The Opsono-Adherence Assay analyzes the presence of functional oposonic antibodies produced in animals after vaccination, which have been shown to correlate with the efficacy of several licensed human vaccines. This assay is preferred for use with biological select agents and other infectious pathogens and can be performed in a BSL-1 or BSL-2 lab after the inactivated BSL-3 agent is transferred. Antibodies against bacterial capsules, including the Bacillus anthracis capsule, have been demonstrated to be efficacious in animal models of infection.
Opsono-Adherence assays functionally evaluate antibodies without infecting animals. Follow all institutional safety and security procedures when working with encapsulated virulent strains of Bacillus anthracis. For this video, we're using an inactivated strain under biosafety level two conditions.
Begin this procedure with preparation of RAW 264.7 cells, as described in the text protocol. To plate the cells for the opsono-adherence assay, replace the flask with fresh medium and use a cell scraper to gently scrape cells off the flask. Pipette up and down to break up the cell clumps for easier cell counting.
Plate 14, 000 cells per well in a 96-well 0.15 micron thick glass flat bottom plate. Allow cells to grow for three days, replacing spent medium one day before performing the OAA. After three days of incubation, the number of cells should be approximately 50, 000 cells per well.
To begin the assay, prepare a master plate of Bacillus anthracis from a spore stock by streaking it for isolation on an NBY agar plate. For demonstration purposes, a sheep blood agar plate was used. Incubate at 37 degrees Celsius for one day before culturing in broth.
Then, inoculate 30 milliliters of culture broth in a vented 250 milliliter flask with several colonies of the bacilli. Shake the broth culture at 125 RPM, 37 degrees Celsius, with 20%CO2 and humidity for 18 to 24 hours. Use negative staining with India ink to ensure sufficient encapsulation, and that Bacilli are in short chains prior to fixation.
To do so, mix five microliters of bacterial suspension with two microliters of India ink on a microscope slide. Then, place a one or 1.5 micron thick cover glass on top of the suspension without creating bubbles. Observe the bacterial suspension using a 40 times to 100 times oil objective lens on a light microscope.
A thick capsule excludes India ink. Ensure that the Bacilli are encapsulated by observing a zone of clearance surrounding each bacterium. Also ensure that the Bacilli chains are short.
To determine Colony Forming Units, or CFUs, serially dilute 100 microliters of culture one to 10 in PBS solution, and plate culture dilutions on sheep blood agar plates. Incubate for 12 to 18 hours at 37 degrees Celsius. Count CFUs, and determine the number of bacteria per milliliter of culture.
After ensuring that the Bacilli are sufficiently encapsulated, diluted, and plated, fix the remaining bacterial culture by adding 10 milliliters of 16%paraformaldehyde to the 30 milliliters of culture at a final concentration of 4%paraformaldehyde. Transfer the volume to a 50 milliliter conical tube. Then, slowly agitate on a shaker at ambient temperature for seven days.
To test for sterility, transfer four milliliters, or 10%volume, of fixed bacterial suspension to a 50 milliliter conical tube. Wash the suspension by adding PBS, and pelleting at a minimum of 3000 G for 45 minutes. Then remove the supernatant, ensuring that the fluffy pellet is not disturbed.
After repeating this wash at least two times, resuspend the fixed sample in four milliliters PBS. It's necessary to remove all fixative during the washes so that the fixative does not interfere with the viability testing. If necessary, increase the number of washes.
For viability testing, inoculate 40 milliliters of a bacterial growth medium with four milliliters of washed suspension and incubate at 37 degrees Celsius for seven days. Check optical density at 600 nanometers in a spectrophotometer before and after seven days to measure turbidity. After seven days of incubation in broth culture, plate at least 100 microliters of broth onto an agar plate and incubate for another seven days.
If no increase in turbidity and no growth on plates are seen, the original culture is considered sterile, and maybe transferred to a BSL-2 laboratory. Follow institutional guidelines for transfer approval. Wash the inactivated bacterial stock three times in PBS to remove fixative.
