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During 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 Addition

When you are first beginning to optimize and develop your assay, you need a testing method that is fast, reliable, and doesn’t require an abundance of material or equipment. The wet conjugate addition testing method satisfies all these requirements. In this method, both the conjugate and sample are in a liquid format. The strip is assembled normally, consisting of a nitrocellulose membrane, sample pad, wick pad, and a conjugate pad. Once the test strip is ready for testing, you can then decide on the order that the sample and conjugate will be added. The two most common additions of sample and conjugate are:

  1. Simultaneous Addition: Simultaneous addition first involves mixing the conjugate and sample together in a separate vile or container. This allows for the formation of a conjugate-analyte complex. 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 will only work if your conjugate is specific for your target analyte, and not sensitive to interferents that may cause aggregation or non-specific binding. Simultaneous addition also doesn’t require a conjugate pad, and thus is an easier test strip to manufacture during development.
  1. Sequential Addition: Sequential addition refers to the adding of the sample and conjugate in successive steps. First, the sample is added to the sample pad and the solution runs up the test strip. Any target analyte in the sample will 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 benefits of this method is that it allows for any interferent to be filtered out first by the sample pad, as well any cross reactive species to have run through the assay without interfering with the conjugate. Another benefit for this method is that it more closely mimics the final lateral flow test format that will have dried down conjugate.  

Later Stage Development: Dried Down Conjugate

As you move further down the development pathway, you will need to start testing in a format that will closely resemble the final product. Typically this will involve a dried conjugate where a solution containing the conjugate and a stabilizing reagent (e.g. Trehalose) will be deposited onto the conjugate pad. Dispensing is performed with an automated dispenser like the IsoFlow system, or manually with a pipette. After dispensing, water will be removed from the conjugate pad via a forced-air convection oven. 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 it is essential that the dried conjugates are stored in a dry room (i.e. <20% RH) or pouched with dessicant. 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 optimization be 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 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.

Alternative Method: Dipstick Assay

A dipstick assay is an alternative form of testing that utilize strips that consist only of the nitrocellulose membrane and wick pad. No sample pad or conjugate pad is be required which creates a system with less 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, eppendorf tube, or a single well of a 96-well plate (see below). 

Lateral Flow Dip Stick Assay in 96 Well Plate

The strip is dipped in the mixture and the solution 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 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 increase/decrease flow speed. This is accomplished with the use of various salts, surfactants, detergents, stabilizing agents, or blocking reagents. For each lateral flow assay, the buffer will need to be optimized for the individual assay by evaluating the buffering component, molarity, salt, detergent, polymers, and/or proteins.  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, however the formulation should be optimized for each assay. 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 inoculation of the test strip. Once a running buffer formulation has been optimized, it is recommended 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 which removes a user step and simplifies the assay. 

Strip Analysis

When analyzing the test strip, choosing the appropriate analysis method will depend on the stage of development and whether the assay is intended to be qualitative or quantitative. For effective optimization it is important that you 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 semi-quantitative or 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, and not for fluorescent assays.
  1. 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 be analyzed in an image analysis program (e.g. ImageJ) resulting in a number that is directly correlated to the test line intensity.  Various commercial readers are available that will 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 approximately 30 seconds.  We also work with a number of cell-phone based reader technologies that are in development.