The overall goal of the following procedure is to rapidly identify a polio virus isolates using a simple protocol. For this assay, we use sensitized gelatin particles coated with chimeric molecules containing the extracellular domain of the human poliovirus receptor connected to IgG two fc. Since these particles have a uniform affinity for all serotypes of poliovirus isolates, agglutination of the particles occurs upon incubation with samples containing any human poliovirus.
However, if antibodies able to recognize the specific viral isolating the sample are present, neutralization occurs and prevents agglutination using this experimental system, rapid identification of poliovirus isolates can be performed in a single step reaction. The main advantage of this technique of existing vessel, like conventional neutralization test is the SIM simplicity of the procedure and interpretation, aiming to reduce the labor and expedite required and shorten the time for results. To reconstitute the anti PV one, PV two, and PV three antibodies, add 0.5 milliliters of water to each vial.
Then transfer each antibody solution into separate tubes containing 4.5 milliliters of maintenance media. Some lots of anti PV antibody are provided in reconstituted form for such lots of anti PV antibody. Addition of water for reconstitution is not necessary.
Next, prepare one-to-one mixes of combined antibody solution with a final volume of 3.6 milliliters in fresh labeled tubes. For example, mix 1.8 milliliters of anti PV one with 1.8 milliliters of anti PV two. To prepare an anti PV one plus PV two combination, repeat this for every possible pairwise combination and also prepare a solution containing all three types of antibodies by mixing together 1.2 milliliters of each antiserum.
Once the four mixes have been made at 720 microliters of FCS to each antibody solution. This will help reduce non-specific agglutination of gelatin particles by antibodies during the assay. The antibody pools can be stored at minus 20 degrees Celsius until they're used in the agglutination assay.
In this assay, we use lyophilized gelatin particles that have been coated with the extracellular domains of the human poliovirus receptor and IgG two a mouse FC domains. Be sure to use a single lot of particles and not different lots in one experiment to ensure consistency of the agglutination image to reconstitute the particles at 1.5 milliliters of reconstitution buffer to each vial. Tap gently to mix well.
The reconstituted particles can be stored at four degrees Celsius for up to two weeks. First, design your experiment in a 96 well microtiter plate. For each sample, well number one will contain virus alone.
Wells number two through five will contain the virus plus one of the four mixed antibody solutions. Be sure to save one well for a negative control containing media only with no virus or antibody. Once the plate has been set up, add 12 microliters of the correct antibody solution to each well from rows two through five and 12 microliters of growth.
Medium to the virus alone, wells in row one, and to the negative control well in row six. Next, add 12 microliters of virus sample in the first five rows and 12 microliters of growth media to the negative control well in row six. Once the antibodies and virus have been plated, resus, suspend the reconstituted gelatin particles by tapping them gently and immediately add 25 microliters of particles per well to all of the wells.
It is important not to let the particles settle before transferring them to the plate in order to obtain equal amounts of particles in each. Well place the assay plate on a plate mixer and mix for 30 seconds. Then place the plate on a sheet of white paper and incubate the plate at room temperature for two hours to allow a agglutination to occur.
It is important not to move the plate at any time during or after the incubation as the agglutination formed is unstable. Once the reaction has occurred, take several pictures of the plate in which the quality of the agglutination in each well can be appreciated. In wells where agglutination has not occurred, as shown here on the left colored gelatin particles will precipitate at the bottom of the well in wells where agglutination has occurred.
Due to the interaction between the virus and the pvr, IgG two A coated gelatin particles, a diffuse colored pattern is visible as shown in the center. In this experiment, we tested the ability of virus solutions, PV one, two, and three to induce glu in the presence of the antibody. Combinations indicated as shown here in the absence of virus or antibodies, no agglutination occurs and the gelatin particles precipitate in contrast in well containing virus, but no antibodies.
Agglutination has occurred due to the interaction of the virus. With the PVR IgG two A covered gelatin particles in the experimental wells, a glut nation of the viruses is prevented in any well containing antibodies that specifically neutralize that virus. For example, a glut nation of poliovirus type one is prevented in wells containing anti PV one antibodies, but not in wells containing only anti PV two and anti PV three antibodies.
The same is true for poliovirus types two and three. Whilst master, this technique can be done in as little as three hours if it is performed properly.
