This work was funded by a grant from PATH to M.H.N. This study was conducted as a Cooperative Research and Development Agreement between the Walter Reed Army Institute of Research and the University of Alabama at Birmingham.

This protocol follows the guidelines of the WRAIR Human Subject&#8217;s Protection Board. Samples used in this study are human serum samples collected as part of WRAIR protocol number 1328, in compliance with all institutional and federal regulations governing the protection of human subjects. Samples were de-identified, and the use of these anonymous samples was classified as non-human subject research by the UAB IRB (protocol number N150115001).
1. Prepare Assay Reagents
<ol>
	<li>Prepare 1% Gelatin by adding 1 g of gelatin to 100 mL of water. Autoclave and store at room temperature.</li>
	<li>Prepare 100 mg/mL TTC (2,3,5-Triphenyltetrazolium chloride) stock solution (1,000x) by adding 5 g of TTC to 40 mL water. When the TTC is fully dissolved, adjust the volume to 50 mL with water and sterile filter using 0.2 &#181;m filter. Store the solution at 4 &#176;C and protect from light. 
	NOTE: TTC colorizes the bacterial colonies and makes them much easier to count. TTC solution has a slight yellow color. If TTC solution develops a red color, discard and prepare fresh.</li>
	<li>Prepare 10% sodium azide (NaN3) stock solution (100x) by adding 5 g NaN3 to 40 mL of water. After complete dissolution, add water to 50 mL. Store at room temperature. 
	CAUTION: Sodium azide is a poison and may be toxic if ingested or absorbed through the skin or eyes. It may react with lead and copper plumbing to form highly explosive metal azides. On disposal of reagents containing sodium azide, flush with a large volume of water to prevent azide build-up or discard in a biohazard bag.</li>
	<li>Prepare LBA plate (LB agar plate) by adding 35 g of LB agar to 1 L of water and autoclave. Add 25 mL to each square petri dish (120 x 120 mm2). Incubate plates at RT for 10-20 min to allow agar to solidify. Place plates back into plastic bags and store at 4 &#176;C for up to 1 month.</li>
	<li>Prepare Overlay Agar by adding 7.5 g of agar to 1,000 mL of water and autoclave. Incubate in 56 &#176;C water bath until needed. Right before use, add 1 mL of 100 mg/mL TTC and 10 mL of 10% NaN3 and mix well. 
	NOTE: Each LBA plate needs 25 mL of Overlay Agar. Overlay Agar can be prepared up to one month in advance and melted in a microwave or on a hot plate as needed for the assay. Ensure that agar temperature is ~55 &#176;C before application to LBA plates.</li>
	<li>Prepare Assay Buffer by adding 5 mL of 10x Hanks&#8217;s Balanced Salt Solution (HBSS) with Ca2+/Mg2+ and 5 mL of 1% gelatin to 40 mL of water. Store at room temperature.</li>
</ol>
2. Prepare Complement and Target Bacteria
<ol>
	<li>Prepare baby rabbit complement (BRC) 
	NOTE: Detailed criteria for complement lot selection can be found here: https://www.vaccine.uab.edu/uploads/mdocs/UAB-MOPA.pdf
	<ol>
		<li>Obtain frozen BRC and thaw with running cold water. Physically mix the BRC every ~10-20 min until completely thawed. Do not subject BRC to repeated freeze thaw cycles.</li>
		<li>While BRC is thawing, label 1.5 mL, 5mL, or 15 mL centrifuge tubes. Place labeled tubes on ice to pre-cool. Aliquot proper volume BRC to the pre-cooled centrifuge tubes (after filling, immediately return each tube to the ice). Store aliquots at &#8804; -70 &#176;C in the freezer. 
		NOTE: Approximately 1 mL of complement is needed for each assay plate. Complement aliquots are for single use and should be aliquoted in volumes appropriate to assay layouts.</li>
		<li>To prepare Heat-inactivated BRC, thaw one aliquot of active BRC. Prepare a 56 &#176;C water bath. After BRC is completely thawed, transfer it to the water bath and incubate for 30 min.</li>
		<li>After incubation, remove heat-inactivated BRC from the water bath and allow to cool at RT for 10-15 min. Mix vigorously, and aliquot ~150 &#181;L to 1.5 mL microcentrifuge tubes. Store aliquots at &#8804; -10 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare target bacteria stock 
	NOTE: The procedure below is used to prepare 48 aliquots of target bacteria stock; if more aliquots are needed the protocol can be scaled up.
	<ol>
		<li>Remove the bacteria master stock vial from the freezer and scrape the frozen bacterial surface to remove a small amount of ice from the vial onto a blood agar plate. Immediately return the master stock vial to the freezer.</li>
		<li>Streak this small aliquot of bacterial stock onto the blood agar plate and cover with the plate lid. Incubate the plate upside down overnight in 37 &#176;C/5% CO2 incubator.</li>
		<li>Transfer ~10 isolated smooth colonies to a 50 mL tube containing 30 mL of LB broth. Incubate for 3-5 h at 37 &#176;C with gentle shaking until the culture broth has an OD600 of ~0.6-0.7.</li>
