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

The overall goal of this methodology is to stabilize antibodies and proteins on a cellulose matrix to facilitate development of a naked-eye colorimetric immunoassay which can be useful for point-of-care diagnostics. This method can help answer key questions in the point-of-care diagnostic field. The main advantage of this technique is that it helps stabilize protein on cellulose which allows the development of low-cost paper-based diagnostic assays.

Demonstrating the procedure will be Gloria Chang, a student from my laboratory. To begin, prepare one piece of square paper with a dimension of one centimeter by one centimeter. Also prepare 100 paper discs made from Whatman grade number one cellulose paper with a diameter of 6.0 millimeters using a hole punch.

To derive amino groups on the paper discs, mix one milliliter of APS and 10 milliliters of acetone in a 50 milliliter glass bottle in the fume hood. Add paper discs to the freshly prepared APS reagent mixture. Then incubate the discs in the mixture for five hours with orbital stirring at room temperature.

Decant excess solution from the 50 milliliter glass bottle into an organic waste container. Add 10 milliliters of acetone to the glass bottle, mix well, and decant completely to remove any unreacted APS and other impurities. Repeat this step two times.

Spread the paper discs on the paper towel and place them in a 110 degree Celsius oven for three hours. After allowing the paper discs to cool, store the discs in a 50 milliliter centrifuge tube at room temperature. Use Fourier transform infrared spectroscopy or FTIR to check the grafting of amine groups on the cellulose square paper.

Open the software for FTIR spectroscopy. Navigate to measurement initialize. Choose data below the rectangles and select percent transmittance for measurement mode.

Then choose Happ-Genzel for apodization, set 45 for the number of scans, 4.0 for the resolution, and set the range as min 400 to max 4, 000. Click measure, then select data file for the background data. Click BKG to get the baseline for the background.

Next, fix the square paper on the film sample holder. Then select data file for the sample data and click sample to obtain the spectrum for the sample. Close the FTIR spectroscopy application and turn off the computer.

Add five milliliters of 0.05%glutaraldehyde solution to a 20 milliliter glass bottle. Immerse 15 amine functionalized medium flow filter paper discs in this solution and keep for one hour with shaking at room temperature. Concurrently, repeat this step to prepare another 15 aldehyde functionalized fast flow filter paper discs.

To remove unreacted glutaraldehyde from the paper discs, first place the medium flow filter paper discs and the fast flow filter paper discs into separate 15 milliliter centrifuge tubes. Then add five milliliters of deionized water to each tube and shake the tubes for 10 seconds. Remove the water by aspirating with a pipette.

Repeat two times to remove any unreacted glutaraldehyde. Then dry the paper discs in a 37 degree Celsius oven. Once dry, add five microliters of 0.025 milligrams per milliliter mouse IgG Fc fragment antibodies to the medium flow filter paper discs and eight microliters to the fast flow filter paper discs.

After incubating for 20 minutes, add 10 microliters of 50 millimolar PBS pH 7.4 to each paper disc without removing the antibodies. Incubate for 40 minutes for the amine aldehyde reaction. Wash the paper discs with 0.2 milliliters of washing buffer on top of a paper towel.

Repeat the wash three times. After drying the paper discs in an oven at 37 degrees Celsius, block the paper discs with 15 microliters of blocking buffer for 10 minutes at room temperature. Then wash each paper disc with 0.2 milliliters of washing buffer on top of a paper towel.

Repeat the wash three times. Next, run the IgG standards. Load 10 microliters of various IgG concentrations onto individual discs in triplicate.

Incubate for one hour at room temperature. Wash the paper discs with 0.2 milliliters of washing buffer on top of a paper towel. Repeat the wash three times.

Following the washes, load 10 microliters of HRP conjugated mouse IgG Fc fragment antibodies and incubate for one hour at room temperature. Wash the paper discs with 0.2 milliliters of washing buffer on top of a paper towel. Repeat the wash three times.

Finally, load a 10 microliter mixture of TMB and hydrogen peroxide onto each disc. Take images of all paper discs with a digital camera or smartphone after five minutes of incubation and analyze as discussed in the text protocol. Shown here is the FTIR spectra of untreated and APS-treated medium flow filter paper and fast flow filter paper.

The increase in intensities at bands of 902 and 1, 170 inverse centimeters and 1, 210 to 1, 500 inverse centimeters for medium flow filter paper as well as the characteristic peaks in the 972 to 1, 180 inverse centimeter range for fast flow filter paper showed that APS has been successfully grafted onto the cellulose paper. The effects of different concentrations of glutaraldehyde on the immobilization of IgG FITC on the paper discs are shown here. The optimal concentration for glutaraldehyde is 0.05%Similarly, the effects of different concentrations of IgG FITC on the immobiliation of IgG FITC on the paper discs are presented.

Increasing the load and concentration of IgG FITC increase the amount of immobilized IgG FITC on the paper discs. However, increased concentration of immobilized IgG FITC does not improve the binding capacity with antigen. Shown here are the calibration curves for the determination of IgG by paper-based ELISA.

Antibodies were detected at a concentration of one nanogram per milliliter IgG with the naked eye using this paper-based assay. The concentration of IgG serum had a linear relationship with colorimetric intensity from zero to 500 nanograms per milliliter when each step was incubated for one hour. After watching this video, you should have a good understanding of how to immobilize antibodies on cellular matrix for the development of immunoassays.

We first had the idea when we were developing low-cost diagnostic assays which has limited sensitivity. After its development, the technique paved the way for researchers in the field of point-of-care diagnostics to explore the development of on-site low-cost diagnostic tests for various pathogens.