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

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

1. 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
   1. 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.
   2. While BRC is thawing, label 1.5 mL, 5 mL, 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)....

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 compou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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/