The Shigella research field currently lacks well defined assays to examine the functional role of antibodies generated by immunization or infection. This assay represents a well defined method to qualify these immune responses and measure bactericidal activity of Shigella specific antibodies. The main advantage of this assay is that it allows for high throughput analysis of multiple samples.
The techniques used in this protocol greatly reduce hands-on time and overall assay time to measure direct bacterial killing of antibodies and serum samples. To begin, incubate the samples in a warm water bath at 56 degrees celsius for 30 minutes to heat and activate the test samples. Obtain an assay plate and 20 microliters of assay buffer to columns one through twelve of rows A through G.Add 20 microliters of assay buffer to columns one and two of row H.Load 30 microliters of each test sample and duplicate to row H of the assay plate.
To begin performing threefold serial dilutions of the test samples, use a multi-channel pipette to remove 10 microliters from wells 3H through 12H. Transfer the samples to the corresponding wells in row G and pipette up and down 8 to 10 times to mix the sample well. Then, remove 10 microliters from these wells.
Transfer to the corresponding wells in row F and pipette up and down 8 to 10 times to mix. Continue this serial dilution process through row A.After mixing the wells in row A, remove and discard 10 microliters from wells 3A through 12A so that the final volume in all wells is 20 microliters. Retrieve one vial of frozen target bacterial stock and thaw it at room temperature.
Then, dilute the bacteria in 20 milliliters of assay buffer. according to the predetermined optimal dilution factor. Using a multi-channel pipette, add 10 microliters of diluted bacteria to each well of the assay plate.
Retrieve one vial of frozen Baby Rabbit Compliment and one vial of frozen heat activated BRC. Use cold running water to thaw the vials to room temperature. Then, mix 100 microliters of heat activated BRC with 400 microliters of assay buffer.
Add 50 microliters of this 20 percent heat activated BRC solution to all wells in column one. Mix one milliliter of native BRC with four milliliters of assay buffer. Add 50 microliters of this 20 percent mixture to all the wells in columns two through twelve.
Place the plate one a plate shaker for 10 to 15 seconds to gently mix the assay plate. Incubate the assay plate in a microbiological incubator for two hours. Meanwhile, remove the lids from the two LB Agar plates and place the plates face up in a biological safety cabinet for 40 to 60 minutes to dry.
When the incubation is complete, transfer the assay plate to wet ice and incubate for 10 to 20 minutes to stop the reaction. Using a twelve channel pipette, mix the wells in row H and spot plate 10 microliters of the reaction mixture onto the bottom of an LBA plate. Immediately tilt the plate and allow the spots to run for about 1.5 to 2 centimeters.
Repeat this procedure for rows G, F, and E, spotting them above the previous row. Incubate the LBA plates at room temperature until the solution is absorbed into the LBA plates. Then, put the lids on the plates and transfer them upside down into a microbiological incubator to incubate overnight.
The next day, add 25 milliliters of overlay Agar at 55 degrees celsius, containing 100 micrograms per milliliter TTC and 0.1 percent sodium azide to each LBA plate. Incubate at 37 degrees celsius for two hours to allow to allow the surviving bacteria to develop a red color. Then, use a digital camera to photograph the plates.
Transfer the images to a computer and use NIST's Integrated Colony Enumerating software to analyze the images as outlined in the text protocol. Representative LBA plates demonstrate that color development has taken place after the overnight incubation and overlay addition, with all the surviving colonies being visible in red. Bacterial killing is clearly seen for all samples tested in the first three dilutions.
A decrease in bacterial killing is seen as samples are diluted further up the plate and the serum is less concentrated. Microcolony counts are then performed using the NIST software. The average CFU count for each dilution of each sample is then calculated, as is the 50 percent killing value.
This 50 percent killing value is then applied to the average CFUs for each serum dilution to determine the values needed to calculate the SBA KI.This protocol relies on consistent and uniform bacterial plating on LB Auger. Good spot plating and tilting technique will allow for the growth of discrete microcolonies and accurate colony counting. This technique uses live virulent Shigella and requires appropriate aseptic technique and PPE to ensure the bacteria are handled safely according to BSL 2 procedures.
