By April 19, 2019 July 1st, 2019 Articles

When beginning any assay development program, you must consider a number of factors before you are on the road to a finished product. Between reagent selections, regulatory requirements, controls, sample collection, cassette design, and end user requirements, details can slip through the cracks. Even the smallest missed detail can halt progress and delay your development timeline. At DCN Dx, we have helped many clients prevent problems with assay developments in order to make assay development as efficient as possible. Whether you are just getting started and want to avoid delays or you are experiencing problems in your current program, our team of scientists and engineers can help you take the right steps. Here we discuss three common pitfalls during each stage of development and the simple steps to avoid them.

Common Assay Development Issue #1: Reagent Selection

Selecting suboptimal reagents for your assay is a pitfall with a long road to recovery. During the Feasibility stage, you must screen and optimize reagents and materials in order to determine the basic performance of the system and decide whether the assay is feasible. This will set the groundwork for the future direction of development. To ensure you choose the right reagents, keep in mind these tips.

Each assay is unique

Antibodies that performed well in other platforms, such as an ELISA, may not translate to lateral flow. Incubation time, washing steps, and sample dilutions can result in significantly different performance than other platforms. Materials and buffers that worked in similar assays may not be optimal for a new assay. To achieve full performance of your system, always screen antibodies, materials, and buffers for each individual assay.

Think long-term

Consider any limitations that will affect your assay in the long-term, such as reagent cost and availability in large quantities for the duration of manufacturing. If cross-reactivity is an issue, test the antibodies with the potential cross reactants early in development. Exchanging even a simple reagent down the road can require extensive re-optimization, adding time and cost to your development program. It is also important to keep the end user in mind. If you use fluorescent particles for maximum sensitivity, the end user will need a fluorescent-based reader. If you need to collect a set volume of sample such as saliva, you will need an efficient and user-friendly method for sample collection. Consider the end user and design the device for usability. Re-evaluation of critical reagents and materials at a later point in development is a significant detour.

Common Assay Development Issue #2: Samples, Samples, Samples

A lack of real samples or waiting too long to test real samples can halt progress and cause a serious setback in development. To avoid this often-underestimated setback, follow these general tips.

Source samples

Before you get started, identify where you will source your samples, how much they will cost, and what quantities and volumes you will require. The difficulty of getting real samples varies significantly based on what you are detecting. There are many sources for obtaining endogenous analytes in normal state samples (i.e., non-disease samples), as well as disease state samples. Certain sample types may be more difficult to obtain than others (e.g., stool vs. plasma), while others may require extremely fresh collection for testing (e.g., saliva). It is critical to make sure you have a verification method for the desired analyte, such as an ELISA or outside lab reference.

Transition to real samples early

Postponing the transition to real samples is another common hindrance to efficient assay development. Tackle this hurdle earlier rather than later in the development cycle. There are times when the purified antigen spiked into buffer or the sample medium does not mimic the native antigen. Introducing real samples also introduces interfering substances, potential cross-reactants, and sample variation issues that can have a significant impact on assay results. Optimization and screening may need to be repeated to mitigate these effects, so it is important to discover this early on in the assay program.

Common Assay Development Issue #3: Transitioning to Manufacturing

Thinking about the transition to manufacturing early in development can avoid many delays, save time, and save money. Considerations include the supply of reagents and materials, transitioning materials to reel-to-reel manufacturing, lot-to-lot variability of materials and reagents, scale-up manufacturing of buffers and assay components, the stability of each component, and the final assay.

Scaling up

Many processes during the R&D stage, from conjugating small volumes to striping a single membrane, are done at a small scale. Although it may seem like a negligible issue, scaling up these processes could alter your assay results and require optimization. To avoid this issue, compare the small-scale to the large-scale procedures side-by-side and determine the appropriate specifications and tolerances for each procedure. Consider the materials that will be used in scaling up to reel-to-reel manufacturing. Make sure the tensile strength of materials, such as a conjugate pad or sample pad, are compatible with reel-to-reel and that the materials are available in roll format.

Variability and stability

The development stage should include thorough evaluation of lot-to-lot variability of all materials used. Before Verification, conduct interim stability studies to identify any issues that may need optimization prior to transfer. Evaluate the stability of the reagents and materials before processing to ensure steady and stable storage of all required components during manufacturing. At DCN Dx, our manufacturing capabilities help transition assays from small-scale to full-scale lot sizes. Our in-house reel-to-reel system can help identify any issues quickly and we help find the optimal solutions.

If you are interested in learning more about lateral flow assay development, join us for our Advanced Lateral Flow Course this October, where we will discuss assay development issues in greater depth.