A novel immuno-molecular strategy for the detection of Streptococcus pneumoniae serotypes in human cerebrospinal and middle ear fluids.

Streptococcus pneumoniae is one of the most common microorganisms causing acute otitis media (AOM) in children. While bacterial culture of middle ear fluid (MEF) is the gold standard to detect the etiological organisms, several host and pathogen factors impact the survival of the organisms resulting in false negatives. To overcome this limitation, we have developed and validated an innovative multiplex immuno-molecular assay to screen and detect the S.

Development and Validation of Two Optimized Multiplexed Serologic Assays for the 9-Valent Human Papillomavirus Vaccine Types.

Two multiplex immunoassays are routinely used to assess antibody responses in clinical trials of the 9-valent human papillomavirus (9vHPV) vaccine. The HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA) and HPV6/11/16/18/31/33/45/52/58 total immunoglobulin G Luminex immunoassay are used for measurements of immunogenicity. Following their initial validation in 2010, both assays were redeveloped, and several parameters were optimized, including the coating concentration of virus-like particles, type of Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the phycoerythrin-labeled antibodies.

Development and Validation of a Sensitive and Robust Multiplex Antigen Capture Assay to Quantify Streptococcus pneumoniae Serotype-Specific Capsular Polysaccharides in Urine.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP) in young children, older adults, and those with immunocompromised status. Since the introduction of pneumococcal vaccines, the burden of invasive pneumococcal disease caused by vaccine serotypes (STs) has decreased; however, the effect on the burden of CAP is unclear, potentially due to the lack of testing for pneumococcal STs. We describe the development, qualification, and clinical validation of a high-throughput and multiplex ST-specific urine antigen detection (SSUAD) assay to address the unmet need in CAP pneumococcal epidemiology.

Development and qualification of a fast, high-throughput and robust imaging-based neutralization assay for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques.

Real-time PCR assay as a simple and efficient tool for viral stability study.

For oncolytic virus trials, regulatory agencies often require pharmaceutical industry to evaluate risks of released viruses from patients to environment. This study was to establish a real-time PCR method to assess viral shedding and viral stability in human urine. Herein, we describe an incubation of viral drug product in human urine and use of real-time PCR as a simple, efficient and high throughput assay to assess the level and stability of a nonenveloped and single stranded RNA virus.

Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue.

Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times.

Serologic assay to quantify human immunoglobulin G antibodies to the Staphylococcus aureus iron surface determinant B antigen.

A direct binding Luminex assay has been developed and validated for the detection of human immunoglobulin G (IgG) antibodies to the Staphylococcus aureus iron surface determinant B protein (IsdB) in serum following natural infection or immunization with investigational Saccharomyces cerevisiae-derived IsdB-based vaccines. To ensure that IsdB-specific IgG antibodies are measured following immunization with S. cerevisiae-derived IsdB, an Escherichia coli-produced IsdB antigen is used in the assay.

Optimization and validation of a multiplex, electrochemiluminescence-based detection assay for the quantitation of immunoglobulin G serotype-specific antipneumococcal antibodies in human serum.

Pneumovax 23 consists of a mixture of highly purified capsular polysaccharides (Ps) from 23 of the most prevalent serotypes of Streptococcus pneumoniae. Testing of vaccine immunogenicity has been historically performed on the enzyme-linked immunosorbent assay (ELISA) platform, validated to measure immunoglobulin G (IgG) antibodies to all 23 serotypes included in Pneumovax 23. In order to significantly improve the throughput of this form of testing, we have developed and validated a direct binding electrochemiluminescence (ECL)-based multiplex assay that can measure the antibody response in human serum to eight serotypes within a single microtiter well.

Complexities of clinical assay development and optimization prior to first-in-man immunization trials - a description of immunogenicity assay development for the testing of samples from a phase 1 Alzheimer's vaccine trial.

Immunogenicity is often a critical clinical endpoint in the assessment of vaccines prior to the submission of data to regulatory agencies. As a result, the assays used to measure immunogenicity must be highly characterized, well-controlled, and statistically supported. These goals are not easily attained, however, when the development of the assay must occur prior to the first-in-man studies.