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. 

Early Development: Wet Conjugate Testing

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

1. Simultaneous Addition: Simultaneous addition first involves mixing the conjugate and sample together in a separate vial or container. This allows for the formation of a conjugate-analyte complex in an extended incubation period. The proportions of sample and conjugate in this mixture will be dependent on each assay and depend on dilution requirements, sample treatment, conjugate concentration, etc.. Once the sample and conjugate have been mixed, they are added to the sample pad of the test strip in a single step. This addition method maximizes sensitivity, but will only work if your conjugate is highly specific for your target analyte, as any interferents  will also have more opportunity to cause aggregation or non-specific binding, .
2. Sequential Addition: In sequential addition, the conjugate is first added to the sample pad at a low enough volume to not run up the strip, followed by an addition of the sample to the pad below the conjugate. This format will more closely mimic the interaction time between the conjugate and sample that is created in a dried down test strip.  Therefore, simultaneous addition may be more useful during initial antibody screen to determine which pairs form a sandwich, while sequential addition yields test lines that are more representative of final assay results. 
3. Sample First: Sample first also refers to the adding of the sample and conjugate in successive steps, but in the opposite order as sequential addition. First, the sample is added to the conjugate pad and run up the test strip. This way, any target analyte in the sample will first bind to the test line. After the sample has had an opportunity to run, the conjugate will be added to the conjugate pad. The conjugate will then wick across the strip and bind to any analyte captured at the test line.  The benefit of this method is that the conjugate never interacts with any cross-reactive interferents which may be present in the sample. If desired, a sample pad may also be incorporated into the test strip, with the sample first applied to the sample pad, and then the conjugate applied to the conjugate pad. This way, interferents may be filtered out first by the sample pad.  Comparing results in this format to the simultaneous and sequential addition methods described above may help determine the degree to which a sample contains components that interfere with the conjugate. If needed, the final dry-down strips can be constructed to utilize a sample first approach. 
4. Immunoprecipitation: In immunoprecipitation, the sample and conjugate are combined with an incubation period, similar to the simultaneous addition method. However, the combined liquid is then centrifuged to form a pellet of the analyte-conjugate complex, the supernatant (containing any interferents from the sample) is removed, and the particles are resuspended in a clean buffer.  These particles, already bound to any analyte in the sample, are then run up the test strip to bind to the test line.  This way, possible interferents in the sample never interact with the test line.  In combination with sample first testing, immunoprecipitation can help the user to understand where interference is taking place, and to what extent.  

Later Stage Development: Dried Down Conjugate

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:

1. Stability: Proteins are susceptible to chemical and physical degradation that will result in changes to its native conformation. This process is accelerated in aqueous solutions. Storing the protein in a lyophilized or freeze-dried format will prolong the shelf life of the protein and the assay.
2. Reduce Complexity: While pre-mixing a liquid conjugate with a sample is not a complex task, any extra steps performed by a user introduces more uncertainty and chance of error. Developing a low risk streamlined testing procedure is essential in designing an assay for R&D or commercial use.

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.

Alternative Method: Dipstick (Half-Strip) 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

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. 

Strip Analysis

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:

1. Eye Test: A first option is to read the assay by eye. This is acceptable for positive/negative scoring but is not useful for quantitative assays.  At nanoComposix we’ve also produced gradient score cards where the strength of the lateral flow line can be measured against a printed line intensity in order give a semi-quantitative score. Since this method doesn’t involve an instrument to capture the results, capturing a photo is a convenient method to record results. This format will only work for absorbent material, not for fluorescent assays.
2. Scanner: Alternatively, a flatbed scanner, a camera set up with controlled lighting, or a dedicated commercial reader can be used to capture an image of the test line. The color density (and thus line strength) can then be analyzed with an image processing program (e.g. ImageJ), resulting in a number that directly correlates to the test line intensity.  Various commercial readers are available that can analyze strips.  At nanoComposix, we use a wide variety of readers from companies such as Lumos Diagnostics and Qiagen that provide a quantitative readout in as little as 30 seconds.  We have also also worked with a number of cell-phone based reader technologies that are in development.