A lateral-flow assay (LFA), or lateral-flow test, is a rapid, convenient test that uses antibodies (or sometimes antigens) to detect specific macromolecules such as antigens, drug substances, or other proteins. They are usually compact, portable, and can be stored at ambient temperatures, which makes them useful for point-of-care (POC), at-home, or remote use.
Meet Klaus
Dr. Klaus Hochleitner is a recognized global expert in membrane-based detection and analysis systems. He has published extensively and is in demand as a speaker on this topic.
Klaus is currently Global Product Specialist for Diagnostics at Cytiva. In this position, he focuses on teaching, training, and supporting diagnostic rapid test developers and manufacturers, sharing his expertise on the materials and methods to develop and produce diagnostic systems.
The anatomy of lateral-flow assays
An LFA starts with the application of a liquid sample (often urine, blood, or saliva) onto a sample pad. The liquid then travels laterally through a conjugate release pad. The conjugate release pad holds and preserves the reagents that interact with the sample. Then the sample travels through a nitrocellulose membrane. The role of the membrane is to immobilize the capture molecules (typically antibodies) at the test and the control lines (T- and C-lines) and to guide the flow of the sample and the detection conjugate to the reaction area. The target molecules may be antigens, drug substances, or other proteins. Finally, the sample travels to an absorption pad or wick. This pad is a sink for the reagents and keeps the liquid traveling through the membrane. (Fig. 1)
Fig 1. The common parts of an LFA
Request samples of LFA and other immunoassay components
Webinar 1: Lateral-flow assays: test development, optimization, and troubleshooting
In the first webinar of the lateral-flow assay series, Klaus gave an overview of the many things that LFA designers need to consider as they develop their tests. He emphasized that before you even start picking out membranes or pads you need to fully understand the biological system that you are working with. And it’s more than just knowing your target molecules and sample type. You also must consider who will be tested, where they live and work, their health and medications, and more. You also have to consider things like your desired sensitivity and specificity levels and even where and how the test might be used.
Next, Klaus walked through what happens in each of the major steps in LFA development:
- Selection of materials and reagents
- Analysis of the LFA with artificial samples
- Analysis of the LFA with real samples
- Validation of the LFA
Only after going through all those steps will your assay be properly ready for manufacturing!
The last section of Klaus’s presentation covered troubleshooting. He went through many common issues encountered while testing the LFA with artificial samples. He discussed possible causes for each problem and gave potential solutions for each cause. Then, Klaus described issues that happen with real samples and how to solve them.
If you would like to hear Klaus’s first talk on lateral-flow assays, you can watch the webinar on demand.
Watch part 1 of the LFA webinar series
Webinar 2: Lateral-flow assays: discover the importance of the nitrocellulose membrane
The second webinar in the series focused on nitrocellulose (NC) membranes: the heart of LFAs. Klaus started by explaining what nitrocellulose is and how NC membranes are made. He also described the difference between backed and unbacked NC membranes. Backed membranes are attached to a plastic film that give the membrane greater strength, which makes them easier to handle. Unbacked membranes have no supporting film and are a little weaker, but they are more versatile. Unbacked membranes have two different surfaces: the air side and the belt side from the manufacturing process. The structure of the belt side is denser than the air side (Fig 2), which influences the amount of protein bound and therefore the test sensitivity. LFA developers can choose the side of the membrane that works the best for their application.
Fig 2. Scanning electron microscope images comparing the air side and belt side of the same NC membrane
Klaus then talked about the specified parameters of both types of NC membranes including thickness, surface structure, and capillary flow time. Capillary flow time is a particularly important parameter because it has a big effect on the effectiveness of an LFA and whether the assay gives false positives or false negatives. Klaus then suggested strategies to use when picking NC during development and validation to optimize assay performance.
The next topic was the use of surfactants and why they are needed in many LFAs to avoid the formation of micelles. Klaus noted that assay developers needed to test antibodies in the presence of various surfactants to optimize assay performance. Approximately 3% of mAbs don’t work in the presence of any surfactant, and many others work only at low surfactant concentrations.
Surfactants can be added to a NC in three different ways: post-treating a wet membrane, post-treating a dry membrane, and adding the surfactant as a component of the casting mix. Klaus described each of these methods and their advantages and disadvantages. Cytiva membranes are available using the last two methods only.
Klaus then discussed the challenge of unspecific (i.e., unwanted) binding of proteins to NC membranes, which is the interaction of sample components and/or detector conjugates with the membrane surface outside of the T- and C-lines. The two ways to address this challenge is to block binding sites either by coating the membrane with blocking agents in a separate manufacturing step or by blocking on the fly. Blocking on the fly means to add the blocking agents to the conjugate release pad so that the agents travel with the liquid in the NC membrane ahead of the target molecules.
Finally, Klaus talked about the many factors that influence the binding of target molecules to the membrane. These factors include membrane properties, molecule properties, dispensing buffer, dispensing equipment, and environmental conditions. He discussed how many of the factors affected binding and how some factors could be controlled to optimize binding.
Klaus had so much to stay about NC membranes, that it’s difficult to summarize all of it here. So, watch the webinar on-demand hear everything he had to say.
Watch part 2 of the LFA webinar series
Webinar 3: Lateral-flow assays: characterizing antibodies using SPR technology
In the third webinar, Klaus focused on screening antibodies for lateral-flow assays. He started by briefly talking about selecting reagents using ELISA. ELISA is the traditional way LFA developers identify capture and detection reagents. With ELISA, you can test all possible reagents with a large checkerboard-type experiment, but the method has its drawbacks. You won’t get numerical data, and the method is time consuming. Furthermore, you can miss some suitable binders because ELISA preferably selects binders that bind analytes for a long time but does not select for fast binding.
