Developing and Running Assay Protocols

Introduction

This module describes the process of testing specimens in the laboratory and key considerations for seroprevalence studies.

Learning Objectives

Provide an overview of laboratory assays and specimen management in the laboratory
Understand key aspects required in a laboratory testing protocol

Topics covered in this module

Operational considerations in handling and managing specimens
Considerations for what to include in laboratory testing protocols

Note: Refer to below modules for related information

Specimen Collection
[MODULE – Specimen processing, storage, and shipment]
Quality assurance for assays and interpretation of results

Overview of Serological Assays
Handling and Management of Specimens
Developing a Laboratory Assay Protocol
References
Toolkit

Overview of Serological Assays

The most common assays for serosurveys are binding assays that use the basic immunological concept of an antigen (e.g., whole virus or viral proteins) binding to a specific antibody (e.g., IgG) to detect the presence of the antibody in a serum specimen. Such binding assays are commonly referred to as enzyme immunoassays (EIAs) or enzyme-linked immunosorbent assays (ELISAs). An alternative is a functional assay, such as a plaque reduction neutralization (PRN) assay.

	Enzyme immunoassays (EIAs)	Plaque reduction neutralization (PRN) assays
Type of assay	Binding assay	Functional assay.
What is measured	Presence or absence of antibodies based on a pre-specified threshold (qualitative output). Antibody concentration (quantitative or semi-quantitative output).	Neutralizing antibody titer based on serial dilutions.
Assay description	Detect antibodies to one or more proteins using a color-based or fluorescent enzyme tag with an optical density (OD) read out. Some EIAs transform OD units into IgG antibody concentrations (e.g., IU/mL) based on a set of standards with known antibody concentrations. Thresholds are used to distinguish individuals with (“seropositive”) and without (“seronegative”) antibodies.	Measures titers of functional antibodies by mixing the virus of interest with a range of dilutions of the test specimen (serum or plasma) in cell culture to determine the dilution at which the virus is neutralized. International reference standards (e.g., WHO Measles 3rd International Standard) may be run simultaneously to generate IgG antibody concentrations (e.g. IU/mL). Measles neutralizing antibody titers are typically calculated from the dilution of serum that neutralizes the virus in cell culture by 50%. For other viruses a higher threshold can be used (e.g. 90%).
Inference of results	Seropositivity or antibody concentration. Designed to test individuals for exposure to a pathogen or vaccine (typically IgG antibodies) or to identify current or recent infection (typically IgM antibodies).	Estimated titer or concentration of neutralizing antibodies.
Time requirements	Approximately 2-4 hours.	Approximately 5-7 days.
Assay requirements	Requires EIA plate reader. Assay can be run using commercial EIA kits or one developed in experienced laboratories (“in-house” assay). Feasible in most laboratories.	Requires experienced laboratory with capacity to do cell culture.
Pros	Widely used, allowing for comparability to other studies, although not all commercial EIA kits are comparable. Potential for high throughput with automated machines.	“Gold standard” assay for inferring immunity. May be used to evaluate EIA test kits or revise EIA thresholds. May be used on a subset of equivocal or negative samples to validate EIA performance.
Cons	Recommended thresholds for diagnostic EIAs are developed to have high specificity, minimizing false positive results. Potential to misclassify seropositive individuals as seronegative. Potential for variability between manufacturers, labs and lot-to-lot variability.	Difficult to standardize, technically demanding and time-consuming, which makes PRN assays impractical for large-scale serosurveys.

More complex binding assays (e.g., multiplex bead assays (MBAs)) are now available that allow measuring multiple antigens in a specimen by using an array of beads.
Other functional assays include hemagglutination inhibition assay (HAI), indirect immunofluorescence assay (IFA), and serum bactericidal activity assay.
Note: For purposes of convenience for this module, an IgG commercial EIA will be assumed to be the assay of choice for the serosurvey.

Serum specimens

Proper maintenance of specimens, particularly serum specimens, is critical to ensuring the antibody levels in the specimen reflect the individuals’ status at the time of collection. Considerations include:

Specimens with labels containing barcodes can be scanned into a specimen management system in the laboratory. This helps organize specimens for easy access during testing and re-testing.

Minimize freeze-thaw cycles, which may impact antibody levels in the specimen. After processing, serum specimens should be stored at -20 °C or colder and should remain at that temperature prior to testing.
- Ideally specimens should be aliquoted to smaller volumes and only one aliquot thawed at the time of testing.
- If there is residual volume after testing and a potential to retest (Refer to Module on Quality assurance for assays and interpretation of results module(link) for more details), the residual volume should be placed at 4-8 °C until it is determined whether or not the specimen needs to be retested to avoid a second freeze-thaw cycle. But the specimen should not be stored at 4-8 °C longer than 7 days.
Carefully monitor the temperature of the freezer where the specimens are stored. Freezer temperature should be tracked daily using a log sheet and any deviations in temperature beyond a specified range should be discussed with laboratory leads.
Maintain clear documentation on the location of each specimen (e.g., freezer box number and position) so that technicians can easily pull a specimen for testing.