		<li>Harvest the top 12.5 mL of the culture and transfer to a fresh 50 mL tube. Centrifuge the culture at 15,000 x g for 2 min using a table top micro-centrifuge. Discard the supernatant and re-suspend the pellet in 25 mL of 15% sterile glycerol-LB.</li>
		<li>Mix well and dispense 0.5 mL aliquots into sterile 1.5 mL micro centrifuge tubes (~48 tubes). Store aliquots at &#8804; -70 &#176;C in freezer.</li>
		<li>Confirm the bacterial identity using the agglutination test before use.</li>
	</ol>
	</li>
	<li>Determination of optimal dilution factor for target bacteria stock 
	NOTE: Each batch of target bacteria stock must be titrated in assay conditions to determine the dilution necessary to yield ~120 CFU/spot on LB plates.
	<ol>
		<li>Get a microtiter plate (Dilution Plate) and add 135 &#181;L of Assay Buffer to well 1A. Add 120 &#181;L of Assay butter to wells 1B-1H.</li>
		<li>Remove a vial of frozen target bacteria from the freezer and thaw at room temperature. Add 15ul of thawed bacterial stock to well 1A to make a 10-fold dilution of the bacterial stock.</li>
		<li>Transfer 30 &#181;L of bacterial solution from well 1A to well 1B to perform a 5-fold serial dilution. Continue 5-fold serial dilutions to well 1H for a total of 8 dilutions (1:10; 1:50; 1:250; 1:1 250; 1:6 250; 1: 31 250; 1: 156 250; 1:781 250).</li>
		<li>Get another microtiter plate (Assay Plate) and add 20 &#181;L of Assay Buffer to all well in columns 1 and 2 in the Assay Plate.</li>
		<li>Transfer 10 &#181;L of diluted bacteria from each well in column 1 of the Dilution plate into the corresponding wells in columns 1 and 2 of the Assay Plate. 10 &#181;L of bacteria is transferred from well 1A of the dilution plate into well 1A and 2A in the Assay Plate, etc.</li>
		<li>Continue with the assay as described in Serum Bactericidal Assay (SBA) below for Control A and Control B, steps 3.6-3.12.</li>
		<li>After the plates have been incubated on ice, use a multichannel pipette with 8 pipette tips to spot 10 &#181;L from the wells in column 1 onto an LBA plate. Also, spot the wells from column 2 onto the LBA plate.</li>
		<li>Continue with the assay as described below in steps 3.14-4.6.</li>
		<li>Determine the bacteria dilution that yields ~120 CFU/spot in Control B, this dilution will be used in the assay.</li>
	</ol>
	</li>
</ol>
3. Serum Bactericidal Assay (SBA)
NOTE: The procedure described below is for one assay plate, but the number of Assay Plates can be increased.
<ol>
	<li>Heat-inactivate test samples by incubating samples in a 56 &#176;C water bath for 30 min. 
	NOTE: Test samples must be heat-inactivated prior to the test to abrogate any endogenous complement activity. This can be done ahead of the assay and inactivated samples can be re-frozen or stored at 4 &#176;C until tested.</li>
	<li>Get an Assay Plate and add 20 &#181;L of Assay Buffer to columns 1 through 12 of rows A through G. Add 20 &#181;L of Assay Buffer to columns 1 and 2 of row H, see Table 1.</li>
	<li>Load 30 &#181;L of each test sample, in duplicate, to row H of the Assay Plate. For example, dispense 30 &#181;L of sample 1 into wells 3H and 4H, and dispense 30 &#181;L of sample 2 into wells 5H and 6H, etc.</li>
	<li>Perform 3-fold serial dilutions of test samples using a multichannel pipette.
	<ol>
		<li>Remove 10 &#181; of the sample from wells 3H-12H and transfer to corresponding wells in row G and mix the sample well by pipetting up and down 8-10 times.</li>
		<li>Then remove 10 &#181; from wells 3G-12G and transfer to corresponding wells in row F and mix well.</li>
		<li>Continue these serial dilutions through row A. After mixing the wells in row A, remove and discard 10 &#181;l from wells 3A-12A so that all wells contain 20 &#181;l. 
		NOTE: Because 20 &#181;L of serum is used in a total assay volume of 80 &#181;L, there is a 4-fold additional dilution in the assay. This dilution must be taken into account during the calculation of an SBA titer by multiplying the dilution of serum by 4. For example, if a starting dilution of 1:2 is used, the actual dilution being tested is 1:8.</li>
	</ol>
	</li>
	<li>Remove one vial of frozen Target Bacteria Stock and thaw at room temperature. Dilute the bacteria in 20 mL of Assay Buffer according to the pre-determined optimal dilution factor (this dilution factor was determined in section 2.3). Add 10 &#181;L of diluted bacteria to each well of the assay plate using a multichannel pipette.</li>
	<li>Remove one vial of frozen BRC and one vial of frozen Heat-inactivated BRC, thaw at room temperature with running cold water or place on the grate of a biological safety cabinet with blowing air to thaw quickly.</li>
	<li>Prepare a 20% solution of heat-inactivated BRC. Mix 100 &#181;L of heat-inactivated BRC with 400 &#181;L of Assay Buffer. Add 50 &#181;L of this 20% heat-inactivated BRC solution to all wells in column 1 (Control A wells). 
