Source: Weerts, H. P., et al. A High-throughput Shigella-specific Bactericidal Assay. 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.
1. Prepare Complement and Target Bacteria
2. Serum Bactericidal Assay (SBA)
NOTE: The procedure described below is for one assay plate, but the number of Assay Plates can be increased.
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
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | Control A | Control B | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 | Dilution 8 |
B | Control A | Control B | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 | Dilution 7 |
C | Control A | Control B | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 | Dilution 6 |
D | Control A | Control B | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 | Dilution 5 |
E | Control A | Control B | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 | Dilution 4 |
F | Control A | Control B | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 | Dilution 3 |
G | Control A | Control B | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 | Dilution 2 |
H | Control A | Control B | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 | Dilution 1 |
Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
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.
Figure 2: NIST'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.
The authors have nothing to disclose.
Gelatin | Sigma | G9391 | Type B, powder, BioReagent, suitable for cell culture |
TTC (2,3,5-Triphenyltetrazolium chloride) | Sigma | T8877 | ≥98.0% (HPLC) |
Sodium azide (NaN3) | Sigma | S2002 | ≥99.5% |
Baby Rabbit Complement | PelFreez | 31061-3 | 3-4 week old |
HBSS with Ca2+/Mg2+ | Invitrogen | 14065-56 | Without Phenol Red |
LB Agar (Lennox) | Sigma | L2897 | Powder microbial growth medium |
Bacto Agar | BD | 214010 | Powdered, (C12H18O9)n |
Glycerol | Sigma | G5516 | For molecular biology, ≥99% |
LB Broth (Lennox) | Sigma | L3022 | Powder microbial growth medium |
Square Petri Dish | Sigma | Z617679-240EA | 120 mm x 120 mm |
Assay Plate | Costar | 3799 | 96 well u-bottom plate with lid |
NICE Software | University of Alabama at Birmingham | ftp://ftp.nist.gov/pub/physics/mlclarke/NICE/ |