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STIMULI TO THE REVISION PROCESS

Stimuli articles do not necessarily reflect the policies
of the USPC or the USP Council of Experts
Assays Using Test Kits: Applications in a Quality Control Environment
20052010 Validation of Research Test Kits Advisory Panel,a Fouad Atoufb
ABSTRACT Commercial test kits and reagents that are developed and labeled for Research Use Only by the
supplier can be used for quality control lot release, stability testing, raw and ancillary material testing, in-process
testing of biological products, and related activities provided that proper risk assessment and validation are
performed. This article explores some of the qualification and validation aspects raised by the use of these types
of test kits and reagents, but also by the use of regulated test kits that are used for a purpose other than
approved use. This Stimuli article concludes that concerns about the use of test kits and reagents in a regulated
environment can be managed if certain principles are followed and if critical items are addressed. The article is
presented here to elicit comments that can assist USP in publishing a future chapter that will address
qualification and validation considerations associated with the adaptation of test kits that will be used in a
regulated environment. Please send suggestions to Fouad Atouf, PhD, fa@usp.org.
INTRODUCTION AND SCOPE
Commercial test kits and reagents that are developed and labeled for Research Use Only (RUO) by the supplier
can be used for quality control (QC) lot release, stability testing, raw and ancillary material testing, in-process
testing of biological products, and related activities provided that proper risk assessment and validation are
performed. In vitro diagnostic (IVD) kits, approved under section 510(k) of the Federal Food, Drug, and Cosmetic
Act, sometimes are used in a QC environment for applications not included in their registration. Risk analysis
strategies will determine the level of qualification and validation needed for the use of such kits outside the scope
of their registration. In this article, reference to test kits means RUO and IVD kits.
Test kits frequently are used in the early stages of development of a licensed product, as well as during testing of
licensed drug products. Issues associated with the use of test kits and reagents in a regulated environment can
be managed if certain principles are followed and if critical items are addressed. This article explores these
concerns and provides recommendations for the selection, qualification, and validation principles to consider
when using a test kit in a QC environment. USP solicits feedback and comments from the public so that a
chapter on the topic can be developed. The intent of the future chapter is to discuss systematic approaches for
the validation of kits used for QC release.
The Table lists examples of test kits and reagents that, with proper risk assessment and validation, can be used
in a regulated environment. These include but are not limited to kits used for assaying nucleic acids, proteins,
polysaccharides, and microorganisms. Binding methods include immunoassays, nucleic acid binding, or
proteinprotein interactions. Most commercial antibody-based assay kits [e.g., radioimmunoassays (RIA),
enzyme-linked immunosorbent assays (ELISA), and luminescent and fluorescence-based immunoassay kits]
are in this category. Commercial nucleic acidbased test kits (NAT) that contain proprietary primers or probes
(see Nucleic Acidbased Techniques 1125 , 1126 , 1127 , 1129 , and 1130 ) often are used
both in early-stage development as well as in testing of licensed drug products. Examples of commercial
reagents include assay reference materials used to calculate the quantitative response of a test sample based
on signal output and comparison to controls. Many test kits are based on enzyme reactions used in sample
preparation or are required to calibrate analytical instrumentation or to conduct systems suitability for an
analytical procedure. The intent of using test kits is to measure an analyte in a manufactured medicine and also
other associated materials such as related compounds or impurities. Some applications are listed in the
following Table.
Table. Examples of Test Kits and Reagents

Type of Kit or Reagent

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Typical Applications
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Immunoassay (RIA, ELISA, etc.)

NAT
Enzyme Assay
Dye/Chemical Kit

Standardization

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Residual host cell proteins

Residual Protein A 131 (chapter in preparation)
Residual bovine serum albumin
ELISA kits for process residuals, e.g., insulin
Flow Cytometry 1027
Virology Test Methods 1237
Transgene expression for a gene therapy product
Residual host cell DNA
Mycoplasma testing
Chromogenic assay for endotoxin determination
Endonuclease digestion (sample preparation)
Protein quantitation assays, e.g., BCA, Bradford
DNA extraction kit
RNA extraction kit
Beads for FACSa applications
Protein molecular weight standards for SDS-PAGEb

a FACS = fluorescence-activated cell sorter.

b SDS-PAGE = sodium dodecyl sulfatepolyacrylamide gel electrophoresis.
CONSIDERATIONS AND ISSUES
All manufactured products display lot-to-lot variability in measured attributes. As with a manufactured medicine,
this variability has several components in test kits used for QC purposes. For example, the analytical procedure
in the test kit will necessarily exhibit variation in results, as well as content of the substance to be measured in
the supplied assay reference materials. Reagents themselves used in execution of the procedure of the test kit
may also add variability. The contribution of these factors to this variability should be considered in the course of
validation of a test kit to a particular use. The validation begins with the selection of the assay kit or reagent and
its supplier. The selection of a specific test kit and vendor will contribute greatly to consistency within and across
assays; see Use of a Test Kit vs. In-House Assay Development: Risk Assessment and Mitigation Strategies
below.
A reagent may exist as a formulation supplied with the test kit. The reagent thus may consist of several
components that can change over time. Subtle changes in reagent formulation, such as buffer, salt, and/or
excipients, can significantly influence the reagents' stability and performance. Thus, the test kit may not give
reproducible results after a series of assays because of unexpected degradation of reagent components.
Changes in reagent components also may significantly alter assay results if a test kit is used for purposes other
than its intended application (e.g., using a kit to determine the concentration of a target protein in tissue culture
media when the kit was developed to determine the concentration of the target protein in human plasma).
Changes in the reagent's formulation may be incompatible with the sample matrix and thus may influence assay
results. Typically, limited information is available regarding stability, purity, or formulation of reagents in
commercial kits. Such uncertainty complicates proper assessment of a kit or reagent. Unless a manufacturer
has established a supply agreement with the vendor obligating the latter to disclose changes made to reagents
or kits, the vendor has no obligation to disclose if changes were made or when.
Supply-chain risks also can contribute to the lot-to-lot variability of reagents in kits and must be considered
before a manufacturer selects a test kit or reagent. Factors such as single-source reagents or test kits, the
components of the test kit, and the manufacturer's stability, size, and location can affect availability.
Analysts also should remember that some test kits have unique or proprietary reagents that cannot be easily (or,
in some cases, legally) reproduced or procured individually.
USE OF A TEST KIT VS. IN-HOUSE ASSAY DEVELOPMENT: RISK ASSESSMENT AND MITIGATION
STRATEGIES
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During the early stages of assay development the two primary options are to purchase test kits from a vendor or
to develop an assay in-house. Manufacturers of therapeutic products (end users) should perform a risk
assessment to evaluate the advantages of developing and validating an in-house assay relative to validating
purchased test kits. Assays that use test kits require validation of the test kit and are relatively straightforward. If
the kit is designated RUO, then more extensive and comprehensive validation of the entire assay is required. The
basic elements for analytical validation are presented in Validation of Compendial Procedures 1225 and in
guidance documents from the International Conference on Harmonization (ICH; see Q2R1). If the kit application
covers an assay or test for which specific compendial guidance or a referee test exists (e.g., Bacterial
Endotoxins Test 85 ), the assay-specific guidance must be considered in the validation.
The risk assessment should consider the applications and requirements of the specific assay. If the assay is
used for the evaluation of a pharmaceutical product, the product's stage of development must be considered as
well. The closer a product is to submission for licensure, the greater the impact of a failed assay validation.
Critical assays must be validated based on their use. Development and characterization assays may or may not
require validation, depending on their use at the time of licensure.
The assessment also should evaluate the availability of facilities, equipment, and expertise to develop an inhouse assay or to adopt a kit. The frequency of testing also should be considered. A test performed daily on a
large number of test samples has a different level of risk compared to an assay performed rarely on a limited
number of test samples. Beyond the frequency of testing, it is also important that an assay is not limiting for QC
release. Also consider the cost/benefit ratio between use of a kit and an in-house assay. In-house assay
development requires more internal resources but offers more control and knowledge of assay components.
Variability in the manufacture of reagents in the kit may lead to unacceptable lot-to-lot variability in performance.
Test kits usually are assigned expiration dates that also can be assigned to the individual kit components. The
kit performance may degrade over time, and stability should be accounted for in the expiry date. This stability
ideally is determined when manufacturers assess test kit performance over time and multiple lots. When
assessing the performance of multiple lots of a test kit, users should understand how the vendor defines a lot. A
lot may merely reflect different dates of manufacture with identical lots of bulk reagents, or it may reflect
manufacture with completely different lots of bulk reagents. Test kits may contain reagents with unknown
composition and/or proprietary components. Mitigating the risk associated with such test kits depends on the
supplier's willingness to discuss the details of the reagents or, at a minimum, to provide advance notice when
components change. Detailed information about the test kit's reagents may be critical for troubleshooting or
validation.
A comprehensive assessment includes evaluating the availability of the kit or reagent. If the test kit or reagent is
based on a single source or if the reagent supply is finite (e.g., a polyclonal antibody), manufacturers face more
risk compared to their use of a test kit or reagent that is widely available from multiple suppliers. This supplychain risk is best mitigated during qualification and validation by evaluating test kits or reagents from several
vendors or contract testing laboratories. An alternative supply source should not be confused with an alternative
distributor that supplies the identical reagent from the same source (e.g., a monoclonal antibody that is produced
by one source but distributed by several vendors). If test kits from multiple vendors are used in testing, then each
test kit must be validated for this use. If a change to a test kit leads to the use of an alternative test kit, the latter
must be compared to the former and must be shown to give equivalent results. The assay also should be
validated using the alternative test kit to demonstrate performance for the intended use and to allow use on a
routine basis.
New or relatively small vendors may pose additional risks. A test kit vendor may be purchased by another
company or may move its manufacturing facility to another location. The performance of the test kit may depend
on a limited number of key employees who produce a critical component. The vendor may undergo changes that
could affect production, customer service, and distribution. Any change in the test kit's manufacture may affect
the validation of the assay performed with the kit and may warrant the assay's revalidation.
The physical location of the test kit vendor can affect its ability to consistently supply test kits. If the supplier is
located in a foreign country, changes or delays in international shipping and customs practices may affect kit
availability and stability. If the vendor is located in an area prone to natural disasters such as earthquakes or
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hurricanes, this also may affect the availability of the test kit.
Once a vendor is identified, manufacturers may find it beneficial to establish a supply agreement. Although a
supply agreement can help control the cost of the kit for a specific period of time, its main benefit is to detail
whether production changes are acceptable, how the customer will be notified of changes, and how much time
the end user needs to assess the influence of changes on test kit performance. Under a supply agreement a
manufacturer may purchase large amounts of a single lot that meets carefully established performance
requirements. Although a customer may prefer to purchase large volumes of a single test kit lot to decrease
potential lot-to-lot variability, this strategy does not address stability risks. A supply agreement with a test kit
manufacturer should clearly define important details such as: (1) definition of a lot; (2) scope of QC tests
performed by the vendor; (3) whether a Certificate of Analysis (CoA) is created for each kit lot; (4) information
included in the CoA; and (5) user notification mechanisms and impact mitigation in the event of product changes.
If a single supplier is available for a test kit or a critical kit component such as a fluorescent probe, monoclonal
antibody, or chromatography resin, the manufacturer can mitigate risk by purchasing a sufficient number of test
kits for the projected lifetime of the assay (if this is feasible based on production quantities and confirmed
stability of reagents). This strategy must consider the expiration and the variability of the test kit or reagents. A
test kit also may be licensed from the vendor. The supply agreement might require the sole-source supplier to
assist in transferring the kit's production process to a qualified vendor of the customer's choosing if the primary
supplier decides to stop production. One additional strategy may be to purchase the supplier outright.
Many test kits that are labeled RUO come with a box license that gives the purchaser the freedom to perform the
test for RUO applications. However, the purchase of a RUO kit does not automatically guarantee the
manufacturer the freedom to use the kit for clinical or commercial applications, and obtaining these rights can be
complicated and expensive. Depending on their components, kits developed in-house also may be subject to
intellectual property and license fees. For example, numerous patents have been assigned for the fluorescent
dyes used to label oligonucleotides, as well as the specific configuration of these dyes on the oligonucleotide. To
determine intellectual property implications associated with use of a purchased test kit, the manufacturer should
read the package inserts and any description of patents or limited uses associated with the kit. Sometimes the
vendor posts this information on the product Web site. The customer can also ask the vendor directly. Legal
counsel should be sought to fully assess all intellectual property implications associated with the use of a
chosen kit.
VALIDATION OF ASSAYS THAT INCORPORATE TEST KITS
Validation ApproachesGeneral Considerations
The goal of assay validation is to demonstrate and document that the assay is suitable for its intended use. To
achieve this goal, clearly delineate the assay's performance requirements and relate them to the intended
application of the assay before validation. The quality of results obtained with test kits is variable and must be
evaluated in relation to the assay's performance requirements. Specific validation strategies depend on the nature
of the kit (quantitative vs. qualitative data), how the kit is employed in the assay, and how the assay results are
used, e.g., product lot release, raw material identification, and other uses.
Test kits can be incorporated into an assay in two basic ways: the test kit can be used directly (or with some
modifications) as the entire assay, or it can be used in a step (or series of steps) within the context of a more
complex assay, e.g., a dye or chemical kit or a standardization kit such as those listed in the Table. This
distinction can be important in guiding the approach to validation. If a test kit covers the entire assay procedure
and is the source of the reported assay results, validation of the assay should be conducted as described in
general chapter

1225

and in applicable ICH guidance documents. If the scope of the assay is to determine

the relative potency, then validation should be conducted according to Biological Assay Validation 1033 .
During assay validation, give special attention to the ruggedness of data generated from test kit reagents and
standards. If test-specific compendial guidance or a referee procedure exists, consider this in the validation.
If a test kit is used within the context of a larger assay (i.e., the data generated from the kit are used to calculate
the assay results but are not reported directly), the assay step using the kit may not require independent
validation. Instead, the kit should be treated as a critical reagent for the larger assay, and different lots of the kit
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should be included in the evaluation of robustness and ruggedness of the overall assay.
Validation of assays using test kits usually follows the general concept of assay validation as described in
1225 . Because end users often have no access to the kit's development history, the major challenge in
validating a kit-based assay is designing meaningful experiments. Before designing the validation study the end
user should analyze all available historical data to identify potential critical factors of the assay, including
procedural parameters, reagents, and lot-to-lot variation.
Test Kit Definition and Configuration
Before validating an assay that uses a kit, define the configuration of the kit. If test kits will be received and used
as complete sets, then each kit lot can be considered a single reagent lot. If kits will be modified after they are
received (e.g., by substitution of one kit component for an in-house component or exclusion of a kit control in the
procedure), then the kit configuration should be further defined. If kits will be disassembled and the reagents
used in an in-house procedure, the reagents can be treated individually. In any case, the assay should be
sufficiently well characterized so that critical assay components are identified before validation.
The definition of the kit and its configuration for use in the assay dictate how the robustness and ruggedness
studies will be designed in terms of reagent lots. The definition also has implications regarding the assignment of
reagent expiration dates (individually assigned to components vs. assigned together as a single kit). The goal is
to ensure that all relevant permutations of reagent combinations are addressed adequately.
Evaluation of Test Kits or Test Kit Reagents in the Determination of Assay Ruggedness
Ruggedness is defined in 1225 as the degree of reproducibility of test results obtained by the analysis of the
same samples under a variety of normal test conditions, such as different analysts, different instruments,
different lots of reagents, different elapsed assay times, different assay temperatures, different days, etc. Test
kits or kit components should be evaluated to identify critical reagents. Ruggedness is the reported variation of
test results across different conditions that are known to influence the reportable value. Statistically designed
experiments provide an efficient, cost-effective way to evaluate the effects of changes in different parameters
such as reagent lots, alone and in combination with other parameters, on test results. Many software packages
perform Design of Experiments (DoE) methodology, but end users must exercise care to apply these
appropriately.
Applications of Design of Experiments
Statistical DoE is an efficient procedure to investigate systematically the effects of many variables on an assay.
It has the advantage of being able to determine many interactions among variables under study. One application
of DoE is to generate data to support storage conditions and assignment of expiration dates to test kit reagents.
This use is important because the quality of test kit reagents can influence the assay's specificity, accuracy,
and precision.
The variability associated with a reagent in a test kit depends on the nature of the reagent, e.g., antibody vs.
chemical, the degree of control applied to its production, and how labile the reagent is under different storage
conditions. Storage conditions include the concentration, formulation (e.g., dry or reconstituted), temperature,
the storage vessel, exposure to light, humidity, and others. End users should understand the factors that affect
the quality of the reagent, including its quality on receipt from the supplier (to evaluate lot-to-lot variation) and how
different handling and storage procedures in the laboratory can affect the reagent's performance in the assay.
Reagent-handling considerations are particularly important for reagents that may be stored as a concentrated
stock under one storage condition and then diluted and aliquoted as a working stock for storage under another
condition. In some cases reagent kits may come with instructions for use and assigned expiration dates. In other
cases, the supplier simply provides the lot number for the reagent. Information about the age of the reagent and
the manner in which the reagent is prepared and stored must be evaluated experimentally to support handling
and expiration assignments. If one storage condition and one expiration date will be assigned to the entire kit,
then the kit can be treated as a single reagent. However, information regarding the manufacture and age of the
reagents used to assemble the kits must be evaluated to ensure that studies of test kit lot variation include a full
range of the ages of component reagents.
Robustness studies should be performed to demonstrate that at the time limits proposed and under the specified
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storage conditions the reagent does not influence the assay parameters of interest (e.g., specificity, accuracy,
and precision). If the outcome demonstrates that the reagent's age can influence assay accuracy or precision,
the impact on the reportable value must be quantified and demonstrated to be acceptable for the intended use.
If a number of reagents will be evaluated, extreme conditions (e.g., newly prepared vs. expired reagents) can be
tested initially. If use of reagents at extreme conditions indicates a strong influence on assay outcome, then a
narrower range of conditions can be re-evaluated. The user should conduct these experiments early in
development and should evaluate the effects on assay readout if the strategy for testing and calculating a
reportable value has not yet been determined.
If a test kit has many reagents, fully evaluating all possible permutations and combinations may be challenging,
even with DoE. An understanding of the assay mechanisms, the role of each reagent in the assay, and its known
or predicted stability in the assay application can help guide the selection of reasonable test conditions.
Although convenience should be a consideration in determining reagent preparation, analysts should exercise
caution because certain steps may contribute to poor reagent stability and performance in the assay.
Retention of Reagents, Assay Controls, and Other Test Kit Components
To enable investigation of changes in assay performance compared to validation results, end users should
archive assay controls and critical components of the test kits. This information serves as a baseline for future
comparison, provided the stability of controls and components is sufficient. Assay controls may be provided in
the test kit or can be prepared in house in a suitable sample matrix. Critical components of the test kit should be
retained. Alternatively, the entire test kit can be retained. If the entire test kit is retained, tracking the lot or batch
number of each critical component, in addition to the lot number of the test kit itself, may be helpful for future
comparisons. Both the assay controls and the kits, or their critical components, should be stored under
conditions that maximize stability. These conditions may be different from standard storage conditions.
Adequate storage ensures preservation of the test kit components' activity, which is important to ensure accurate
comparisons. In order to avoid simultaneous expiration, the expected expiry dates for the assay controls and the
test kit components should be distinct. In any comparison study, the assay control and representative test
article(s) should be tested on both the retained kit and the newly acquired kit. The extent of the comparison
study, including the number of assays, should be determined based on the intended use and the assay precision
profile. If the influence of the test kit variability was underestimated when the assay was validated, revalidation
may be required, and the impact of any changes should be evaluated.
Test Kit or Component Changes or Discontinuation after Assay Validation
When a test kit is discontinued, the end user must choose whether to identify suitable alternatives or to develop
an in-house assay. In either case, the assay should be validated using the new test kit unless the difference
between the old and new kits can be clearly identified and shown to have minimal impact on assay performance,
thus justifying a reduced validation effort. When key components of a kit are discontinued and replaced by the
vendor or by the end user, the nature of the components and their functions in the assay define the work needed
to support their inclusion.
Many test kits include components that contain undisclosed ingredients, and vendors are not always obligated to
reveal changes in the ingredient(s) or the production process for these components so users may not be
informed. In practice, a user may first notice that the performance of a kit has changed. If details of the change
are disclosed and the difference in assay performance is relatively small or modification of the assay protocol
corrects the difference, a limited validation study may be sufficient to demonstrate equivalence. For instance,
changing the blocker in an immunoassay kit may have demonstrably minimal impact on assay performance. If
assay performance has significantly changed but remains suitable for the intended use, then analysts should
consider a full validation effort. When the change renders the assay no longer suitable for the intended use or the
desired range, manufacturers must conduct extensive work to identify suitable modifications, including possible
replacement of critical reagents or, perhaps, replacement of the entire kit.
Examples
This section recapitulates and shows a specific application of the general concerns and approaches discussed
earlier. Each analyst's challenge may be unique, but the purpose of this section is to offer a specific application
of the general principles, namely validation of an ELISA kit. When an ELISA kit is used to quantify an analyte,
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whether it is a drug substance or an impurity, the assay must be validated for its specificity, accuracy, precision,
limits of quantitation, linearity, range, and robustness, and the specific properties that must be validated depend
on the kit's use (see Table 2 in 1225 ).
The initial activity of the validation is to formally demonstrate the stability of the test kit's critical reagents. The
test kit's critical reagents have been identified before validation. In the case of an ELISA kit these include the
antibody reagents, the assay standard(s) or calibrator(s), and any included control reagent(s). One or more of the
antibody reagents may be modified with a label, e.g., ruthenylation or biotinylation, or may be coupled to an
enzyme, e.g., horseradish peroxidase or alkaline phosphatase. The critical reagents are characterized, typically
with regard to concentration, purity including lack of aggregated forms, and activity or function, e.g., binding
ability or enzymatic activity. Labeled antibody reagent(s) also are characterized to assess the level of label
incorporation. Characterization of these features is performed over the course of the reagent stability study in
conjunction with evaluation of test kit performance and with more than one lot of critical reagent. Because
antibodies typically are stable when stored frozen, i.e., at 20 or lower, the focus of critical reagent stability
studies is usually on the assay standard(s) or calibrator(s) and on the modified antibody reagents. If reagents will
be stored frozen until time of use, a formal reagent study might include evaluation of the reagent after 3 freezethaw cycles, followed by short-term storage at 2 10 , typically for 1 to 10 days. In this case the thaw
conditions should simulate those the laboratory will use routinely.
For specificity, the assay must demonstrate a high degree of selectivity toward the target analyte in a relevant
sample matrix. This usually is studied in two respects: 1) to show that this kit provides a positive signal only
when the target analyte is present (i.e., establish the false positive rate) and that the signal is proportional to the
amount of target analyte and 2) to prove that the kit is capable of distinguishing the target analyte from similar
compounds or proteins in a relevant sample matrix (i.e., establish the false negative rate). The latter study may
be omitted from the validation study for an ELISA developed in-house because the immunological cross-reactivity
often is extensively studied and documented during antibody generation. Although manufacturers usually screen
for such cross-reactivity during selection of an ELISA kit, good practice is to quantitatively re-evaluate crossreactivity and document the results for the validation study. For an ELISA kit used to quantify a biological drug,
the focus of the specificity study may be to demonstrate that the ELISA kit will not detect other biological drugs
that will be handled in the same laboratory. The specificity test would include samples spiked with 50- to 1000fold greater concentrations of the other, nonrelevant, biological drugs.
The validation study to establish the assay kit quantitation rangeincluding the lower limit of quantitation, lower
limit of detection, and upper limit of quantitationusually involves performing the assay using multiple replicates
of the assay standard(s) or calibrator(s), including the zero or negative calibrator per ELISA plate and using
multiple ELISA plates and ideally is performed over a number of days and by more than one analyst. A typical
design might include 6 replicates of each assay calibrator per ELISA plate, 6 or 12 zero calibrators per plate, and
6 plates. The 6 by 6 setup then is performed by 3 analysts over 3 days (3 by 3). The confidence interval for the
lower and upper range of the assay then is based on the number of replicates for each calibrator. In an ELISA the
lower limit of detection generally is calculated based on the standard deviation of the zero calibrator. The lower
and upper limits of quantitation usually are calculated based on the standard deviation of the relevant calibrator
signal and the slope of the fitted calibration curve (see 1225 ), but a confidence limit approach also can be
used. The lower and/or upper limit of quantitation should be confirmed further with samples or blank sample
matrix spiked with the analyte.
The validation study designs used to establish the accuracy (recovery) and precision (repeatability and
intermediate precision, plus reproducibility if multiple laboratories are involved) for ELISA kits are similar to those
for other quantitative assays. The same set of samples with known amounts of analyte are tested in multiple
replicates (usually 6) to determine accuracy (percentage of observed values against the expected ones) and
repeatability (variation of observed values within an assay) and testing the same samples in 6 independent
assays for accuracy and intermediate precision (variation of observed values across multiple independent assays
conducted, e.g., on different days, on different equipment, and/or by different analysts). One also can calculate
the limits of quantitation and/or detection, linearity, and quantitation range from the standard curves used in the
accuracy and precision experiments. System suitability samples, which usually are one or more controls with
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known amounts of analyte within the linear range, should be included in every assay. When results fall within an
acceptable range, they support assay validity. These system suitability samples must be included in the
validation study to ensure that every assay is valid before results are used to conclude the validation study.
When lot-to-lot variation is negligible or a stringent qualification plan is in place to ensure the performance of new
lots is similar to that of existing lots, multiple lots of the kit usually are tested only for robustness in the
validation study. When lot-to-lot variation contributes to a detectable but acceptable bias, using multiple lots to
determine key assay performance characteristics (accuracy, precision, limits of quantitation/detection, linearity,
and range) and reporting averaged values provides a more accurate picture of assay performance. For instance,
when some lots of an ELISA kit are not as sensitive or precise as other lots in detecting the analyte at a
concentration near the expected limit of detection or quantitation, the limit of detection or quantitation may
require reporting based on data from the less sensitive lots.
As described in Application of DoE (above), statistical considerations and DoE should be applied to robustness
experiments. Understanding how a vendor defined and/or qualified a kit aids in the design of a study to evaluate
the tolerance of the assay to potentially small but deliberate deviations. For example, if a vendor specifically
pairs a lot of coating antibody and a lot of detecting antibody in an ELISA kit, each pair of the coating and
detection antibodies should be used together. Lack of detailed knowledge about proprietary reagents represents
another challenge in choosing meaningful ranges during good robustness studies. A proprietary reagent may
require exposure to different conditions to generate an appropriately stressed reagent. For instance, if a coated
ELISA plate in the kit is sealed under partial vacuum and stored at 2 8 C, the performance of this ELISA
should be evaluated after the plate has been stored at 2 8 C with vacuum for slightly beyond the shelf life and
after it is stored at room temperature, with and without vacuum, for a time that is relevant to the assay procedure.
In order to fully test the assay's tolerance to potential deviations, one should also take into account potential
interactions among critical factors demonstrated by prior knowledge of similar assays and/or data analysis. For
an ELISA kit these critical factors could be reagent stability and incubation conditions such as time and
temperature for antigen and antibody binding. Assay robustness related to these factors is best evaluated by
DoE.
SUMMARY
Regulatory guidelines for the validation of test kits have not been established. Current industry practices rely
heavily on establishing a risk assessment strategy to mitigate risks associated with use of kits vs. assays
developed in house, but also to control the supply chains for this type of reagents. A future USP chapter on
validation of test kits should provide approaches to determine the level of validation needed when using a test kit
and also a stepwise approach for validation that takes into account current regulatory and compendial
approaches. Readers are invited to submit comments about the content of this article and also about the scope
and goal of a potential chapter on this topic. Recommendations for examples of test kits that should be
discussed in a future chapter are strongly encouraged. Please send suggestions to Fouad Atouf, PhD,
fa@usp.org
a USP 20052010 Validation of Research Test Kits Advisory Panel: Chairperson: Beth Hutchins, PhD; Members: Maria
A. Croyle, PhD; Lori Rinckel, PhD; Nancy Sajjadi, MS; Shian-Jiun Shih, PhD; William E. Tente, MS; Wesley E. Workman,
PhD; Seeven Vydelingum, PhD.
b Correspondence should be addressed to Fouad Atouf, PhD, Senior Scientific Liaison, US Pharmacopeial Convention,
12601 Twinbrook Parkway, Rockville, MD 20852-1790; tel. 301.816.8365; e-mail fa@usp.org.

www.usppf.com/pf/pub/index.html

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