NALF and Beyond: Lateral Flow Goes Molecular

NALF, nucleic acid lateral flow, LFA, lateral flow assay, molecular diagnostic

Antibodies and other immunoreagents are so tied to lateral flow assays that we frequently use the terms LFA (lateral flow assay) and LFIA (lateral flow immunoassay) interchangeably. However, lateral flow assays may have no immunoreagents whatsoever onboard.

Over the past 15 years, a subset of lateral flow testing using nucleic acids as targeting molecules or analytes, including nucleic acid lateral flow or NALF, has quietly emerged and entwined principles of both immuno- and molecular recognition systems. Nucleic acid techniques, immensely sensitive because of enzymatic exponential amplification, offer unrivaled performance. However, their detection comes at a cost. Described here are some assay architectures that incorporate nucleic acids to take advantage of the many assets LFAs offer, such as low cost, simplicity, and low labor requirements.

Nucleic Acid Lateral Flow (NALF)

The strictest definition of nucleic acid lateral flow (NALF) incorporates no immunoreagents on the nitrocellulose membrane. Signal originates from a sandwich hybridization assay occurring at the test line, at which a DNA probe is commonly immobilized by a terminal biotin that interacts with streptavidin or NeutrAvidin on the nitrocellulose. As in lateral flow immunoassays, signaling moieties can be oligonucleotide-decorated gold or latex particles, or simply a fluorescent dye that modifies the tag sequence.

One potential drawback of NALF is that recognition of the target strand by the probe and tag strands must occur quickly and is highly dependent on Watson-Crick base pairing hybridization kinetics. Any secondary structure in any of the three strands will greatly destabilize these interactions, as might common additives (e.g., surfactants) that promote flow up the membrane. These parameters must be individually optimized for each discrete sequence.

Nucleic Acid Lateral Flow Immunoassay (NALFIA)

The nucleic acid lateral flow immunoassay (NALFIA) hybrid format incorporates together the selectivity of Watson-Crick base pairing, the sensitivity of molecular techniques, and the well-known and well-characterized immunochromatographic assay. NALFIA is commonly used to detect amplicons in a point-of-care setting and eliminates the need for time-consuming agarose gels or the expensive optics that accompany a real-time system.

In a NALFIA scenario, base modifications are incorporated into the primers, and therefore, the amplicons, using a given amplification technique. After the cycles are complete, the amplicon then acts as an antigen in a lateral flow assay, with species on the nitrocellulose membrane and the conjugate able to bind the amplicon in a sandwich via the modifications on the primers.

Nucleic acid modifications for NALFIA are widely commercially available and include fluorescent dyes (FITC/FAM, Cy5, Texas Red) and small molecules (biotin, digoxigenin, DNP). Antibodies against these modifications are widely available from numerous sources.

Because detection is independent of sequence and instead relies upon recognition of tagged primers by antibodies, universal lateral flow tests can be applied to the detection of multiple different amplicons. Anti-digoxigenin, anti-FAM, and anti-biotin are common capture antibodies, and these strips can be purchased from various vendors or produced in-house.

This universal recognition also means that NALFIA can be applied towards the detection of amplicons produced by many different amplification scenarios. Common methods include:

  • Symmetrical and asymmetrical polymerase chain reaction (PCR)
  • SNP detection via ligase chain reaction (LCR)
  • Isothermal techniques, such as loop-mediated isothermal amplification (LAMP), rolling-circle amplification (RCA), and recombinase polymerase amplification (RPA)

Newly-emerging isothermal techniques are especially attractive, as they eliminate thermocycling and can potentially be executed at room temperature, greatly simplifying equipment requirements. These methods paired with the speed of LFIA move molecular diagnostics more towards a point-of-care scenario and a cheaper price tag.

Lateral Flow with Aptamer Technologies

Though lateral flow tests developed with aptamers do not fall strictly into the definition of molecular diagnostics, they do offer a nucleic acid-based alternative to antibodies, with some notable advantages.

Aptamers represent a class of nucleic acid probes that are specifically engineered to adopt a certain conformation and bind targets of interest. The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, developed by the teams of Tuerk and Gold and also Ellington and Szostak in 1990, enabled the discovery of the first RNA aptamer probes against T4 DNA polymerase and organic dyes, respectively. A random library of DNA or RNA is incubated with the target for interaction to occur and then the bound sequences are separated from the unbound sequences and amplified. This process is cycled numerous times, with higher-affinity sequences emerging at the end of each cycle.

Due to the varied nature of the targets available (small molecules, proteins, peptides, cells), aptamer-target affinities can vary from sub-nanomolar up to micromolar, making Kd values potentially on-par with antibody-antigen interactions. Further, aptamers offer a number of potential advantages over antibodies, including automated and reproducible synthesis, increased stability, and ease of labeling and scale-up during the fabrication process.

Assay architectures with aptamer lateral flow are similar to those found in traditional antibody-based LFIA. If two aptamers are available for a given large molecular weight target, and the aptamers bind to different regions of the analyte (dubbed “aptatopes”), a sandwich assay can be designed. Competitive assays are also popular, and because aptamers can bind either their target or their nucleic acid complement, there is more than one option for a test line. Signal-on assays can also be designed by incorporating a complementary and sacrificial quencher sequence that is freed from a fluorophore-labeled aptamer once the target is bound.

While lateral flow cannot currently rival the sensitivity of molecular techniques such as PCR, one could easily argue that given resource, time, and cost constraints, ultrasensitive detection is not lateral flow’s space. Lateral flow shines in its ability to supplement and support molecular techniques in a point-of-care scenario.

DCN Dx’s scientists and engineers are skilled in the development of many different immunological and molecular assay architectures, including NALF. Contact us to find out how the vision for your assay can be realized.