheme (top) and magnetically prompted sandwich immunoassay using useing antibody coated FF beads (bottom). 18

19.  To verify the feasibility of the newly devised system: BNP, which is a hormone secreted by the heart and a basic biomarker of heart failure, as the target antigen The BNP concentration in the blood plasma of healthy persons is usually 100 pg/mL BNP are associated with heart disease Magnetic collection of anti BNP antibody–coated FF beads in an immuno reaction allowed dose dependent detection of BNP within 1 min (Fig. 3A). By contrast, the assay without magnetic collection of FF beads could detect only 200 pg/mL BNP (Fig. 3B) Without magnets, the fluorescence intensity derived from the FF beads held on the plate through the antigen–antibody reaction was highly variable (Fig. 3B) 19

20. Fig. 3, C–E, show the detection of BNP at single incubation times of 1, 2, and 3 min These graphs indicate that the magnetic force clearly enhanced the detection of BNP Notably, a specific antigen–antibody reaction could be detected rapidly by only 1 min magnetic collection of FF beads without the use of any enzyme and enzymatic reaction 20

21. Fig. 3. 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. 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. 21

22.  Next investigated detection of PSA, Healthy individuals generally have low concentrations of PSA ( 4.0 ng/mL; hence, clinical examination of prostate cancer requires detection of PSA in serum ranging from 0.1 to 10 ng/mL Using the magnetic system, we successfully detected as little as 0.02 ng/mL PSA within 5 min of sample addition. Fig. 4 22

23. Fig. 4. 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. 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 mean (SD) of 3 measurements. 23

24.  LIMIT OF DETECTION, LIMIT OF QUANTIFICATION, AND REPRODUCIBILITY IN THE SANDWICH IMMUNOASSAY WITH FF BEADS The limit of detection (LOD) was defined as the lowest measured value 5.0 pg/mL for BNP in plasma and around 0.005 ng/mL for PSA in serum 24

25.  MAGNETICALLY PROMPTED RAPID IMMUNOHISTOCHEMISTRY USING FF BEADS FF beads coated with antibodies recognizing specific carcinoma cell surface antigens are added onto fixed samples of carcinoma cells, and a magnet is attached beneath them to enhance the antigen–antibody reaction (10 min) Fig. 6 25

26. Fig. 6. Schematic of standard staining (top) and magnetically prompted immuno histochemical staining of carcinoma cells using antibody coated FF beads (bottom). 26

27.  To confirm the magnetically promoted immunohistochemical staining with FF beads Fig. 7 27

28. Fig. 7. Immunohistochemical staining of EGFR (A); Lung cancer cell (B) ;breast cancer (C) Left, HE staining; middle, paraffin embedded staining; right, immune histo-chemical staining with magnetic collection of FF beads 28

29. Discussion Conventional immunoassays exhibit low level signals due to the use of enzyme modified detection antibodies Magnetic particles are attractive functional materials in the development of immunoreactions, because magnetic force often improves efficiency and analytical sensitivity conventional immuno histochemical staining generally requires overnight incubation of samples with primary antibodies for high analytical sensitivity The FF beads were quite stable and could be stored for at least 6 months without any damage and loss of fluorescence intensity 29

30. 3–10 min immuno reaction with the magnet seemed to be most effective for the detection of the biomarkers The successful rapid and analytically sensitive detection of target molecules (BNP, PSA, and EGFR) by magnetic collection of FF beads in the immuno reaction 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 30

31. The immunoreaction systems with FF beads described here used magnetic force, not to remove the magnetic beads but to enhance the signal for detection of biomarkers enables improvements in both analytical sensitivity and total assay time 31

32. summary  In summary, they successfully developed a rapid immuno reaction system using uniquely developed fluorescent magnetic beads  Two distinguishing properties strong magnetic force and high fluorescence intensity enable the shortening of assay time dramatically without loss of sensitivity and eliminate the signal amplification step  Immunoreaction systems with FF beads would be feasible conventional immunoassays immuno histochemical staining 32

33. Conclusion  This proof of principle system demonstrates that immunoreactions involving the magnetic collection of antibody coated FF beads allow acceleration of the antigen– antibody reaction  The simple magnetic collection of antibody coated FF beads to a specific space enables rapid detection of disease related biomarkers and identification of carcinoma cells 33

34.  Magnetically Promoted Rapid Immunoreactions Using Functionalized Fluorescent Magnetic Beads 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 Published January 16, 2014 doi: 10.1373/clinchem.2013.211433 Clinical Chemistry April 2014 vol. 60 no. 4 610-620 Link: http://www.clinchem.org/content/60/4/610.long 34

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