Throughout all stages of the development process, it is necessary to measure the performance of prototype lateral flow assays. Here we discuss three testing methods that are used at various points during development.
In the early stages of development a liquid conjugate method is typically used where the conjugate and the sample are mixed together before being added to the strip. Four variants of wet conjugate testing are listed below. In each method, chase buffer may be added following the sample and conjugate to help fully develop the test strip
As you move further down the development pathway, you will need to start testing in a format that closely resembles the final product. Typically this involves a dried conjugate in which a solution containing the conjugate and one ore more stabilizing reagents (e.g. Trehalose) are deposited onto the conjugate pad. Dispensing is performed manually with a pipette, or more precisely with an automated dispenser like an IsoFlow system. After dispensing, water is removed from the conjugate pad via a forced-air convection oven. For example, a conjugate pad can be dried at 37°C for one hour. Following this, it can be cured further by storing in a dry environment, such as a dry room (eg: <20% RH) or pouched with desiccant. Different assays may require different cure times. Once dried, the conjugate will be stable for a longer period of time (typically many months or even years), as long as it is not exposed to moisture. All dried material will readily absorb moisture from the environment, so dry storage is essential. During the testing phase, the conjugate can be dried in multiple formats aside from a typical conjugate pad. If you are having trouble optimizing the conjugate to the conjugate pad, or would like a simpler strip design, it is possible to dry the conjugate down in sample collection tubes, pipette tips, or the sample port of the test strip housing. In all cases, a dried conjugate provides the following benefits:
Nearly all finalized lateral flow assays for commercial sale use a dried down conjugate approach, and it is recommended that all final optimizations are performed with a dried down prototype. The drying process requires planning to ensure that there is consistent solubilization of the conjugate and conjugate buffer constituents before application. If any of the components do not solubilize, this can affect conjugate release from the conjugate pad, or otherwise impact the assay quality. Proteins may also undergo conformational changes during the drying procedure that may result in an increase in non-specific binding or specific signal. These changes will have to be tested empirically for each assay.
A dipstick assay is an alternative form of testing that utilize strips which consist only of the nitrocellulose membrane and wick pad. No sample pad or conjugate pad is required, which creates a system with fewer variables and facilitates rapid testing. To begin, the sample and conjugate are pre-mixed in a container in a process very similar to the simultaneous addition method for wet conjugate. The container for mixing can be a test tube, an eppendorf tube, or a single well of a 96-well plate (see below).
The strip is dipped in the mixture and the solution is allowed to wick up the strip. This format is excellent for screening various optimization conditions associated with the test line interaction. By removing the sample pad and conjugate pad, potential sources of variations are eliminated creating a simple system for directly comparing conditions. Parameters that are often optimized in this format include dispensing related conditions (e.g. dispense speed, dispense rate, dispense volume), antibody pairs, and membrane treatment buffers. After you have narrowed down your conditions, it is important that you test the conditions in the fully assembled format with a sample pad and dried down conjugate.
Running buffer, or “chase buffer,” is an essential component of a lateral flow assay. A well formulated running buffer will allow you to buffer sample pH, minimize non-specific binding, neutralize interferents, and control flow speed. This is accomplished with the use of various salts, surfactants, detergents, stabilizing agents, or blocking reagents. These components and their concentrations will need to be optimized for each individual assay. Always keep in mind that the simpler the running buffer is, the easier it will be to manufacture, and the longer the shelf life will be. 1X PBS with 1% tween 20 is a good starting place for a running buffer. The introduction of the running buffer can be done sequentially or simultaneously, depending if the sample needs to be exposed to the running buffer constituents prior to development of the test strip. Once a running buffer formulation has been optimized, one option is to dry down the running buffer constituents on the sample pad. In some cases, this can eliminate the necessity of separately applying the running buffer, removing a user step and simplifying the assay.
When analyzing the test strip, the appropriate analysis method will depend both on the stage of development and whether the assay is intended to be qualitative or quantitative. For effective optimization, it is important to have an objective means of quantifying the output of the test strips. Overall, you will be observing the sample and conjugate flow through the strip, the presence of any non-specific binding at the test line, and the intensity of binding at the test line when running a true positive sample. There are two methods for strip analysis: