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

Summary

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.

Abstract

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.

Introduction

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 20^th century, when paper chromatography was first invented in 1952. The most prevalent example is immunochromatographic tests³, such as pregnancy and diabetes test ....

Protocol

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) at room temperature³².
   Caution: Handle APS and acetone in the fume hood.
3. Decant excess solution from the 50 ml glass bottle into....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cellulose filter paper, Grade 1 (medium flow filter paper)	GE Healthcare Pte Ltd Singapore	1001 110
Cellulose filter paper, Grade 113 (Fast flow filter paper)	Sigma-Aldrich, Singapore	1113-320
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
NaCl	CALBIOCHEM	567441
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
Equipment
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
ImageJ	RGB, free download	N/A