Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Our research focuses on optimizing antigen preparation for the hemagglutination inhibition assays. To improve NDV antibody detection accuracy, we provide a method for the quick and accurate preparation for the 4-HAU antigen solution. The HI assay remains the most common method for NDV antibody detection.

However, new technologies, including automated liquid handling systems, high-throughput serological platforms, and the advanced imaging tools can also be integrated into the HI assay. They enable faster and more accurate analysis, especially when handling large batch sizes. We established an efficient method for more accurate detection of HA titers of antigen stock solution, combined with a rigorous back-titration method and a well-defined adjustment process.

This approach provides an accurate method and minimize computational tasks, increasing test efficiency and reliable, and contributing to improved disease surveillance and control in poultry populations. The chicken red blood cells suspension and the visual readout of HA HI results still contribute to variability. Our lab will focus on standardizing the RBC suspension and exploring computer vision or other methods to digitalize the HA HI assay results, reducing subjectivity and enhancing consistency.

To begin, add three milliliters of Alserver solution into a sterile, 15 milliliter conical centrifuge tube. Collect one milliliter of blood from the wing vein of each of the three non-immune chickens. Then, immediately transfer the collected blood into the anticoagulant tube and gently mix the contents by inversion.

Fill the tube with sterile PBS to bring the total volume to 12 milliliters. Mix the solution gently to ensure an even distribution of components. Then, place the tube into a centrifuge equipped with a horizontal rotor and centrifuge at 500 G for 10 minutes.

After centrifugation, carefully remove the supernatant and the white blood cell layer from the tube. Using a pipette, reverse pipette one milliliter of the RBC pellet into 99 milliliters of sterile PBS solution to prepare a 1%RBC suspension. To reconstitute the lyophilized antigen, add two milliliters of sterile PBS to the ampoule containing it.

Gently shake the ampoule to dissolve the contents completely and let it stand for two to three minutes to minimize bubble formation. Aliquot reconstituted solution into 1.5 milliliter centrifuge tubes and store it in a freezer set at minus 20 degrees Celsius until use. To begin, dispense 40, 80, 120, and 160 microliters of PBS respectively into four adjacent wells of a disposable 96-well V-bottom microtiter plate.

Add 20 microliters of the reconstituted antigen to each well and pipette up and down 10 times to create dilution. Then, label a new microtiter plate clearly for subsequent steps, and dispense 25 microliters of PBS into each well in rows one to five. Using a multichannel pipette, place 25 microliters of the diluted antigen solutions into the first wells of rows one to five.

Pipette the contents up and down at least five times to ensure uniform distribution. Then, transfer 25 microliters from the first well of each row to the second well, mixing thoroughly. Discard 25 microliters of the final solution after the 11th column.

Next, add 25 microliters of PBS and 1%chicken RBC suspension respectively to each well, starting with the wells containing the lowest antigen concentration. Place the microtiter plate on the microplate shaker to mix thoroughly. Leave the plate undisturbed on the benchtop at room temperature for approximately 30 minutes to allow the RBCs in the 12th column to settle completely.

Calculate the dilution factor for preparing the 4-HAU antigen solution using the formula shown here. Determine the total volume of the 4-HAU solution needed and the required volumes of the antigen stock solution and PBS using the given formula. Using a pipette, transfer the calculated volume of PBS into a container.

Add the corresponding volume of antigen stock solution and mix thoroughly to prepare the 4-HAU antigen solution. Then, dispense 25, 50, 75, 100, 125, and 150 microliters of PBS into six adjacent wells of a microtiter plate. Add 25 microliters of the 4-HAU antigen solution to each well, and pipette up and down 10 times to ensure proper dilution.

Using a multichannel pipette, transfer 25 microliters of each diluted antigen solution from the initial row into the wells of another row. Then, add 25 microliters of PBS to each well, followed by 25 microliters of a 1%chicken RBC suspension in each well. Place the plate on a microplate shaker for approximately 20 seconds to thoroughly mix the contents.

Carefully observe the hemagglutination patterns in the wells, and record the results.