A Paper-Based Immunoassay for the Detection of Immunoglobulin G
Published: March 29, 2024
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 …
開示
The authors have nothing to disclose.
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 |
