A Paper-Based Immunoassay for the Detection of Immunoglobulin G

Abstract

Source: Peng, Y. et al., Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays. J. Vis. Exp. (2016)

The video demonstrates a paper-based immunoassay, involving amine-functionalized cellulose discs cross-linked with glutaraldehyde. Covalent immobilization of IgG-Fc-specific capture antibodies, followed by the introduction of immunoglobulin G and horseradish peroxidase-conjugated detection antibodies, results in a color change confirming the presence of immunoglobulin G.

Protocol

1. Grafting Amine Functional Groups on Cellulose Paper Discs 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. To derive -NH2 groups on the paper discs, mix 1 ml 3-aminopropyltrimethoxysilane (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,…

Representative Results

Figure 1. 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&#160…

Materials

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-Aminopropyltrimethoxysilane	Sigma-Aldrich, Singapore	T1255	>96%
Glutaraldehyde	Sigma-Aldrich, Singapore	G6257	Grade II, 25% in H2O
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
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
Oven	NUVE FN500	N/A
Turbo mixer VM-2000	MYC LTD	N/A
Image J	RGB, free download	N/A