Klaus then discussed the many benefits of using surface plasmon resonance (SPR) technology to characterize antibodies. It’s label-free and contact-free, and you can watch binding happening in real time. The whole process can reduce your workload and save you time. What’s more, SPR provides kinetic data that you cannot gather using ELISA. With SPR you’ll get a measure of
- kon (on-rate): how fast a binder binds its target analyte
- koff (off-rate): how fast a binder releases its analyte back into solution
- KA (affinity constant): kon/koff
Next came an overview of how SPR works and how to read the outputs of an SPR assay (Fig 3). Klaus showed example results and described what a LFA developer should look for when choosing reagents. Ideally, on-rates should be fast for efficient binding at low concentrations (and therefore higher sensitivity) and so that fast membranes can be used in an assay. Conversely, off-rates should be slow because they indicate a more stable complex. Slow off rates allow for use of slow membranes (if needed) and are good for multiline and quantitative assays.
Fig 3. The graph compares analyses from two SPR runs. On-rates are calculated from the green parts of the curves and off-rates are calculated from the blue parts of the curves.
Finally, Klaus walked through a case study in which a developer used SPR to select antibodies for a lateral-flow test. The developer had 16 antibodies, but very limited data about them. Cytiva worked with the developer to determine antibody concentration, perform a kinetic analysis of antigen binding, build pairs of antigens, and select a membrane surfactant.
Watch this webinar to learn how you can use and benefit from SPR technology when developing your own assay.
Watch part 3 of the LFA webinar series
Webinar 4: Lateral-flow assays: optimizing fiber pads in test development and manufacture
While the second webinar was all about the nitrocellulose membrane in LFAs, the fourth webinar was all about the other components: the fiber pads. Klaus explained that pads serve multiple purposes in LFAs.
- Sample pad and/or blood separator pad: receives the sample and, if the sample is blood, separates plasma from the other components in the blood; sometimes contains sample-adjusting compounds
- Conjugate release pad: holds and preserves the test reagents and then releases them when the sample passes through; often contains blocking reagents
- Absorption pad or wick: absorbs sample liquid to prevent backflow into the membrane
Then Klaus compared the properties, benefits, drawbacks, and uses of cellulose fibers (CF) and glass fibers (GF), the two materials commonly used to make the various LFA pads. He also described the manufacturing process for both CF and GF pads and explained that due to the process, the pads are asymmetrical. One side of the pad (the belt side) is denser than the other (the air side). This difference is similar to what is observed in nitrocellulose membranes (Fig 2). The difference between the sides means that the absorption rate also differs, and a test developer should test both orientations of pad while optimizing their tests.
Klaus then listed the various parameters that are usually defined by a pad’s manufacturer and pointed out which parameters are most important to consider when developing LFAs. In general, capillary flow time, tensile strength, and parameters that affect liquid uptake (such as thickness and grammage) are most relevant to an assay’s use and manufacturing.
At the end of the webinar, Klaus described Fusion 5, a glass fiber matrix available from Cytiva. Fusion 5 is designed to be an all-in-one test strip that can simplify LFA manufacturing (Fig 4).
Fig 4. Fusion 5 matrix combines five functions into one strip of material.
Watch the webinar to learn more about blocking on the fly, the importance of pad thickness, and additives usually found in CF and GF pads.
Watch part 4 of the LFA webinar series
Webinar 5: Lateral-flow assays: the rapid rise in molecular diagnostics
For the fifth webinar, Klaus was joined by Nina Garrett, CTO of Abingdon Heath. Together, they discussed the emerging field of point-of-care molecular diagnostics. They specifically talked about nucleic acid lateral flow (NALF), which is the use of lateral-flow tests to detect the products of nucleic acid amplification.
Molecular diagnostics is defined as diagnostics based on the analysis of nucleic acids. It is historically done by amplifying nucleic acids from a sample using PCR and then detecting through gel electrophoresis and blotting. This process was usually centralized in diagnostic labs because it requires expensive, specialized equipment and trained personnel.
Klaus talked about why decentralization of molecular diagnostics would benefit public health. Citing the COVID-19 pandemic, he reminded us of the large number of PCR tests that were needed in a short period of time and the often inconveniently long wait times to receive the results. Then Klaus enumerated the requirements needed to decentralize including simple sample collection and nucleic acid isolation; affordable, small-footprint, isothermal amplification equipment; and rapid detection of amplified nucleic acids.
This led to the next part of the webinar in which Nina described the molecular lateral-flow assay that her company has developed. This assay is a universal nucleic acid, lateral-flow immunoassay that Abingdon Health calls PCRD. It combines the sensitivity of PCR tests with the speed of lateral-flow tests. Nina described in detail how PCRD works on a molecular level.
Nina also talked about the variety of ways PCRD could be read. Of course, it can be read by simple visual inspection by the end user, but it could also be read using a smartphone app. The app could interpret the results and even upload data to a centralized server for regional, national, or even global tracking of diseases.
Klaus wrapped up the webinar by describing the holy grail of NALF: a single device that integrates nucleic acid amplification and lateral-flow detection. This product doesn’t exist yet, but we’ll be there to support you with its development.
To learn more about point-of-care molecular diagnostics and PCRD, watch the webinar.
Watch part 5 of the LFA webinar series