	NOTE: Heat-inactivated BRC is used as a control to monitor non-specific killing (NSK) in the assay.</li>
	<li>Prepare a 20% solution of native BRC. Mix 1 mL of native BRC with 4 mL of Assay Buffer. Add 50 &#181;L of this mixture to all wells in columns 2 through 12 (Control B and test sample wells). 
	NOTE: The final concentration of BRC in the reaction mixture is 12.5%.</li>
	<li>Briefly mix Assay Plate by shaking gently for 10-15 s on a plate shaker or mix by pipetting up and down 8 times using a multichannel pipette.</li>
	<li>Put the Assay Plate in 37 &#176;C microbiological incubator for 2 h (without shaking).</li>
	<li>Dry 2 LBA plates by removing lids and placing plates face up in biological safety cabinet for 40-60 min.</li>
	<li>When the 2 h incubation is complete, move the Assay Plate to wet ice and incubate for 10-20 min to stop the reaction.</li>
	<li>Using a 12-channel pipette, mix the wells in row H, and spot plate 10 &#181;L of the reaction mixture onto the bottom of an LBA plate. Immediately tilt the plate and allow the spots to run for ~1.5-2 cm. Repeat this procedure for row G, F, and E, spotting them above the previous row on the LBA plate. Row E, F, G, and H are spotted onto one LBA plate, and row A, B, C, and D are spotted onto a second LBA plate in the same manner, see Figure 1.</li>
	<li>Incubate the LBA plates at room temperature until the solution is adsorbed into the LBA plates (10-15 min). Put the lids on the LBA plates and place the LBA plates in the microbiological incubator upside-down to incubate overnight (~16-18 h). Incubate S. flexneri 2a and 3a at 29 &#176;C and incubate S. sonnei is at 26 &#176;C. 
	NOTE: These temperatures yield smaller &#8220;micro-colonies&#8221; with sizes suitable for accurate counting by a colony counter9.</li>
	<li>After overnight incubation, add 25 mL of Overlay Agar (at ~55 &#176;C) containing 100 &#181;g/mL TTC and 0.1% NaN3 to each LBA plate.</li>
	<li>Incubate the LBA plates at 37 &#176;C for 2 h to allow the surviving bacteria to develop red color, see Figure 1 below.</li>
	<li>Photograph plates using a digital camera and transfer images to a computer where NIST&#8217;s Integrated Colony Enumerating Software (NICE) has been installed, see Figure 2. 
	NOTE: NICE colony-counting software is available at no charge. See Materials List for details and installation instructions.</li>
</ol>
4. Count Bacterial Colonies
<ol>
	<li>Open NICE Software and input Operator Name, Experiment information, and any assay Notes into the empty fields. Once this data is entered click the Done button.</li>
	<li>Import photographed plates by clicking the Open button and selecting the correct files from the computer.</li>
	<li>Adjust the assay parameters by setting the number of Rows to 4 and the number of Columns to 12. Adjust the Background setting to -3 sigma and the Resolution to Low. See Figure 2.</li>
	<li>Move the Regions of Interest (ROIs) by clicking and dragging so that each ROI is directly over one sample spot. Ensure that all bacterial colonies are inside the ROI with no overlap between samples. Once one plate is complete, double click the next plate in the Stored data Images list and adjust the ROIs. See Figure 2.</li>
	<li>When all plates have been adjusted, click the green Count button, see Figure 2 and Figure 3. When counting is finished click the Export button to export the data in .xls/.xlsx format. Name and save the .xls/.xlsx file for data analysis.</li>
	<li>Arrange exported data into a table format so that counts are organized in a table representing the 96-well assay plate, see Table 2.</li>
</ol>
5. Calculate SBA Titer (KI) and Non-specific Killing (NSK)
NOTE: SBA titer, or killing index (KI) is defined as the reciprocal of the serum dilution that kills 50% of the target bacteria.
<ol>
	<li>Calculate the 50% killing index (KI) cutoff value by averaging the CFU of active complement control wells (Control B) and dividing by 2. Calculate the average CFU for each dilution of each sample that was run in duplicate, see Table 3.</li>
	<li>Because a serum dilution will rarely yield exactly this 50% KI value, it can be interpolated from two sequential serum dilutions, one that kills less than 50% and one that kills more than 50%, see Figure 4. The formula for calculating the interpolated SBA KI is shown below: 
	<img alt="Equation" src="/files/ftp_upload/59164/59164eq1.jpg" /> 
	NOTE: The bactericidal titer, or KI, can also be calculated automatically using Opsotiter software developed by UAB. To Request Opsotiter, contact Dr. Moon Nahm or Mr. Rob Burton. See https://www.vaccine.uab.edu for contact information.</li>
	<li>Calculate the NSK value by taking 1 minus the average of Control B divided by the average of Control A.</li>
</ol>