Dried blood spots

Handling of dried blood spot (DBS) specimens:

Serum from dried blood spots (DBS) need to be extracted prior to testing. For spots obtained on filter papers a “punch” is removed from the spot (see figure) then sera extracted from the punch.

Not all assays are designed for use with DBS specimens so it is important to select the assay with the specimen type in mind.
If using DBS as the primary specimen, it is recommended to also collect venous specimens from a subset of participants to use for validating the DBS results and to develop any adjustment factors to improve comparability (e.g., DBS may systematically result in antibody concentrations lower than venous blood specimens).¹
- When testing specimens from the validation subset, paired specimens should be treated similarly and run in the same assay (i.e., on the same EIA plate) to minimize the influence of run-to-run variability.
Serum from the DBS specimens collected on a filter paper or device will need to be extracted prior to testing. Typically, this process occurs just prior to testing. There are examples of elution protocols publicly available.^2,3 However, it is recommended to evaluate and optimize the extraction protocols for your selected assay with specimens of known antibody concentrations prior to testing serosurvey specimens. Key factors to consider in the extraction protocol include the volume of whole blood in the specimen, contents of the buffer, and dilution factors.

Developing a Laboratory Assay Protocol

There are multiple steps and considerations in developing a laboratory assay protocol. Refer to the below tabs for more information about each step.

Developing a laboratory assay protocol. ULOD, upper limit of detection.

Choosing an Assay

Choosing an Assay

Since assays are often validated for specific specimen types, it is important to consider your specimen type when selecting the assay. If using an assay that is not validated for your specimen type, conduct validation experiments prior to testing your specimens.
Review of potential assays and selection of an assay should be performed by study leadership in collaboration with experienced lab personnel and should include persons familiar with performing EIAs and electronic data management.
The assay should meet the following requirements for the serosurvey:
- Address the study objectives (e.g., testing for exposure or correlates of immunity)
- Produce results in the format needed for the planned analyses. Some assays produce only qualitative or semi-quantitative results, whereas others produce quantitative antibody levels.
An assay should be selected based on available laboratory equipment and capacity.
- This should consider not only the costs of running an assay protocol but also the supply-chain, personnel, and equipment needs that come with collecting, processing, and running the volume of specimens required.
- Decisions will also need to be made regarding the sites at which assay will be performed.

Considerations for laboratory equipment and capacity

Will assays be run at one laboratory or several?
Will specimens need to be shipped out of the country to a laboratory?
Will specimens be processed completely prior to being sent to the lab or will additional processing be required at the lab prior to testing?

Depending on the antigen of interest there may be multiple commercial assay kits to choose from.
- Select a kit that has an established performance record and published evidence on sensitivity and specificity.

Considerations for selecting and purchasing commercial assay kits

How the kit will be sourced (e.g., local distributor who sells the kits)?
Will there be the potential for shipment delays or supply constraints?
Can a sufficient number of kits be purchased to ensure the same lot across all specimens for the serosurvey, without kits expiring?
If multiple lots are purchased, what quality assurance step should be implemented to identify potential variability between lots? (see Quality Assurance module).

Establishing a Protocol

Establishing a Laboratory Assay Protocol and Performing the Assay

Once an appropriate assay is selected for the serosurvey, an SOP should be drafted to guide laboratory personnel.

Examples of details to include in the laboratory testing protocol

Proper technique for performing the assays
Necessary supplies and equipment for performing the assays
Use of electronic templates for recording specimen information, processing assay output, and storing and disseminating results
Calibrating the assay based on reference IgG standards to enhance assay quality
Development of internal controls to be run with every assay
Retesting protocols to ensure reliability of the results both at the beginning of the study and throughout the course of the study

Refer to the manufacturer’s recommendations for step-by-step instructions on performing the assay when developing the SOP. Related Toolkit materials include the ELISA Lab Testing One-Pager, and examples of test kit inserts from Euroimmun and Enzygnost.
Estimate the number of test kits required based on the number of specimens and the assumed percentage of specimens to be retested. Ensure there are sufficient test kits and materials available to test all specimens, ideally from the same lot.
Develop a testing schedule and estimate the testing timeline based on the number of assays to be run and the number of technicians who will be testing specimens, which is demonstrated in the Lab WorkPlan.
It is important that roles and responsibilities of lab personnel are well established to minimize systematic errors (misclassification, measurement, etc.) that bias the assay results.
- All lab personnel conducting the assay should be fully trained on testing procedures prior to testing specimens.
- The number of technicians testing specimens should be limited to minimize impact of variability between technicians.
An example assay SOP using a commercially available rubella IgG EIA is presented here, EIA Standard Operating Procedure, as an example of how to run a serological assay.

Recording Procedures

Recording Procedures and Data Storage for Assays

Ensuring the testing results are properly documented and linked to the correct participant is critical to the quality of a serosurvey.
A standardized template for reporting results should be developed prior to testing specimens. Ideally, this template will be in electronic format and developed in consultation with the data management team and statisticians who will be compiling and analyzing results. The template must be consistently used for all assay runs to prevent data entry errors (see details below).

