eoe sub articles

EoE Sub Articles

1. Prepare Complement and Target Bacteria
<ol>
	<li>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
	<ol>
		<li>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.</li>
		<li>While BRC is thawing, label 1.5 mL, 5 mL, or 15 mL centrifuge tubes. Place l...

a serum bactericidal assay for the complement-mediated bactericidal activity of antibodies

Source: Weerts, H. P., et al. <a href="https://app.jove.com/v/59164">A High-throughput Shigella-specific Bactericidal Assay.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21785/21785_Figure1.jpg" /> 
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...

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

Source: Weerts, H. P., et al. A High-throughput Shigella-specific Bactericidal Assay. J. Vis. Exp. (2019).
This video demonstrates a technique to evaluate ...

Take a serially diluted serum sample containing monoclonal antibodies against lipopolysaccharides, or LPS &#8212; an outer membrane component of gram-negative bacteria.
Introduce gram-negative bacteria. Serum antibodies bind to bacterial LPS.
Add complement proteins. C1 complex binds to LPS-bound antibodies, forming an enzymatically active C1.
Activated C1 cleaves C4 and C2 to form C3 convertase, which cleaves C3 to form C5 convertase. The convertase cleaves C5 to produce C5b, which recruits C6 and C7. The resulting complex inserts into the outer membrane and associates with C8 and multiple C9, forming a pore.
Outer membrane damage destabilizes the inner membrane, causing cell lysis.
Spot the mixture onto an agar plate and spread. Incubate for bacterial growth. Overlay the plate with agar containing Triphenyl tetrazolium chloride, or TTC, which gets reduced by microbial dehydrogenases, imparting a red color to bacterial colonies.
Colony number increases with sample dilution, indicating reduced bactericidal activity with lower antibody concentration.

a-serum-bactericidal-assay-for-complement-mediated-bactericidal

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

JoVE Journal

Bioengineering

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Immunology and Infection

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

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

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