<ol><li>Riddle, M. S., Sanders, J. W., Putnam, S. D., Tribble, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Incidence%2C+etiology%2C+and+impact+of+diarrhea+among+long-term+travelers+%28US+military+and+similar+populations%29%3A+a+systematic+review%5BTitle%5D)+AND+%22The+American+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">Incidence, etiology, and impact of diarrhea among long-term travelers (US military and similar populations): a systematic review.</a> The American Journal of Tropical Medicine and Hygiene. 74 (5), 891-900 (2006).</li>
<li>Tickell, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+management+of+Shigella+infection+in+children+with+diarrhoea%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22The+Lancet+Global+Health%22%5BJournal%5D)">Identification and management of Shigella infection in children with diarrhoea: a systematic review and meta-analysis.</a> The Lancet Global Health. 5 (12), e1235-e1248 (2017).</li>
<li>Livio, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shigella+isolates+from+the+global+enteric+multicenter+study+inform+vaccine+development%5BTitle%5D)+AND+%22Clinical+Infectious+Diseases%22%5BJournal%5D)">Shigella isolates from the global enteric multicenter study inform vaccine development.</a> Clinical Infectious Diseases. 59 (7), 933-941 (2014).</li>
<li>Barry, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+and+pitfalls+in+Shigella+vaccine+research%5BTitle%5D)+AND+%22Nature+Reviews+Gastroenterology+%26+Hepatology%22%5BJournal%5D)">Progress and pitfalls in Shigella vaccine research.</a> Nature Reviews Gastroenterology & Hepatology. 10 (4), 245-255 (2013).</li>
<li>Noriega, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategy+for+cross-protection+among+Shigella+flexneri+serotypes%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Strategy for cross-protection among Shigella flexneri serotypes.</a> Infection and Immunity. 67 (2), 782-788 (1999).</li>
<li>Levine, M. M., Kotloff, K. L., Barry, E. M., Pasetti, M. F., Sztein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+trials+of+Shigella+vaccines%3A+two+steps+forward+and+one+step+back+on+a+long%2C+hard+road%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road.</a> Nature Reviews Microbiology. 5 (7), 540-553 (2007).</li>
<li>Sayem, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+host+immune+responses+to+epidemic+and+endemic+strains+of+Shigella+dysenteriae+type+I%5BTitle%5D)+AND+%22Journal+of+Health+Population+and+Nutrition%22%5BJournal%5D)">Differential host immune responses to epidemic and endemic strains of Shigella dysenteriae type I.</a> Journal of Health Population and Nutrition. 29 (5), 429-437 (2011).</li>
<li>Rahman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+zinc+supplementation+as+adjunct+therapy+on+the+systemic+immune+responses+in+shigellosis%5BTitle%5D)+AND+%22The+American+Journal+of+Clinical+Nutrition%22%5BJournal%5D)">Effects of zinc supplementation as adjunct therapy on the systemic immune responses in shigellosis.</a> The American Journal of Clinical Nutrition. 81 (2), 495-502 (2005).</li>
<li>Nahm, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((interlaboratory+evaluations%2C+and+application+of+a+simple%2C+high-throughput+Shigella+serum+bactericidal+assay%5BTitle%5D)+AND+%22mSphere%22%5BJournal%5D)">interlaboratory evaluations, and application of a simple, high-throughput Shigella serum bactericidal assay.</a> mSphere. 3 (3), (2018).</li>
<li>Burton, R. L., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+a+fourfold+multiplexed+opsonization+assay+%28MOPA4%29+for+pneumococcal+antibodies%5BTitle%5D)+AND+%22Clinical+and+Vaccine+Immunology%22%5BJournal%5D)">Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies.</a> Clinical and Vaccine Immunology. 13 (9), 1004-1009 (2006).</li>
<li>Tribble, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+and+immunogenicity+of+a+Shigella+flexneri+2a+Invaplex+50+intranasal+vaccine+in+adult+volunteers%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">Safety and immunogenicity of a Shigella flexneri 2a Invaplex 50 intranasal vaccine in adult volunteers.</a> Vaccine. 28 (37), 6076-6085 (2010).</li>
<li>Kim, H. W., Kim, K. H., Kim, J., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high+throughput+serum+bactericidal+assay+for+antibodies+to+Haemophilus+influenzae+type+b%5BTitle%5D)+AND+%22BMC+Infectious+Diseases%22%5BJournal%5D)">A high throughput serum bactericidal assay for antibodies to Haemophilus influenzae type b.</a> BMC Infectious Diseases. 16, 473(2016).</li>
<li>Maslanka, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardization+and+a+multilaboratory+comparison+of+Neisseria+meningitidis+serogroup+A+and+C+serum+bactericidal+assays%5BTitle%5D)+AND+%22The+Multilaboratory+Study+Group.+Clinical+Diagnostic+Laboratory+Immunology%22%5BJournal%5D)">Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays.</a> The Multilaboratory Study Group. Clinical Diagnostic Laboratory Immunology. 4 (2), 156-167 (1997).</li>
<li>Jang, M. S., Sahastrabuddhe, S., Yun, C. H., Han, S. H., Yang, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+bactericidal+assay+for+the+evaluation+of+typhoid+vaccine+using+a+semi-automated+colony-counting+method%5BTitle%5D)+AND+%22Microbial+Pathogeneis%22%5BJournal%5D)">Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method.</a> Microbial Pathogeneis. 97, 19-26 (2016).</li>
<li>Necchi, F., Saul, A., Rondini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+high-throughput+method+to+evaluate+serum+bactericidal+activity+using+bacterial+ATP+measurement+as+survival+readout%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout.</a> PLoS One. 12 (2), e0172163(2017).</li>
<li>Riddle, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Safety+and+immunogenicity+of+a+candidate+bioconjugate+vaccine+against+Shigella+flexneri+2a+administered+to+healthy+adults%3A+a+single+blind%2C+randomized+phase+I+study%5BTitle%5D)+AND+%22Clinical+and+Vaccine+Immunology%22%5BJournal%5D)">Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella flexneri 2a administered to healthy adults: a single blind, randomized phase I study.</a> Clinical and Vaccine Immunology. , (2016).</li>
<li>Shimanovich, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+and+Antigen-Specific+Serum+Antibody+Levels+as+Correlates+of+Protection+against+Shigellosis+in+a+Controlled+Human+Challenge+Study%5BTitle%5D)+AND+%22Clinical+and+Vaccine+Immunology%22%5BJournal%5D)">Functional and Antigen-Specific Serum Antibody Levels as Correlates of Protection against Shigellosis in a Controlled Human Challenge Study.</a> Clinical and Vaccine Immunology. 24 (2), (2017).</li>
</ol>

