TITLE: Bangladesh National Serosurvey Vibriocidal Protocol (96-well plate, OD600, mAb)
AUTHORS: Taufiqur Bhuiyan, Jason Harris, Owen Jensen, Daniel Leung, Andrew Azman, Firdausi Qadri

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol provides the details on how vibriocidal assays were perfomed on serum from the nationally representative serosurvey for </span><span style = "font-style:italic;">V. cholerae</span><span> O1.</span></div></div>

[STEPS]
?. Streak V. cholerae (O1 Ogawa or Inaba) onto blood agar and incubate at  overnight (approx )
37 °C
?. Inoculate a loopful of bacteria from the blood agar plate (1-2 colonies) into 15mL culture tube from previous step
?. Incubate in an orbital shaker-incubator at  and 220 rpm for  -
37 °C
?. While the bacterial culture is growing in the orbital shaker/incubator, aliquot  of the serum samples to be tested into Eppendorf tubes and place them in a  water bath for  to heat-inactivate the native complement system proteins
20 µl
56 °C
?. Dilute the heat-inactivated sample sera 1:10 ( + ) before beginning the assay (this is the starting titer of 10), and keep on ice until ready to add samples to 96 well plate (below)
[serum]
[0.9% NaCl saline]
?. Take guinea pig complement serum aliquot (300 μL plate) out of the -20°C freezer and let it begin thawing at 4°C (refrigerator)
?. When the bacterial culture has finished growing, transfer it into a 15 mL centrifuge tube
?. Centrifuge at 3000 rpm and - (room temperature) for
22 °C
25 °C
?. Discard supernatant into 1:10 bleach solution or biohazard waste
?. [Wash 1]
Wash cells by resuspending the cell pellet in
[saline]
?. [Wash 1]
Centrifuge again at 3000 rpm and  - (room temperature) for
22 °C
25 °C
?. [Wash 1]
Discard supernatant into 1:10 bleach or biohazard waste
?. [Resuspending cells post-wash]
After washing cells 2 times, resuspend them in 1 mL 0.9% NaCl.
?. Transfer  into 96 well plate and measure Optical density (OD) at 600 nm, OD600. Based on OD600 reading prepare 1 ml of V. cholerae working dilution diluted to approximately OD600 0.3 with 0.9% NaCl. (Ex. if OD600 =1.0, add 300 μL bacterial solution + 700 μL 0.9% NaCl).
[solution]
?. Add  of 1:10 diluted serum samples from Step 5 to wells in column 2 (for 96 Well Plate Layout  ).	Each sample should be done in duplicate; e.g. Sample 1 in 2A and 2B, Sample 2 in 2C and 2D, and Sample 3 in 2E and 2F.
50 µl
?. Use a multichannel pipettor to perform 2-fold serial dilutions of the samples starting from column 2 and going to column 12, i.e. transfer  of the sample from column 2 into column 3, gently pipette up and down a few times, then transfer  from column 3 into column 4, and so on down the rows, removing g from the last column (12) and discarding. Check the volume of the sample/dilution wells after you are finished to make sure they are uniform before proceeding
25 µl
25 µl
25 µl
?. Prepare the Growth Indicator solution as follows for a single 96-well plate:•	 saline•	 diluted bacteria (working dilution as determined in Step 15).•	 guinea pig complement serum
2.85 ml
150 µl
300 µl
?. Pour the Growth Indicator solution into a  reagent reservoir, add  of it to wells 1A, 1B, 1C, and 1D (Growth Controls), and  to all sample/2-fold dilution wells (columns 2-12; for plate layout  ). Be careful not to cross-contaminate any of the wells. Mix wells by gently pipetting up and down with the multichannel pipettor, be sure to change tips between columns to avoid cross-contamination!
25 ml
25 µl
25 µl
?. Prepare a 96 well plate by first adding 25 μL of saline to all columns, except column 2 (See 96 Well Plate Layout below)
?. Incubate plate on orbital shaker-incubator at  and 50 rpm for
37 °C
?. Take the plate out of the shaker/incubator and add  BHI to all wells (including controls), being careful not to cross-contaminate.
150 µl
?. Incubate at   without shaking for  and then measure the OD595. Make sure the Growth Control wells are between 0.20 and 0.28. If below, continue to grow and measure OD595 every half hour (can take up to 4 hrs total).
37 °C
?. Read the OD of the entire plate at 595 nm with a plate reader. Subtract the average OD of 4 negative control wells from all wells to get a 'blanked' value (if this brings any values below 0, the value should be set to zero).
?. Fill 15 mL culture tube with  sterile brain heart infusion broth (BHI) that has been left at ( - ) for at least 1 hour
10 ml
22 °C
25 °C
?. [Wash 2]
Wash cells by resuspending the cell pellet in
[saline]
?. [Wash 2]
Centrifuge again at 3000 rpm and  - (room temperature) for
22 °C
25 °C
?. [Wash 2]
Discard supernatant into 1:10 bleach or biohazard waste
?. Add of V. cholerae,  Inaba or Ogawa monoclonal antibody in PBS (1 μg/ml) into wells 2G and 2H, which will serve as the assay control.
50 µl
?. Add  saline to 1E, 1F, 1G, and 1H (Negative Controls)
25 µl
?. Check to make sure monoclonal antibodies are within acceptance range. If mAb assay control readings are not in range (50% Kill titer for Ogawa of 0.0625-0.03125 (Col 5 or 6), and for Inaba of 0.125-0.0625 (Col 4 or 5), plate should be rejected.
?. Take the average of OD of the four Growth Controls and divide by 2 to get the 50% kill OD.If ≥ 2 Growth control wells are out of range (0.2-0.28) the plate should be rejected. If only one growth control well is out of range, that well will be rejected and the other three averaged to get the 50% kill OD.
?. Determine the titer for each sample/row by taking the reciprocal of the dilution factor for which the OD595 reading is less than the 50% kill OD. If this is not achieved by a sample, assign a titer value of 5 to indicate below limit of detection.Example: If the average Growth Control OD595 = 0.24, and the sample in row B (above) has an OD595 of 0.11 in column 7, then the titer is reported as 320.
?. Check to make sure technical replicates for each sample have a titer within one 2-fold dilution of one another.If the technical replicate titers exceeds more than one 2-fold dilution, the sample should be rejected.If the technical replicates titers differ by one 2-fold dilution, the sample should be assigned the lower of the two titers