A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies
Published: November 30, 2023
Abstract
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.
Protocol
1. Prepare Complement and Target Bacteria 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 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. While BRC is thawing, label 1.5 mL, 5 mL, or 15 mL centrifuge tubes. Place l…
Representative Results
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. <img alt="Figure 2" src="/files/ftp_upload/21785/21785_Figure2.j…
Declarações
The authors have nothing to disclose.
Materials
| 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/ |
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A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies. J. Vis. Exp. (Pending Publication), e21785, doi: (2023).