R.W.K is an employee of the U.S. government and as such the views expressed in this publication are those of the authors and do not necessarily reflect the official policy or positon of the Department of the Army, Department of Defense, nor the U.S. Government.

The protocol described here demonstrates a functional immune assay to assess the shigellacidal activity of antibodies in serum. In the assay demonstrated for this protocol monoclonal antibodies specific for S. flexneri 3a were used9 along with control human sera from a previous Shigella vaccine study11. The source of serum tested in this assay may vary widely from pre-clinical animal samples to human clinical samples, and the shigellacidal activity of the serum sample will be impacted by immunizations and exposures that the individual has experienced. Some cross-reactivity may be expected between closely related serotypes, specifically S. flexneri 2a and S. flexneri 3a but little cross-reactivity has been seen in these strains in comparison to S. sonnei9. The basis of the SBA focuses on the activation of the complement cascade by antibody-antigen binding. Therefore, the handling of the BRC reagent is one of the many critical steps involved in the execution of this protocol. BRC was selected for use in this assay because of its consistent performance and low levels of NSK in other bactericidal assays12,13,14. &#160;The activity of BRC is temperature sensitive and appropriate measures must be taken to ensure that freeze thaw cycles are minimized, that the BRC is aliquoted into single use volumes, and that the BRC aliquots are thawed quickly, immediately prior to use in the assay. The consistency of complement activity will impact the reproducibility of this assay. Another critical step that impact assay reproducibility is the production and dilution of bacterial stocks. It is important that before beginning the assay the appropriate dilution of bacterial stocks is determined, as assay success is depending on the consistent production of controls A and B having CFU counts averaging ~120 CFU per spot. In order to get spots that are countable by the software, it is also imperative that the technique used to plate bacteria is executed successfully. The deposition of bacterial solution and tilting of the plate so that spots run ~2-3 cm is critical for producing colonies of the right size and correct distribution for accurate counting by the NICE software. Mastering all of these steps will ensure that accurate, consistent results are produced by this protocol
Even when all critical steps are executed well there may still be instances where it is necessary to modify or troubleshoot this protocol. Modifications of this protocol to evaluate other bacteria may require optimization of the overnight LBA plate incubation temperature, to ensure the formation of micro-colonies. Other strains of bacteria or antibody sources, other than serum, may require optimization of complement concentration. NSK values and KIs of control sera should also be monitored to ensure that the assay is working appropriately. NSK should not rise over 70%. The KI of control sera should not vary more than mean &#177; 2SD. To achieve successful and consistent results when using this protocol, it will be necessary to perform all steps as described here with special attention to the critical steps outlined above.
While this protocol fills an important need in the Shigella research community, it is not without its limitations. This protocol relies on biological materials and, therefore, will always be some variability that is difficult to control. Variations in complement activity from different lots and sources may contribute to variability in assays. To mitigate this, it is important to handle complement appropriately and test new lots of complement for activity before purchase. It may also be helpful to create pools of complement lots with known activity to have a homogeneous supply. This protocol is simple by design and does not require any specialized equipment and the automated colony enumerating software is freely available. While this simplicity is an advantage, allowing this protocol to be used in virtually any laboratory, it does still require an overnight incubation. Recent assays have been described that have much shorter incubation requirement, but do require specialized, preparatory reagents15. Another limitation of this assay is that in its current form it is only capable of investigating a single bacterial species at once. In the Shigella field there is a desire to create a multivalent vaccine, and having immunological assays that can assess pathogens in a multiplex manner is of great value. This assay could be modified in the future to meet this need, but in its current form, it is a single-plex assay.
While this assay has some limitations, it still has many advantages over existing or alternative methods. These advantages include many improvements that combine to make the execution of this method much less labor-intensive and more high-throughput than traditional SBAs. The use of frozen bacterial stocks, a 96-well plate assay format, the plating on larger square petri dishes, the coloration of the colonies that allows for photographing and automatic colony-counting, all help to reduce the materials and time needed to complete this assay. This assay also has advantages over other high-throughput methods because it does not require any specialized reagents or equipment. The protocol described can be performed with basic reagents and freely available software, allowing for its application in any laboratory setting.
All of the advantages this protocol provides supports its use in many future investigations. The assay is ideally suited for the examination of immune responses after vaccination or natural infection. This application allows for the SBA to be a valuable tool in Shigella vaccine research and has already been used to evaluate vaccine immunogenicity in Shigella bio-conjugate vaccines where it has demonstrated the ability of these vaccines to induce the production of functional antibodies16. This protocol has been extensively tested by multiple laboratories and has been shown to produce reliable, reproducible results9. This protocol also produces comparable results when the same samples are tested using other bactericidal assays17. The consistency in the data generated by this assay, and its compatibility with other older methods makes it a robust tool for accurately assessing bactericidal activity in serum samples. The assay can also easily be adapted to evaluate additional sample types. While serum is readily available in adult clinical trials, it can be difficult to get sufficient serum in trials focusing on infants and small children; one of the eventual target populations for Shigella vaccines. In these trials, whole blood is routinely collected on filter paper and dried. There has been some preliminary success with this type of sample format using the SBA. In addition to whole blood, mucosal samples (such as saliva, fecal extracts, and urine) are also a target that is relevant in Shigella vaccine research. Currently, this protocol has been evaluated for three of the most clinically relevant serotypes of Shigella but it can also be adapted for additional Shigella spp. as well as other bacterial pathogens. Future work will focus on the production of a multiplex assay with many of the same characteristics as the assay described by this protocol. A multiplexed assay will allow for evaluation of multiple Shigella serotypes simultaneously, further conserving sample volumes and hands on assay time. There is also work underway to transfer this assay to laboratories across the globe. These global evaluations will generate more data towards qualifying the assay on a larger research scale, while at the same time increasing number of microbiology and immunology laboratories that have access to this SBA to evaluate bacteria and serum samples collected from different endemic locations. The assay described here is simple and high-throughput and has the ability to improve immunological assessment in the Shigella field, as well as broader applications to assessment of other bacterial pathogens.

