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…

開示

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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記事を引用
A Serum Bactericidal Assay for the Complement-Mediated Bactericidal Activity of Antibodies. J. Vis. Exp. (Pending Publication), e21785, doi: (2023).

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