The overall goal of this salivary antibody bead-based multiplex immunoassay is to identify the etiological agents responsible for environmentally associated symptomatic and asymptomatic illnesses by measuring the IGG antibody responses to multiple pathogens simultaneously. This method can help answer key questions in the field of exposure science. For example, which pathogens are responsible for causing illnesses in particular populations.
The main advantage of this technique is that it can be used to quickly and cost-effectively measure antibodies to multiple analyze at one time using very small simple volumes. This method can provide insight to etiological agents and their sources of exposure, including foodborne, waterborne and airborne pathways. Official demonstration of this procedure is critical because if you don't set it up properly, the results can be unreliable.
Begin by vortexing and sonicating the stock mixtures of beads coupled to the antigens of interest for 20 seconds at a time to generate homogeneous bead suspensions. Then dilute the coupled bead stocks to a final concentration of 100 beads per microliter of each unique bead set in PBS, supplemented with 1%BSA. Next, set up at least seven two-fold serial dilutions of anti-species IGG primary antibody, in PBS plus BSA in a 96 well round bottom plate according to the manufacturers recommendations.
Pre-wet a separate eight 12 column of a 96 well filter bottom plate for each antigen coupling confirmation test with 100 microliters of wash buffer and remove the supernatant by vacuum. Then, add 50 microliters of the antigen coupled beads to the pre-wet wells at 50 microliters of the serially diluted antibody to rows one through seven of the 96 well filter plate, and add 50 microliters of PBS plus BSA to row eight as the diluted antibody background control. Use a multi-channel pipette to mix the contents of each of the wells up and down five times.
Then cover the plate, and incubate the antibodies with the antigen coupled beads in the dark at room temperature on a microplate shaker at 500 rpm. After one hour, aspirate the supernatant and wash the wells with two 100 microliter aliquots of wash buffer. After the second wash, replace the buffer with 50 microliters of PBS plus BSA.
Now dilute the biotinylated anti-species specific IGG secondary detection antibody, to 16 micrograms per milliliter in PBS plus BSA and add 50 microliters of diluted secondary antibody to each row. Incubate the beads and antibodies at room temperature in the dark for 30 minutes on a plate shaker. At the end of the incubation, aspirate the supernatant, and wash the beads with two 100 microliter aliquots of wash buffer.
After the second wash re-suspend the beads in 50 microliters in PBS plus BSA and dilute the streptavidin r-phycoerythrin reporter to 24 micrograms per milliliter in PBS plus BSA. Add 50 microliters of the reporter to each well and mix by pipetting. Incubate the plate in the dark for 30 minutes at room temperature on the shaker, followed by two 100 microliter rinses in wash buffer as demonstrated.
Then re-suspend the beads in 100 microliters of PBS plus BSA and evaluate the antigen coupled beads on the analyzer. After re-suspending the antigen coupled bead stocks by vortexing and sonication for 20 seconds, dilute the coupled bead stocks to a final concentration of 100 beads per microliter of each unique bead set in PBS plus BSA. Then prepare a one to four dilution of room temperature saliva in PBS plus BSA in a 96 well deep well plate.
Pre-wet a filter plate with 100 microliters of wash buffer and aspirate the supernatant. Next, add 50 microliters of antigen coupled beads and an equal volume of the diluted saliva to 95 wells of the 96 well filter plate for one to eight final dilution. At the same time, add 50 microliters of antigen coupled beads, plus 50 microliters of PBS plus BSA alone to the control well.
Incubate the beads in the dark at room temperature for one hour on the microplate shaker at 500 rpm. Then aspirate the supernatant and wash the wells two times with 100 microliters of wash buffer. After the second wash, re-suspend the beads in 50 microliters of PBS plus BSA.
Dilute the secondary antibody, and add 50 microliters of it to each well. After mixing the well contents with a multi-channel pipette incubate the plate on the shaker in the dark for 30 minutes at room temperature, then wash the wells two times with 100 microliters of wash buffer and re-suspend the beads in 50 microliters of fresh PBS plus BSA. Now dilute the reporter and add 50 microliters to each well.
Mix thoroughly with a multi-channel pipette. After 30 minutes at room temperature on the shaker in the dark, wash the wells two times as demonstrated, then re-suspend the beads in 100 microliters of PBS plus BSA and evaluate the samples on the analyzer. To ensure that the antigen coupled beads are capable of approaching the dynamic range of the assay, proper coupling is defined as a mean fluorescence intensity, greater than or equal to 18, 000.
As demonstrated for these representative coupling confirmations of the antigens in the assay for C.jejuni and T.gondii. Based on the established cut-off point, the immunoprevalence rates for the samples ranged from about 2%for T.gondii to nearly 50%for Norovirus geno group 1.1 indicating that the immunoprevalence rate was highest for the Noroviruses, followed by Hepatitis A and H.Pylori. While 32%of the samples were immuno-negative for all of the pathogens in the assay, 68%were immuno-positive to one or more pathogen.
Once mastered, this procedure can be completed in about four hours if performed properly. While attempting this procedure it's important to keep the beads in the dark to avoid photo-bleaching. Following this procedure, other methods like ELISAs and Western Blotting can be used to answer additional questions about sensitivity and specificity.
As well as cross-reactivity of the antibodies. After its development, this technique paved the way for researchers in the fields of epidemiology, exposure science, and clinical microbiology, to explore human risks from environmental exposures. After watching this video you should have a very good understanding of how to develop, analyze, and interpret the results of a multiplex immunoassay, measuring antibodies to multiple pathogens simultaneously.
Don't forget that working with bodily fluids and sodium azide can be extremely hazardous, and that precautions such as wearing appropriate personal protective equipment and proper reagent disposal should always be taken when performing this procedure.
