Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

The overall goal of the following experiment is to detect antibodies against adventitious infectious agents in laboratory animals. This is achieved by incubating test serum with antigen coated microspheres. Next, the serum is incubated with a biotinylated tagged species specific anti immunoglobulin, which detects any antigen antibody immune complexes.

Then a streptavidin labeled FICO eryn Fluor force solution is added in order to detect any biotinylated immunoglobulin results are obtained that show positive or negative antibody status for common agents in lab rodents based on multiplex fluorescence, immunoassay net score, fluorescent sample values. The main advantage of using this technique over the existing method like Eliza, is that MFIA is a multiplex assay. This enables a researcher to screen up to hundred different assays in a single test.

Well, this technique decreases the amount of serum regions and disposal disposables used in the existing methods while increasing the amount of information generated from a single test. Well, we first had the idea of using this method when we were testing a large number of Sera samples for multiple agents and wanted to reduce labor as well as testing time. Demonstrating the procedure will be Donna Cohen, a senior technologist from our lab To begin, have ready two buffers required for the multiplex, fluorescence, immunoassay or MFIA procedure.

The first is a primary diluent, which is available from Charles River and contains proprietary blocking agents to decrease nonspecific interactions for the assay wash buffer. Prepare a solution of PBS with 1%BSA pH 7.4 to remove particulates. Filter the buffer through a 0.2 micron bottle top unit into sterile labeled containers using assay wash buffer.

Prepare two x concentrations of biotinylated antis species, IGG or BAG and strep AVID and FICO erything or SPE, which will be used to label the antigen antibody complexes formed during the test serum incubation step to prepare samples of the following materials and disposables assembled multi and single channel micropipets and tips, and 96 well low protein binding microtiter plates for protein dilution. After collecting blood samples and isolating serum vortex the serum briefly before diluting the samples in primary diluent in a 96 well microtiter plate to ensure proper and uniform well evacuation pre-wet. Every well of the 96 well test plate with assay wash buffer slowly aspirate the solution throughout the assay.

Aspiration should take five to 10 seconds to prevent bead aggregation and compacting beads into the filter membrane, both of which can slow read times at the end of the assay. Verify that liquid drainage has stopped by blotting the underside of the test plate with paper towels. It is important to blot the test plate after every wash step to prevent wicking out of the wells during incubation.

To prepare the beads begin by vortexing the stock coupled bead suspension. Then briefly sonicate them in a bath. It's crucial to resuspend the beads properly to prevent aggregated bead clusters, which can result in longer read times.

Next, dispense the 20 x stock suspension to a two x working bead solution in primary diluent. Then dispense 50 microliters of the two x bead suspension into each assay well of the pre wetted test plate. Pipette 50 microliters of each two x test and control serum to the test plate based on a predefined plate map.

Secure the lid to the plate. Cover it with aluminum foil and incubate the test plate for 60 minutes on an orbital shaker at room temperature To avoid evaporation of solutions which can prevent success of the assay. Keep the test plate covered with the plate lid during all incubation steps.

In addition, the shake of speed should be greater than 400, but less than 700 RPMs so that the beads remain in suspension to allow the serum antibodies access to all surfaces of the coupled antigens. After washing the plate in sequential incubations of the beads with BAG and SPE, wash the samples again. Then resuspend the beads by adding 125 microliters of assay, wash, buffer, and shake for two minutes to read the plate.

Place it into the assay reader within 10 minutes of resus suspending the beads, the beads pass one at a time through a detector where they're exposed to two lasers. One laser excites the internal dyes that identify the beads color set and the other excites the FICO erything reporter dye. The intensity of the FICO erything fluorescence for a predetermined number of beads is reported as the FM FI read the first well to verify that the selected bead panel matches the bead profile in the test wells.

If there are beads falling outside of their designated bead region on the protocol display, an incorrect profile selection has been made. Beads that have been properly resuspended will fill in their specified regions as indicated in the assay reader software. If the beads are aggregated, they will fill their regions at a slow rate.

You can remove the test plate from the reader and manually resuspend the wells to separate the beads. Remove the test plate from the reader and using a multichannel pipetter tri rate each well three to four times. Then return the test plate to the reader and continue examine the results.

Report for errors such as inadequate bead counts or falling IgG anti IgG bead scores, and repeat the assay for these samples. After exporting the data to Excel and calculating the net MFI determine whether any of the test play assay controls excluding the high range immune serum. Any failed controls are unacceptable and the assay must be repeated.

This table represents data for test serum and assay controls from a typical assay plate. Were all IgG controls passed. The test results for this play indicate that numerous tissue reactive and IgG control samples failed as seen in orange wells.

A1C one and F1 indicate a TC tissue reactive failure where the TC score is represented in parentheses, Wells B one and E one illustrate the two types of IgG internal control failures, insufficient test antibody or test reagents, BAG or SPE respectively. The test plate results are interpreted only if the system suitability control and serum control meet the acceptance criteria shown here. Microspheres coated with species specific anti test serum immunoglobulin can show failing scores due to the addition of insufficient sample too high, a sample dilution, incorrect species, or testing serum from an immunodeficient host.

If the test play assay control results are satisfactory, individual assay scores are classified according to the following. Following this procedure, we should use additional methods like Eliza, IFA, and or Western blot to confirm the initial unexpected or positive findings. It's crucial to verify these results before making any animal management decision.

This can be done by using different methodologies on the same sample or additional samples from the same colony. After watching this video, you should have a good understanding of how to perform the multiplex fluoro, metric immunoassay, and to interpret the results obtain at your facility. This will enable you to reduce labor while gathering more information from each test sample well.