Shigella serotypes, Shigella flexneri 2a, S. flexneri 3a, and S. sonnei, demonstrate epidemiological prevalence globally. The diarrheal disease caused by these Shigella species impacts military, travelers1, and is a leading cause of diarrheal death among children under the age of 5 in developing countries2. There are currently no licensed vaccines to protect against Shigella, however, there are multiple candidate vaccines at various stages of the development. Many of these vaccines, and other prophylactic measures currently in development, focus on antibodies produced against Shigella lipopolysaccharide (LPS). LPS is an attractive vaccine candidate because it is a major surface antigen and natural infection with Shigella induces LPS-specific antibodies that can be protective against re-infection in a serotype-specific manner. Therefore, a successful Shigella vaccine will likely need to be multi-valent and target 3-4 Shigella serotypes to induce immunity against 70-80% of globally circulating strains3,4,5,6. This requires that assays to evaluate Shigella vaccine candidates be specific for multiple different serotypes.
Current immunological assays for evaluating vaccine candidates focus on quantifying levels of antibody titers, but there are few well-characterized assays to evaluate functional antibodies. The examination of antibody functional ability is important because pathogen specific antibodies are responsible for combating infection through a number of functional mechanisms including binding bacteria surface antigens, and preventing adhesion to and infection of epithelial cells, targeting bacterial cells or opsonization and phagocytosis, and directly killing pathogens by binding and initiating the complement cascade. The direct killing of bacteria by antibodies occurs when antibodies bind to surface components of the target bacteria and initiate the complement cascade leading to the activation of many zymogens that ultimately result in pore formation in the bacterial cell that causes lysis and bacterial death. This direct killing of bacteria by circulating antibodies and complement may be a crucial early line of defense during infection.&#160;
Individuals who are naturally infected have antibodies with shigellacidal activity in their sera. These Shigella-specific antibodies were detected using traditional complement-mediated killing assays 7,8. This indicates that there may be a role for bactericidal antibodies in protection against Shigella. Traditional bactericidal assays are simple in their execution: serum is heat-inactivated (to destroy endogenous complement activity) and mixed with the bacteria of interest. Exogenous complement is added to this mixture at a specific concentration. The reaction mixture is incubated to allow for bacterial killing and then plated to confirm colony-forming units (CFUs). Once CFUs are counted, a 50% killing index (KI) can be calculated and an SBA titer determined. While this procedure is relatively simple, these assays can be labor-intensive and time-consuming to perform and the results can be highly variable. In addition to these limitations, no well characterized functional assays currently exist for Shigella. Therefore, we have successfully developed and qualified a simple, high-throughput assay to measure Shigella bactericidal activity for three of the most clinically relevant strains9. This protocol describes an SBA with modifications that improve assay efficiency and reproducibility. The first of these modifications is the use of frozen bacterial stocks. The production of single use stocks eliminates the need to culture fresh bacteria for each assay while also reducing assay-to-assay variability. Another time and labor saving advantage of this protocol is the use of a 96-well plate assay format. This allows for serial dilution of samples so that a range of concentrations can be tested. &#160;It also allows for the use of multichannel pipettes for plating samples on square Petri dishes. When these square Petri dishes are used in conjunction with a culture system that produces micro-colonies, the number of agar plates required for the assay is reduced. This, in combination with freely available colony-counting software, originally developed for the pneumococcal multiplexed opsonophagocytic killing assay (MOPA)10, allows for the rapid, automated, and reliable colony enumeration. All of these improvements significantly reduce hands-on assay time and creating a high-throughput system allowing for multiple plates to be run at once.
While this protocol has been optimized for three of the most clinically relevant serotypes of Shigella, the SBA described here can be easily applied to many other bacterial pathogens. In addition to this protocol&#8217;s potential use with other bacteria, this protocol has the potential to expand beyond using only serum as a starting material, which could include the analysis of antibodies in other relevant sample types such as mucosal samples, including saliva and fecal samples. The use of this assay to investigate immunological responses after vaccination may give broader insight into immune responses generated by vaccination leading to the rational design of vaccines, and aid in the understanding of how natural immunity develops.

<table><tbody><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td>Type B, powder, BioReagent, suitable for cell culture</td></tr><tr><td>TTC (2,3,5-Triphenyltetrazolium chloride)</td><td>Sigma</td><td>T8877</td><td>&amp;ge;98.0% (HPLC)</td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S2002</td><td>&amp;ge;99.5%</td></tr><tr><td>Baby Rabbit Complement</td><td>PelFreez</td><td>31061-3</td><td>3-4 week old</td></tr><tr><td>HBSS with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Invitrogen</td><td>14065-56</td><td>Without Phenol Red</td></tr><tr><td>LB Agar (Lennox)</td><td>Sigma</td><td>L2897</td><td>Powder microbial growth medium</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td>Powdered, (C&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;O&lt;sub&gt;9&lt;/sub&gt;)&lt;sub&gt;n&lt;/sub&gt;</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For molecular biology, &amp;ge;99%</td></tr><tr><td>LB Broth (Lennox)</td><td>Sigma</td><td>L3022</td><td>Powder microbial growth medium</td></tr><tr><td>Square Petri Dish</td><td>Sigma</td><td>Z617679-240EA</td><td>120 mm x 120 mm</td></tr><tr><td>Assay Plate</td><td>Costar</td><td>3799</td><td>96 well u-bottom plate with lid</td></tr><tr><td>NICE Software</td><td>University of Alabama at Birmingham</td><td></td><td>ftp://ftp.nist.gov/pub/physics/mlclarke/NICE/</td></tr></tbody></table>

