MICROSPHERE-BASED MULTIPLEXING FOR THE DETECTION OF SEROTYPE-SPECIFIC PNEUMOCOCCAL ANTIBODIES
Overview
Streptococcus pneumoniae remains one of the most significant causes of morbidity and mortality worldwide . Vaccination against pneumococcal infection has been highly effective in reducing the incidence of disease and mortality. However, the efficacy of pneumococcal vaccines varies depending on the serotype of the infecting strain, making serotype-specific detection of antibodies crucial in evaluating vaccine efficacy .
The principle of xMAP technology is based on the concept of a liquid (suspension) array. the xMAP technology uses different sets of microspheres in a liquid suspension to determine the analyte specificity. Beads are colored internally with two different fluorescent dyes (red and infrared). Different concentrations of red and infrared dyes are used to generate up to 100 distinct bead regions. Each microsphere contains functional carboxyl groups which can be used to covalently conjugate an analyte, antibody or probe to the microsphere.(a) followed by binding with a biotinylated detection antibody (b) and a reporter dye, streptavidin-conjugated phycoerythrin (c).
The analysis of beads is performed by two lasers. The red classification laser/LED (635 nm) excites the inner fluorescent dyes of the microspheres, thus identifying a specific microsphere set according to its spectral signature. If the analyte of interest is present, the green reporter laser/LED (525–532 nm) recognizes the fluorescent reporter bound to the captured analyte on the microsphere surface.
INTRODUCTION:
Streptococcus pneumoniae remains one of the most significant causes of morbidity and mortality worldwide (1). Vaccination against pneumococcal infection has been highly effective in reducing the incidence of disease and mortality (2). However, the efficacy of pneumococcal vaccines varies depending on the serotype of the infecting strain (3,4), making serotype-specific detection of antibodies crucial in evaluating vaccine efficacy (5).
METHODS FOR THE DETECTION OF PNEUMOCOCCAL ANTIBODY
Serological assay, Enzyme-linked immunosorbent assay (ELISA) is the “gold standard” for quantifying the concentrations of antibodies to pneumococcal serotype-specific polysaccharides and has largely replaced earlier radioimmunoassay technology (6). However, ELISA has limitations, including low sensitivity and cross-reactivity, which can lead to inaccurate results, is suited for screening the samples against a single analyte, needs a long duration of up to 72 hours for completion, and require a greater volume of serum, which is already limited, with additional infrastructure, manpower, and consumables (7). To overcome these limitations, Microsphere-based Luminex immunoassay has been developed and is gaining popularity in the field of serological testing (8,9). This assay involves the use of microspheres, or beads, coated with antigens specific to a particular serotype of pneumococcus. The microspheres are labelled with a fluorescent dye, allowing for the quantitation of antibody binding (10,11).
FACTORS | ELISA | MULTIPLEXING |
Cost (Multiple analytes) | Expensive | Less expensive |
Time required | Time consuming | Minimal hands on time |
Labor | High intensive | Efficient: simultaneous readout |
Analytes(estimate) | 1 | Upto 100 |
Sample required | 10-100μl per analyte | 25μl for >40 analytes |
Reagent needs | Significant volumes | minimal |
Reporter technique | Spectrophotometer | Laser |
Time to read | Several hours | 45 minutes |
- There are different types of microspheres commercially available and their selection is generally determined by the type of instrumentation used for detection and the particular analyte of interest.
- beads are composed of polystyrene, divinyl benzene, and methacrylic acid, which provide surface carboxylate functionalities for covalent attachment of polysaccharides.
- Basic microspheres are 5.6 µm polystyrene beads whose surface is covered by approximately 10to the power 8 carboxyl groups (COOH) for covalent coupling of capture reagents
- Magnetic microspheres differ in size and structure through the addition of a magnetite layer., The usage of magnetic beads improves washing efficiency as the magnetic separation step enables the elimination of unwanted sample constituents.
Ref: Reslova, N., Michna, V., Kasny, M., Mikel, P., & Kralik, P. (2017). xMAP Technology: Applications in Detection of Pathogens. Frontiers in microbiology, 8, 55. https://doi.org/10.3389/fmicb.2017.00055
HOW DOES THE MICROSPHERE-BASED IMMUNOASSAY WORK?
The microsphere-based immunoassay for the detection of serotype-specific pneumococcal antibodies is a highly sensitive and specific method that utilizes the Luminex xMAP technology. This assay allows for the simultaneous detection of multiple antibodies against pneumococcal serotypes in a single sample, making it a highly efficient and cost-effective tool for serotyping pneumococcal infections.
- X = Biomarker or disease panel to be tested
- M = Multiple
- A = Analyte
- P = Profiling
The assay involves the following steps:
- Coating of Microspheres: Microspheres are coated with specific pneumococcal serotype-specific antigens, which are known to elicit an antibody response in humans.
- Incubation with Serum: The coated microspheres are incubated with human serum samples. Antibodies in the serum will bind to the corresponding pneumococcal serotype-specific antigens on the microspheres.
- Detection of Bound Antibodies: Unbound serum components are washed away, and a secondary antibody that recognizes human IgG is added. This secondary antibody is labeled with a fluorescent dye, allowing for detection by a flow cytometer.
- Analysis: The microspheres are analyzed in a flow cytometer, which quantifies the amount of fluorescent signal associated with each microsphere. The amount of signal corresponds to the amount of bound antibodies in the serum sample.
WORK FLOW
CONCLUSION:
Microsphere-based immunoassays are a promising tool for the rapid and specific detection of serotype-specific pneumococcal antigens in clinical specimens. They offer several advantages over traditional methods of pneumococcal serotyping, including high specificity, rapid turnaround time, and multiplexing capability. As such, they have the potential to improve the diagnosis and management of pneumococcal disease, ultimately reducing its burden on public health.