scholarly journals Intralaboratory evaluation of luminescence based high-throughput Serum Bactericidal Assay (L-SBA) to determine bactericidal activity of human sera against Shigella

2020 ◽  
Author(s):  
O. Rossi ◽  
E. Molesti ◽  
A. Saul ◽  
C. Giannelli ◽  
F. Micoli ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
High Throughput ◽  
Bactericidal Activity ◽  
High Specificity ◽  
Effective Vaccine ◽  
Correlate Of Protection ◽  
Bactericidal Assay ◽  
Immune Correlate ◽  
Human Sera ◽  
Serum Bactericidal Assay

ABSTRACTDespite the huge decrease in deaths caused by Shigella worldwide in the last decades, shigellosis is still causing over 200,000 deaths every year. No vaccine is currently available, and the morbidity of disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not been established yet, the demonstration of bactericidal activity of antibodies induced upon vaccination may provide one means of functionality of antibodies induced on protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA).Here we present the development and intra-laboratory characterisation of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against homologous strain without any heterologous aspecificity detected against species-related and not species-related strains. We assessed linearity, repeatability and reproducibility of L-SBA on human sera.This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine bactericidal activity of any non-clinical and clinical sera that rely on complement mediated killing.IMPORTANCEShigella is an important cause of diarrhoea worldwide and antimicrobial resistance is on rise, thus efforts by several groups are ongoing to produce a safe and effective vaccine against shigellosis. Although a clear immune correlate of protection has not been established, demonstration of bactericidal capacity of sera from patients immunised with Shigella vaccines may provide one means of protecting against shigellosis. We have developed and fully characterised a novel high-throughput L-SBA method for evaluation of functionality of antibodies raised against S. sonnei in human sera. This work will allow the clinical testing of human sera raised against GMMA-based and potentially all vaccines producing antibodies than can work via complement mediated manner.

scholarly journals Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella

2020 ◽  
Vol 9 (2) ◽  
pp. 14 ◽  
Author(s):  
Omar Rossi ◽  
Eleonora Molesti ◽  
Allan Saul ◽  
Carlo Giannelli ◽  
Francesca Micoli ◽  
...  
Keyword(s):  
High Throughput ◽  
Bactericidal Activity ◽  
High Specificity ◽  
Laboratory Evaluation ◽  
Effective Vaccine ◽  
Bactericidal Assay ◽  
Laboratory Characterization ◽  
Homologous Strain ◽  
Human Sera ◽  
Serum Bactericidal Assay

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

scholarly journals Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)

BioTech ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 19
Author(s):  
Maria Grazia Aruta ◽  
Martina Carducci ◽  
Francesca Micoli ◽  
Francesca Necchi ◽  
Omar Rossi
Keyword(s):  
High Throughput ◽  
Bactericidal Activity ◽  
High Performance ◽  
Traditional Methods ◽  
Bactericidal Assay ◽  
Colony Forming Units ◽  
Serum Bactericidal Assay ◽  
Live Bacteria

Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens.

scholarly journals Opsonophagocytosis of Fluorescent Polystyrene Beads Coupled to Neisseria meningitidis Serogroup A, C, Y, or W135 Polysaccharide Correlates with Serum Bactericidal Activity

2002 ◽  
Vol 9 (2) ◽  
pp. 485-488 ◽  
Author(s):  
Joseph Martinez ◽  
Tamara Pilishvili ◽  
Suzanne Barnard ◽  
Joseph Caba ◽  
Willie Spear ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Bactericidal Activity ◽  
Simultaneous Measurement ◽  
Meningococcal Vaccine ◽  
Specific Flow ◽  
Polystyrene Beads ◽  
Serum Bactericidal Activity ◽  
Bactericidal Assay ◽  
Flow Cytometric ◽  
Serum Bactericidal Assay

ABSTRACT We developed a polysaccharide-specific flow cytometric opsonophagocytic assay (OPA) for the simultaneous measurement of functional antibody to Neisseria meningitidis serogroups A, C, Y, and W135. OPA titers significantly correlated with serum bactericidal assay titers for all serogroups tested (mean r = 0.96; P < 0.001). OPA could be used in meningococcal vaccine evaluation.

scholarly journals Detection of Complement-Mediated Antibody-Dependent Bactericidal Activity in a Fluorescence-Based Serum Bactericidal Assay for Group B Neisseria meningitidis

2000 ◽  
Vol 38 (8) ◽  
pp. 2878-2884 ◽  
Author(s):  
Kenneth T. Mountzouros ◽  
Alan P. Howell
Keyword(s):  
Neisseria Meningitidis ◽  
Bactericidal Activity ◽  
Fluorescent Dye ◽  
Bactericidal Assay ◽  
Viable Bacteria ◽  
Serum Bactericidal Assay ◽  
Functional Antibodies ◽  
Group B ◽  
Oxidative Respiration ◽  
Human And Mouse

Serum bactericidal assays (SBAs) for Group B meningococci are considered the methods of choice for the evaluation of functional antimeningococcal antibodies. Many investigators regard SBAs as time- and labor-intensive. Variations in SBA protocols among different laboratories make interpretation of results difficult. Here we describe a fluorescence-based serum bactericidal assay (fSBA) and compare the results obtained with the fSBA to the results obtained with a more conventional SBA. The results generated by both assays were dependent upon the surviving bacteria after incubation, and the assay mixtures contained identical components. Differences between assays lie in how the surviving bacteria are quantified. The fSBA described in the paper uses the fluorescent dye alamarBlue (M. V. Lancaster and R. D. Fields, U.S. patent 5501959, March 1996). The fluorescent signals generated in the fSBA correlate to the oxidative respiration of surviving bacteria. Viable bacteria were detected between 6 and 8 h directly from reaction mixtures in 96-well plates by the fSBA, whereas colonies isolated on semisolid media could be counted after 24 h of incubation. The bactericidal titers generated by both assays were nearly identical. The fSBA described here can be used as an assay for the screening of large quantities of individual sera as complement sources or as a method for the detection of functional antibodies directed against group B Neisseria meningitidisin both human and mouse antisera.

scholarly journals Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00128-18 ◽  
Author(s):  
Danka Pavliakova ◽  
Peter C. Giardina ◽  
Soraya Moghazeh ◽  
Shite Sebastian ◽  
Maya Koster ◽  
...  
Keyword(s):  
Human Serum ◽  
Lower Limit ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Correlate Of Protection ◽  
Immune Correlate ◽  
Relative Standard ◽  
Limit Of Quantitation

ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

scholarly journals HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

2022 ◽  
Vol 8 ◽  
Author(s):  
Ephraim Fass ◽  
Gal Zizelski Valenci ◽  
Mor Rubinstein ◽  
Paul J. Freidlin ◽  
Shira Rosencwaig ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Cost Effective ◽  
Developed Countries ◽  
Spike Protein ◽  
Medical Community ◽  
Effective Vaccine ◽  
Gene Variants ◽  
Spike Gene ◽  
Field Samples

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in &lt; 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Sign in / Sign up

Export Citation Format

Share Document