a high-throughput shigella-specific bactericidal assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A 96-well plate layout used in a typical assay is shown in Table 1. This layout has the active complement control wells (Control B), the heat-inactivated complement control wells (Control A), and five samples in duplicate. Samples are serially diluted 3-fold up the plate from Row H to Row A allowing for 8 dilutions of each sample to be tested at once. Figure 1 shows two LBA plates after the overnight incubation and overlay addition. Color development has taken place and all surviving colonies are visible in red. Bacterial killing is clearly seen for all samples tested in the first three dilutions (rows F-H) and as samples are diluted further up the plate, a decrease in bacterial killing is seen where serum is less concentrated. NICE software interface can be seen in Figure 2. Micro-colony counts from the NICE software can be seen in Figure 3, and these counts have been organized into Table 2. The average CFU count for each dilution of each sample is calculated and a 50% KI value is calculated in Table 3. This 50% KI value can be applied to the averaged CFUs for each serum dilution to determine the values needed to calculate the SBA KI according to the formula described in Figure 4. The final result of the assay is shown in Table 4.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>7</td>
			<td>8</td>
			<td>9</td>
			<td>10</td>
			<td>11</td>
			<td>12</td>
		</tr>
		<tr>
			<td>A</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
			<td>Dilution 8</td>
		</tr>
		<tr>
			<td>B</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
			<td>Dilution 7</td>
		</tr>
		<tr>
			<td>C</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
			<td>Dilution 6</td>
		</tr>
		<tr>
			<td>D</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
			<td>Dilution 5</td>
		</tr>
		<tr>
			<td>E</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
			<td>Dilution 4</td>
		</tr>
		<tr>
			<td>F</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
			<td>Dilution 3</td>
		</tr>
		<tr>
			<td>G</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
			<td>Dilution 2</td>
		</tr>
		<tr>
			<td>H</td>
			<td>Control A</td>
			<td>Control B</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
			<td>Dilution 1</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td colspan="2">Sample 1&#160;</td>
			<td colspan="2">Sample 2</td>
			<td colspan="2">Sample 3</td>
			<td colspan="2">Sample 4</td>
			<td colspan="2">Sample 5</td>
		</tr>
	</tbody>
</table>
Table 1: Assay plate layout. Columns 1 and 2 contain the complement control wells. Control A is located in column 1 and is the heat-inactivated complement control, containing SBA buffer, bacteria, and heat-inactivated complement. Control B is located in column 2 and is the active complement control, containing SBA buffer, bacteria, and complement. Columns 3-12 contain serum samples. Each sample is run in duplicate and serially diluted 3-fold from row H to row A
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59164/59164fig1.jpg" /> 
Figure 1: LBA plates after color development. Representative S. flexneri 3a bacterial micro-colonies have grown overnight to the appropriate size. Overlay agar has been added and colonies have developed a red color by reduction of the TTC compound in overlay agar. <a href="https://www.jove.com/files/ftp_upload/59164/59164fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59164/59164fig2.jpg" /> 
Figure 2: NIST&#39;s Integrated Colony Enumerator (NICE) software interface. Graphical representation of the NICE software interface. Regions of Interest (ROIs) are centered over colonies for each spot before counting. Data can be exported directly from the NICE window. <a href="https://www.jove.com/files/ftp_upload/59164/59164fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59164/59164fig3.jpg" /> 
Figure 3: LBA plates counted in NICE Software. The color photograph images are loaded into NICE software and colony forming units (CFUs) are counted automatically. This image shows two representative LBA plates with their colony count information. <a href="https://www.jove.com/files/ftp_upload/59164/59164fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>185</td>
			<td>167</td>
			<td>145</td>
			<td>151</td>
			<td>122</td>
			<td>134</td>
			<td>119</td>
			<td>142</td>
			<td>124</td>
			<td>115</td>
			<td>113</td>
			<td>123</td>
			<td>1:17496</td>
			<td>Dilution 8</td>
		</tr>
		<tr>
			<td>186</td>
			<td>152</td>
			<td>145</td>
			<td>138</td>
			<td>132</td>
			<td>135</td>
			<td>108</td>
			<td>116</td>
			<td>100</td>
			<td>119</td>
			<td>105</td>
			<td>109</td>
			<td>1:5832</td>
			<td>Dilution 7</td>
		</tr>
		<tr>
			<td>179</td>
			<td>160</td>
			<td>145</td>
			<td>135</td>
			<td>109</td>
			<td>120</td>
			<td>54</td>
			<td>55</td>
			<td>71</td>
			<td>75</td>
			<td>79</td>
			<td>89</td>
			<td>1:1944</td>
			<td>Dilution 6</td>
		</tr>
		<tr>
			<td>193</td>
			<td>153</td>
			<td>146</td>
			<td>138</td>
			<td>87</td>
			<td>105</td>
			<td>5</td>
			<td>6</td>
			<td>6</td>
			<td>9</td>
			<td>19</td>
			<td>30</td>
			<td>1:648</td>
			<td>Dilution 5</td>
		</tr>
		<tr>
			<td>184</td>
			<td>129</td>
			<td>121</td>
			<td>143</td>
			<td>6</td>
			<td>7</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1:216</td>
			<td>Dilution 4</td>
		</tr>
		<tr>
			<td>180</td>
			<td>145</td>
			<td>22</td>
			<td>24</td>
			<td>2</td>
			<td>3</td>
			<td>3</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>1:72</td>
			<td>Dilution 3</td>
		</tr>
		<tr>
			<td>207</td>
			<td>129</td>
			<td>1</td>