Add FITC solution at a final concentration of 75 micrograms per milliliter. Slowly agitate the mixture for 18 to 24 hours at four degrees Celsius. Wash the bacteria as before to remove excess FITC.
After the final wash, resuspend the Bacilli in PBS in the same start volume. and store Bacilli in minus 20 degrees Celsius until use. Heat-inactivate a small volume of test sera from non-human primates at 56 degrees Celsius for 30 minutes in a heat block or PCR machine.
Fall and wash FITC-labeled bacteria with PBS two times by pelleting at 15, 000 times G for three to five minutes. Resuspend in PBS in the same start volume. In a 96-well round-bottom plate, serially dilute the test sera in cell medium.
In another 96-well round-bottom plate, add 10 microliters of diluted test sera into each well. Then, add 10 microliters of freshly reconstituted baby rabbit compliment. To plate number three, add the appropriate volume of FITC-labeled Bacilli for a multiplicity of infection of 20.
For example, at a volume containing 50, 000 times 20 Bacilli per 50, 000 cells. Top off each well in plate number three with the appropriate volume of cell medium to a total of 105 microliters. Incubate plate number three at 37 degrees Celsius for 30 minutes in the presence of 5%CO2 with humidity to opsonize Bacilli.
Place plate number one, which contains adherence cells, on a 37 degrees Celsius heat block. Use a multi-channel pipetter to remove spent medium from wells, and quickly replace with 100 microliters of opsonize bacteria from plate number three, ensuring no bubbles are generated in the wells during pipetting. To not disturb the cell monolayer, add volume to the sides of the wells.
Incubate plate number one at 37 degrees Celsius with 5%CO2 and humidity for 30 minutes for adherence. Wash each well five times with 150 microliters of PBS, on top of the 37 degrees Celsius heat block, to remove unattached Bacilli. Pipette to the side of the wells to ensure that the cells are not accidentally dispersed during washing.
Add 150 microliters of 4%paraformaldehyde to each well, and fix the cells for one hour. After washing the cells twice with PBS, mix two microliters of high-content screening orange cells stain in 10 milliliters of PBS. Then, add 150 microliters of this staining solution to fix cells, and incubate for 30 minutes at ambient temperature.
Place plate number one on the stage and focus on the cells using brightfield or phase contrast. In the Acquisition tab, select 96-well format as the plate setting. Then, select Channel settings.
Next, select setting to Tile mode. Indicate the number of images to be acquired. Change the Acquisition mode to black and white or monochrome, and set the Binning to at least two by two.
Turn on the Autofocus or focusing module. Indicate how often the autofocusing function is to be performed. Next, turn on the Z-Stack function, if available.
Indicate the number of Z-Stacks that will capture the thickness of the cell monolayer and adherent Bacilli. Designate a file folder to save the images to. Now, run the automated imaging program.
Perform data collection and analysis as described in the text protocol. The Ames strain of bacillus anthracis was negatively stained with India ink to examine encapsulation. The state of encapsulation must be verified, as little to no encapsulation causes them to adhere to host cells without antibodies, producing a high background.
Fluorescence intensity and photo bleaching characteristics of the Bacilli was examined using Confocal Microscopy. It is critical to verify that the fluorescence of the Bacilli do not significantly fade over time, because the Bacilli will undergo long exposures to light during imaging. The maximum projection images of Bacillus anthracis attach to the cell monolayers are shown here.
These are representative fields of view that were counted and scored for the opsono-adherence assay. The increase in attachment of Bacilli incubated with capsule conjugate antiserum is due to the presence of anti-capsule antibodies that opsonized the Bacilli. The serum titers of non-human primates vaccinated with 10 and 50 micrograms of capsule conjugate are significantly higher than the titers for conjugate and capsule alone.
We are developing a similar assay that uses flow cytometry and human neutrophils, which are nonadherent cells, to measure opsonic activity and also to quantitate capsule on the surface of bacteria. After watching this video, you should have a good understanding of the opsono-adherence assay. Don't forget that working with BSL-3 pathogens is extremely hazardous.
Institutional guidelines and safety precautions must be strictly followed.
