Cell-Free Dot Blot as a Practical and Adaptable Immunoassay Platform for the Detection of Antibody Response in Human and Animal Sera

This protocol demonstrates the cell-free dot blood technique, an adaptable, accessible, and affordable immunoassay platform developed in response to the COVID-19 pandemic. The existing gold standard immunoassay methods include variations of the ELISA technique and lateral flow assays. There has been a significant interest in the development of novel immunoassay using technologies such as split reported proteins, electrochemical interfaces, and colloidal nanoparticles.

The challenge is that most of the gold standard immunoassay are inaccessible to non-centralized communities due to the reliance on expensive and sophisticated molecular components and equipment. This novel CFDB immunoassay technique can be quickly adapted to emerging outbreaks. It uses basic laboratory equipment and relies entirely on molecular components that can be produced in-house and in low resource settings.

To begin, obtain the protein sequence from the UniProt database using the accession code P0DTC9. Code on optimize the sequence for ecoli based expression using the IDT code on optimization tool. Insert the optimized sequence in a SnapGene template for the expression construct backbone before ordering the optimized sequence for commercial synthesis as a single stranded DNA fragment re suspended in water.

Perform an initial five microliter scale expression test by adding 10%PCR product to the ecoli BL21 cell-free reaction in a PCR tube. Incubate the reaction without shaking at 30 degrees Celsius for 15 hours. Assess the expression quality of the nucleocapsid protein or NP antigen by loading one microliter of the cell-free reaction on 12%SDS page gel and transfer it to a nitrocellulose membrane for western blot.

Assemble a one milliliter scale cell-free reaction using 15 nanomolar purified linear expression template product for the cell-free dot blot assay Incubate the expression mixture in a 15 milliliter conical tube with shaking at 80 RPM for 15 hours at 30 degrees Celsius. After assessing the expression quality using western blot and aliquoting 50 microliters of the expression mixture, store the aliquots at minus 20 degrees Celsius. Use a master grid image as shown featuring a six by six centimeter pattern containing 12 by 12 circles each with a diameter of two millimeters designed to match the spotting capacity of a 96-well plate.

After printing the master grid, sandwich the printed grid firmly between two layers of adhesive PCR plate ceiling film, cut the sandwich to size along the outer grid borders. Using a two millimeter biopsy punch, hollow each marked circle on the grid. Cut a 6.5 by 6.5 centimeter piece of nitrocellulose membrane, position it under the master grid on a clean surface, and secure it using adhesive tape.

Using a marker pen, mark the outermost circle positions on the nitrocellulose membrane to be used as a guide to cut the membrane after sample spotting. Dilute the serum samples one to 10 in PBS. Using a micro pipette, dispense triplicate 0.4 microliter volumes of each sample onto the nitrocellulose membrane at predetermined grid positions.

Allow the spotted samples to bind and dry at ambient temperature for 10 minutes. Using tweezers, carefully retrieve the nitrocellulose membrane and cut it along the marked outer circles. Transfer the membrane to a 10 centimeter petri dish containing 10 milliliters of blocking solution.

Incubate at room temperature for 30 minutes while shaking gently at 100 RPM. Remove the 50 microliter aliquot of the cell-free antigen expression mixture from the freezer and, after thawing, add it to five milliliters of blocking solution in a 10 centimeter petri dish. Transfer the membrane directly to this antigen containing solution and incubate at room temperature for one hour, shaking at 100 RPM.

After one hour, rinse and wash the membrane in TBST containing 0.05%Tween 20. Next, add 10 milliliters of blocking buffer containing 10 micrograms per milliliter of purified SpyCatcher, two apex, two protein, to the membrane, and place the membrane into a dish containing SpyCatcher apex. Incubate for one hour with shaking at 100 RPM After rinsing the membrane with tris-buffered saline or TBS wash twice for five minutes in TBST followed by a final rinse in TBS.

Stick and secure a piece of parafilm on a clean work surface near the blot imaging instrument. Remove excess liquid by tapping the membrane and using tweezers, place it on top of the parafilm. Immediately add three milliliters of enhanced chemiluminescence or ECL solution on top of the membrane and incubate at room temperature for exactly 90 seconds.

After tap drying the membrane, transfer it to a chemiluminescence compatible imaging instrument. Use the instrument's image analysis function to obtain spot intensities including for three blank positions on the nitrocellulose membrane. Maintain a constant measurement volume per spot.

Export the data to a spreadsheet, ensuring correct labeling of each spot position. Calculate the mean and the standard deviation of triplicate spot intensities. Subtract the nitrocellulose membrane background from all sample measurements.

Then, use the given equation to obtain a cutoff value for the interpretation of positive or negative results. Finally, plot the cutoff subtracted data into a bar graph. A cell-free dot blot assay was performed on the pre characterized commercial human Sarah, and an image of the blot, along with the corresponding signal intensity chart, is presented in this figure.

The cell-free dot blot assay successfully identified all negative control samples and most positive samples. Independent testing of the cell-free dot blot assay at the National Microbiology Laboratory correctly identified all 12 negative and 12 positive patient samples, confirming its robustness. The cell-free dot blot assay also detected antibody responses in SARS-CoV-2 infected hamster Sarah, with positive signals observed in 18 out of 24 post infection samples collected five days after reinfection while all pre infection samples remained negative.