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Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

Published: October 21st, 2016



1School of Applied Science, Temasek Polytechnic, 2University of Applied Sciences Utrecht

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

The point-of-care testing (POCT) diagnostic study is important for the development of new strategies for therapeutics, personalized medicine, and home care1. Cellulose papers are widely used as platforms in immunoassays, as they are cheap, accessible, and familiar to users2. In addition, the porous structure of cellulose paper possesses the power to drive liquid flow without additional energy impact. Records of paper-based bioanalysis can be found as early as the 20th century, when paper chromatography was first invented in 1952. The most prevalent example is immunochromatographic tests3, such as pregnancy and diabetes test ....

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1. Grafting Amine Functional Groups on Cellulose Paper Discs

  1. Prepare one piece of square paper with a dimension of 1 cm × 1 cm, and 100 paper discs made from grade No. 1 cellulose paper with a diameter of 6.0 mm (medium-flow filter paper) using a hole punch.
  2. To derive -NH2 groups on the paper discs, mix 1 ml APS and 10 ml acetone in a 50 ml glass bottle in the fume hood. Add paper discs to the freshly prepared APS reagent mixture, and incubate for 5 hr with orbital stirring (200 rpm).......

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Figure 3
Figure 3. Fourier transform infrared (FTIR) spectra of untreated and APS-treated medium-flow filter square paper (A) and fast-flow filter square paper (B). A. The spectra for untreated medium-flow filter square paper was similar to that of APS treated medium-flow filter square paper. The increase in intensities at bands of 902-1,170 cm-1 and 1,210-1,500 cm.......

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Direct coating of affinity purified goat anti-Mouse IgG-Fc capture antibody on unmodified cellulose paper discs was performed to detect IgG concentrations. The results indicated that, further fixation of the capture antibodies is required for reproducibility. The silane technique was successfully used to introduce amine functional groups to the cellulose paper discs34. The concentration of APS affects the immobilization of antibodies. Therefore, the amount of APS in acetone was also optimized. 1 ml of APS in 1.......

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This work was financially supported by the Ministry of Education, Singapore through the Translational and Innovation Grant (MOE2012-TIF-2-G-009).


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Name Company Catalog Number Comments
Cellulose filter paper, Grade1 (medium flow filter paper) GE Healthcare Pte Ltd Singapore  1001 110
Cellulose filter paper, Grade113 (Fast flow filter paper) Sigma-Aldrich, Singapore 1113-320
3-​Aminopropyl​trimethoxysilane Sigma-Aldrich, Singapore T1255 >96%
Glutaraldehyde Sigma-Aldrich, Singapore G6257 Grade II, 25% in H2O
Surfactant Tween-20, Sigma-Aldrich, Singapore P2287
Bovine serum album  Sigma-Aldrich, Singapore A2153
Skimmed milk powder Louis François  Packed by Kitchen Capers, Singapore
Tris base Promega H5135
Sodium periodate Merck 106597
Na2HPO4 Merck 106585
KH2PO4 Merck 104873
NaOH Merck 106462
HCL Merck 100317
phosphate buffer saline (PBS) N/A N/A PBS, containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 8.0 mmol/L Na2HPO4 and 1.5 mmol/L KH2PO4, is prepared with water and adjusted to pH 7.4 with 0.1 mol/L NaOH or 0.1 mol/L HCl
Acetone Tee Hai Chem Pte Ltd Singapore 9005-68
Mixture of TMB and hydrogen peroxide solution  1-Step ultra TMB-ELISA solution , Thermo Scientific Pierce 34029 1 L
Rabbit anti-human IgG-FITC TWC/Bio Pte Ltd Singapore sc-2278
Peroxidase conjugated goat anti-rabbit IgG TWC/Bio Pte Ltd Singapore sc-2030
Affinity purified goat anti-Mouse IgG-Fc coating antibody Bethyl Laboratories, Inc A90-131A
Mouse reference serum Bethyl Laboratories, Inc RS10-101-5 9.5 mg/mL
HRP conjugated goat anti-mouse IgG-Fc detection antibody Bethyl Laboratories, Inc A90-131P
Fourier transform infrared spectrophotometer Shimadzu IR Prestige-21  N/A
Fluorescence molecular imager Pharos FXTM plus molecular imager, Bio-Rad, Singapore N/A
Oven NUVE FN500 N/A
Turbo mixer VM-2000 MYC LTD N/A
Image J RGB, free download N/A

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