Multiplex Laboratory Methods – Serosurvey Tools

Overview

The development and use of multiplex bead immunoassays (MBIAs) can be challenging compared to monoplex enzyme-linked immunosorbent assays (ELISAs). Many Good Laboratory Practice principles that are relevant to conducting monoplex assays are also relevant for MBIAs. Additional detailed information on MBIAs can be found in PAHO’s Toolkit for Integrated Serosurveillance of Communicable Diseases in the Americas.

Instrument selection

Multiple instruments can be used for MBIAs for integrated serosurveillance, with well-known companies including Luminex (a Diasorin company), Biorad, and Meso Scale Diagnostics. Instrument selection may be informed by factors including affordability, availability of the instrument and reagents, maintenance services, compatibility with laboratory capabilities, and familiarity. Researchers should consider:

the capabilities of the instrument and how they map to their needs,
how frequently they plan to use an instrument,
durability and transportability of the instrument, and
the availability of maintenance services or ease of user-guided maintenance.

Assay development

The multiplex nature of MBIAs can present technical difficulties that must be addressed and validated before an assay is used for serosurveillance. Participants in the 2023 Serosurveillance Summit estimated that it takes approximately 300-5,000 hours to develop an assay. Using or building upon existing assays that have been validated by others may be preferable, especially when more guided technology transfers, rather than simply sharing protocols, can take place. In-person training, whether by bringing personnel to the lab that originated the protocol or having originators or other trained individuals (i.e., train-the-trainer approaches) visit labs that wish to use the protocol, can facilitate smoother transfer. The use of validation assays is especially important for MBIAs, given the complexity of the assay.

Challenges

Some challenges that those wishing to develop an assay themselves may consider include:

Cross-reactivity

Cross-reactivity occurs when antibodies or other proteins bind to antigen-coupled beads which the user does not intend them to bind to. This can occur when antigens are similar in structure (e.g., from similar pathogens, such as the flaviviruses) or when antibodies in serum samples have broad activity against multiple antigens.
Cross-reactive antigens do not always have to be separated from one another into different panels, especially for antigens which have lower levels of cross-reactivity. Inclusion of cross-reactive antigens on the same panel can be mitigated by including and characterizing non-cross-reactive antigens, and some modeling techniques can be used to account for these artifacts.

Controls

Both positive and negative controls should be included on each plate to allow for the classification of seropositivity or, where characterized, the quantification of antibody concentration through the generation of standard curves. Ensuring the appropriate use of positive and negative controls is essential for correctly separating signal from noise.
The use of local controls can provide important context on the effects of potential cross-reactivity in a population exposed to multiple diseases.
Negative controls with antigen-coupled beads only (no serum) and uncoupled beads can prove useful in adjusting for noise from off-target binding (e.g., some populations may have protein in their sera that naturally binds to beads, producing erroneous signal).
Where possible, the use of gold standard reference kits and international standards can assist in validation of new assays and controls. The use of monoclonal antibodies as controls could also prove valuable for long-term sustainability and reliability. In general, using the same controls as other lab groups can assist in comparability and standardization.

Bead coupling

The buffer conditions, such as pH, necessary to couple antigens to beads vary by the antigen type. Because of this, assay developers may need to experiment with different conditions, especially if there is limited literature on coupling for a specific antigen.

Disease experts

Experts who have used multiplex or monoplex immunoassays that detect antibodies of interest can provide valuable insight in the assay development process. This is especially true for settings in which monoplex immunoassays such as ELISAs have been and continue to be used for a specific pathogen, to encourage the interpretation of results between assays. These experts can also help to identify pathogen-related considerations that may focus the target population.
For example, both yaws and syphilis are caused by Treponema pallidum, so antibody responses to antigens from this pathogen should be contextually informed by age group and disease distribution. Considering local epidemiology and the age of individuals from which specimens are used can aid in contextualizing and addressing this issue.

Assay variability

Researchers should also monitor and rule out or adjust for effects linked to changing any parts of the assay, whether through the depletion of materials, instruments becoming outdated, or planned modifications of an existing assay.

Differences in instruments

Changes to the instrument that is being used to measure MBIAs can lead to changes in the signal produced by the same antigen-coupled beads and controls.
Using a new or different instrument requires validation to ensure comparable results or to guide adjustments.

Differences in antigen-coupled beads

The switch between batches of the same antigen-coupled beads could introduce variability that should be accounted for. This could affect the validity of previously established serological cut-offs.

Differences in controls

Planning for assay development should include consideration of the volume of controls available.
If using positive or negative controls for which limited volumes are available, users should consider implications for the validation of new controls, ideally before the preceding controls have been depleted.

Differences in general supplies

The other materials used to perform the assay can also play a role in the results of the assay.
Attention should be paid to the potential for beads to bind to plastic when certain antigens are used, and whether any error resulting from this is acceptable or should be corrected for by adding more beads or correcting the signal.
Care should be taken to limit the exposure of beads to light, such as by using black 96-well plates and covering plates when not in use.

Other considerations

Magnetic beads clump to one another, requiring thorough vortexing durations of 15-30 seconds or more.
Splitting the same samples across plates can allow users to identify inter-plate variability.

Instrument maintenance and preparation

As with any instrument, to ensure that instruments function properly, regular use and maintenance is advised where possible. However, given the mechanics of some MBIA plate readers, such as the MAGPIX RUO, users should give special care to some of the following considerations:

Use of equipment for other purposes

Instruments capable of performing MBIAs may be commonly used for other purposes, such as cytokine assays, involving the flow of highly concentrated protein through the machine. These proteins can bind to the machine’s inner tube and subsequently bind beads, causing blockages and irregular flow rates, producing unreliable results.
If machines are used for these purposes, more intensive bleach and water rinses may be necessary to dislodge beads from the tube before use.

Calibration kits

Instruments like the MAGPIX RUO use companion calibration kits and compact discs (CDs). Users should be aware that expiration dates applying to the kits are encoded on the CD; thus, if the calibration kit expires, the machine may prevent the user from using the instrument.
Sufficient planning is necessary to avoid this issue if calibration kits cannot be easily and quickly purchased.

Maintenance

The specialized use of these instruments compared to ELISA plate readers can make finding experts who can service the machines difficult or delayed.
Building one’s own familiarity with the machine, or leveraging the expertise of others, can enable troubleshooting of some issues, but proper routine maintenance is essential for preventing larger issues from occurring.

Quality Control

There are currently no standardized approaches to cleaning raw laboratory data and establishing seropositivity thresholds, as this varies by antigen, the availability of controls, and population. This first stage of analysis includes performing quality control checks, evaluating serial dilution standard curves, and ensuring that steps to normalize data are appropriately applied. CDC has developed a novel R Shiny application to expedite the MBA QC processes and reduce the risk of user error.

Resources

https://github.com/CDCgov/shinyMBA (quality control of MBA output based on Luminex xMAP technology)