Example of details to include in the laboratory assay data management template

Assay run details: Date, initials or staff ID of the technician who performed the assay, plate number, and lot number (for commercially available kits)
Expected and observed results for calibrators and control (positive/negative) specimens
Participant ID
Raw results (qualitative or quantitative, such as optical density values)

The laboratory, data management team, and statisticians should also agree upon a standard filename structure for each set of results, a method for sharing results (e.g., via email; uploaded to a shared folder on the cloud), and a method for backing up data.
Plate maps are a useful way to organize the results and ensure the raw output is linked to the correct participant ID (Figure 1). Use the tools: EIA template, blank and EIA template, example.
Despite efforts to minimize data entry errors (e.g., using a standardized template and plate maps), the results should be reviewed for potential errors (e.g., confirming that the specimen IDs from the lab results correspond to IDs with a specimen collected based on the field data).

The top map indicates what sample was in each well (i.e., where the calibrator, controls, and specimens are located). The bottom map is where the technician pastes the raw results from the assay run (e.g., OD values).

Handling Equivocal Specimens

Handling Equivocal Specimens

Since the classification of an equivocal specimen is indeterminate, it is strongly recommended that any equivocal specimens be re-tested on either the same assay or a different assay (e.g., PRN) to determine the final result that will be used in analyses.

What does an equivocal mean?

Unlike a positive result (antibody detected) or a negative result (no antibody detected), an equivocal result is indeterminate and no classification or categorization can be made about the presence or absence of antibodies or antigens.

An approach to dealing with equivocal results should be established prior to beginning the serosurvey. The approach should consider the following:

The number of times the specimen should be re-tested (e.g., run in duplicate on a second EIA plate).
Handling of specimens that remain equivocal upon retest (i.e., if any further testing is required using a different assay or if these specimens will be classified as positive or negative).
Refer to Module on Quality assurance for assays and interpretation of results module(link) for more details on interpreting results from equivocal specimens

Use this tool, Example Protocol for Assay Retesting.

Handling Specimens Above the Upper Limit of Detection

Handling samples above the upper limit of detection

Every assay will have a lower and upper limit of detection (for commercial assays refer to the kit insert for details). These are typically based on the highest and lowest standard curve points. Estimates outside this range are qualitatively correct (i.e., seropositive or negative) but the quantitative value is not valid and should not be used in quantitative analyses.

To obtain a valid quantitative result for these specimens, it is recommended to dilute the original specimen and re-test, then multiply the calculated result by the dilution factor to obtain the final quantitative result for the specimen.

Use this tool, Example Protocol for Assay Retesting.

Example of a standard curve with 4 calibrators (50, 250, 1000, 5000 mIU/ml). The red asterisk represents a specimen with an optical density (OD) reading that is above the upper limit of detection. In this scenario the specimen would be diluted and retested in order to obtain an OD value within the standard curve.

References

General

World Health Organization Guidelines on the Use of Serosurveys in Support of Measles and Rubella Elimination, Chapter 4 Laboratory Methods

World Health Organization Manual for the Laboratory-based Surveillance of Measles, Rubella, and Congenital Rubella Syndrome

Chapter 9. Laboratory testing for determination of population immune status

Measles EIA Test Kit Inserts (Enzygnost and Euroimmun). Note: Specific details may vary from lot to lot.

International Reference Standards

Dried blood spots

Riddell, Michaela A, Graham B Byrnes, Jennie A Leydon, and Heath A Kelly. 2003. “Dried Venous Blood Samples for the Detection and Quantification of Measles IgG Using a Commercial Enzyme Immunoassay.” Bulletin of the World Health Organization 81 (10): 701–7. http://www.ncbi.nlm.nih.gov/pubmed/14758429.
Mei, Joanne V., J. Richard Alexander, Barbara W. Adam, and W. Harry Hannon. 2001. “Use of Filter Paper for the Collection and Analysis of Human Whole Blood Specimens.” The Journal of Nutrition 131 (5): 1631S-1636S. https://doi.org/10.1093/jn/131.5.1631S.
Mercader, Sara, David Featherstone, and William J. Bellini. 2006. “Comparison of Available Methods to Elute Serum from Dried Blood Spot Samples for Measles Serology.” Journal of Virological Methods 137 (1): 140–49.https://doi.org/10.1016/j.jviromet.2006.06.018.

Toolkit

Section	Toolkit material	Context
Running the assay	ELISA Lab one-pager	Summary of test kit instructions used in the laboratory for Euroimmun ELISA using serum.
Running the assay	Toolkit Lab Work Plan	Example workplan for a serosurvey testing for measles and rubella
Running the assay	Toolkit-EIA Standard Operating Procedure	Serosurvey with DBS specimen, testing for measles and rubella using Siemens Enzygnost ELISA (including elution)
Recording Procedures and Data Storage for Assays	Toolkit-Data Procedure EIA Assay Template Blank	Template for use with Euroimmun measles or rubella EIA kits
Recording Procedures and Data Storage for Assays	Toolkit-Data Procedure EIA Assay Template Example	Template for use with Euroimmun measles or rubella EIA kit