			<td>0</td>
			<td>2</td>
			<td>2</td>
			<td>1</td>
			<td>0</td>
			<td>4</td>
			<td>1</td>
			<td>0</td>
			<td>1</td>
			<td>1:24</td>
			<td>Dilution 2</td>
		</tr>
		<tr>
			<td>201</td>
			<td>140</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>0</td>
			<td>4</td>
			<td>4</td>
			<td>1:8</td>
			<td>Dilution 1</td>
		</tr>
		<tr>
			<td>&#160;Control A</td>
			<td>&#160;Control B</td>
			<td colspan="2">Sample 1</td>
			<td colspan="2">Sample 2</td>
			<td colspan="2">Sample 3</td>
			<td colspan="2">Sample 4</td>
			<td colspan="2">Sample 5</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Counts of bacterial colonies. CFU counts are exported from the NICE software to excel format. These counts can be arranged into a table showing the bacterial counts for all duplicate samples and control wells.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Control A</td>
			<td>&#160;Control B</td>
			<td>148</td>
			<td>128</td>
			<td>131</td>
			<td>120</td>
			<td>118</td>
			<td>1:17496</td>
			<td>Dilution 8</td>
		</tr>
		<tr>
			<td>189</td>
			<td>147</td>
			<td>142</td>
			<td>134</td>
			<td>112</td>
			<td>110</td>
			<td>107</td>
			<td>1:5832</td>
			<td>Dilution 7</td>
		</tr>
		<tr>
			<td colspan="2">NSK (1-CtrB/CtrA):&#160;&#160; 22%</td>
			<td>140</td>
			<td>115</td>
			<td>55</td>
			<td>73</td>
			<td>84</td>
			<td>1:1944</td>
			<td>Dilution 6</td>
		</tr>
		<tr>
			<td colspan="2">50% KI (CtrB/2):&#160;&#160;&#160;&#160;&#160;&#160;&#160; 73&#160;</td>
			<td>142</td>
			<td>96</td>
			<td>6</td>
			<td>8</td>
			<td>25</td>
			<td>1:648</td>
			<td>Dilution 5</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>132</td>
			<td>7</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1:216</td>
			<td>Dilution 4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>23</td>
			<td>3</td>
			<td>2</td>
			<td>1</td>
			<td>0</td>
			<td>1:72</td>
			<td>Dilution 3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>1</td>
			<td>2</td>
			<td>1</td>
			<td>3</td>
			<td>1</td>
			<td>1:24</td>
			<td>Dilution 2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>0</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>4</td>
			<td>1:8</td>
			<td>Dilution 1</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Sample 1</td>
			<td>Sample 2</td>
			<td>Sample 3</td>
			<td>Sample 4</td>
			<td>Sample 5</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3: Calculation of 50% KI cutoff value and duplicate sample averages. The 50% KI cutoff value was calculated by taking the average of all Control B wells and dividing by 2. Averages of duplicates were calculated for each dilution of each sample that was run in duplicate. NSK value is also calculated.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59164/59164fig4.jpg" /> 
Figure 4: Schematic of linear interpolation. The number of surviving bacteria (y-axis) at each dilution of serum tested (x-axis) is plotted (black diamonds), and individual points are connected by the thin, dashed black line. The solid and dashed horizontal lines indicate 0% and 50% killing, respectively. The serum dilutions above (Dilution 5) and below (Dilution 4) the 50% killing line are connected by a red line, and bactericidal titer (KI) is indicated. <a href="https://www.jove.com/files/ftp_upload/59164/59164fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>SBA KI</td>
		</tr>
		<tr>
			<td>Sample 1</td>
			<td>72</td>
		</tr>
		<tr>
			<td>Sample 2</td>
			<td>216</td>
		</tr>
		<tr>
			<td>Sample 3</td>
			<td>1994</td>
		</tr>
		<tr>
			<td>Sample 4</td>
			<td>1994</td>
		</tr>
		<tr>
			<td>Sample 5</td>
			<td>648</td>
		</tr>
	</tbody>
</table>
Table 4: SBA results showing bactericidal titer (KI). Final SBA KI values have been determined for each sample and are shown. These values are calculated using the average CFUs, the 50% KI cutoff value, and the linear interpolation formula.

Watch this Scientific Journal Video about A High-throughput Shigella-specific Bactericidal Assay at JoVE.com

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

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Research

JoVE Journal

Immunology and Infection

11.7K Views.  Walter Reed Army Institute of Research. Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Video: A High-throughput Shigella-specific Bactericidal Assay

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Protocols for Analyzing Shigella Infection in Colonic Epithelial Cells

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Author Spotlight: Advancements in Understanding and Combatting Shigella Infections

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies

Source: Weerts, H. P., et al. <a href="https://app.jove.com/v/59164">A High-throughput Shigella-specific Bactericidal Assay.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate the complement-mediated bactericidal activity of antibodies present in serum. This approach combines isolated serum with pathogenic bacteria and exogenous complement proteins. Upon incubation, the bacteria are grown on an agar plate, and the produced colonies are quantified to measure the serum bactericidal titer.

Source: Weerts, H. P., et al. A High-throughput Shigella-specific Bactericidal Assay. J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate ...

A High-throughput Shigella-specific Bactericidal Assay

Summary

Explore More Videos

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Imaging Ca²⁺ Responses During Shigella Infection of Epithelial Cells

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Epithelial Cell Infection Analyses with Shigella

A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies