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Lateral Flow Immunoassays

Before embarking on the development of a lateral flow assay, it is best to take the time to consider the critical requirements of the assay you are going to produce. Assays are designed based on these requirements, and are commonly referred to as “Design Requirements” or “Design Inputs”. Design Requirements typically describe the function, performance, usability, and safety requirements of the assay in relation to its intended use. Ultimately, these requirements will provide a framework for all future development work. A typical assay development pathway follows this figure:

In this module we will be focusing on the first section, “Establish General Design Requirements”, and the role it will play with the “Assay Optimization” step.

What Are Assay Design Requirements?

Assay design requirements are claims that are to be comprehensive, unambiguous, non-conflicting, and measurable. Developing design requirements requires a comprehensive understanding of what the assay purpose is. The purpose will hinge greatly on whether the assay is being developed for commercial or academic use as there will be a significant difference in requirements. For the development of a commercial test, it is recommended that a Customer Needs Report is first drafted. This document will detail the functional needs, handling needs, as well as the diagnostic requirements of the test after feedback from the target market. This type of assay will typically have to pass scrutiny from a review board, like the FDA, so having detailed requirements with measurable outputs will be critical. A test developed for academic use will have requirements that are far less stringent. The assay design requirement for this type of test will be more general. In both cases, the assay is being designed for a specific need, and this need is to be translated into design requirements. These requirements will need to cover what the assay will deliver but does not necessarily have to state how to deliver them. Once the assay is developed, the performance of the assay will be measured against these requirements. This is accomplished through verification studies, design specifications, assay standardization, as well as other methods.

General Assay Design Requirements

Before any feasibility tasks begin, the first thing that needs to be done is to develop a comprehensive list of the general requirements for the assay based on the intended use. These requirements are very basic, and will serve to provide guidance during the development process. Requirements are written by listing out specific requirements (e.g. Assay Run Time), followed by the description/claim (e.g. the test will produce a reliable result for the concentration of hCG in < 25 minutes). When nanoComposix helps develop Design Requirements with our customers, we begin with the following:

Design Characteristics & Functionality

  • Definition: Requirements that specify what the product is expected to do. This will be a bulk of the requirements.
  • Example Requirements: Analysis method, target analyte, test format, assay run time, sensitivity limit, specificity limit, analytical range, precision, interference, limit-of-detection, clinical performance, etc.

Physical Properties

  • Definition: Physical properties that affect the design. These can be associated with the test strip themselves, as well as the cartridge the strip will be placed into. These can be particularly important if you have a specific reader or analyzer your test must work on.
  • Example Requirements: Cartridge dimensions, cartridge features (i.e. conjugate and sample port), overfill volume, reader requirements, etc. 

Handling Requirements

  • Definition: Requirements on the environment in which the test can be used, where the test is to be stored prior to use, and how long the test can be stored.
  • Example Requirements: Operating temperature/humidity (i.e. humidity does terrible things to protein stability), storage temperature, storage duration, equilibration time, etc.

Sample Requirements

  • Definition: All things relating to the sample collection, preparation, transport, and storage
  • Example Requirements: Sample type, sample storage temperature, sample treatment (i.e. centrifuged)

There are many more requirement categories not listed above which should also be given some consideration (e.g. Packaging, Shipping, Labeling, Regulatory, and Safety). Some of these sections may not be relevant to particular projects, and do not need to be included at the onset of the project. It is never too early to give thought to all these sections though. You may find that without consideration early on, some requirements may cause substantial delays later if they need to be added.

Assay Optimization & Feasibility

Once design requirements have been defined, you will now have a more characterized development plan with measurable milestones. The next step will be to begin the optimization of an assay to meet these requirements. Additional NCXU modules have been written to help you through this early development phase. During the optimization process, you may find that new requirements need to be added while others will need to be adjusted or removed all together. This is very typical of the process, and important to developing the right requirements moving forward. The list of design requirements should be a living document, that is continuously evolving along with the optimization. Optimization will eventually lead you to a point where you can effectively demonstrate that you have developed an assay that can meet all the requirements needed for a successful product.