Considerations for Lateral-Flow Immunoassay Development

A lateral-flow immunoassay, or immunochromatographic assay, is a rapid, convenient test that uses antibodies (or sometimes antigens) to detect specific macromolecules such as antigens, drug substances, or other proteins. They can also be called lateral-flow devices (LFDs) or lateral-flow assays (LFAs). In this interactive brochure, we highlight some things you should consider when selecting and working with the different components of your lateral-flow immunoassay and the relevant products to help optimize your test. > 3 With a diverse array of products, Cytiva is one of the leading suppliers in lateral-flow components. Our component offering includes a wide range of blood separators, sample pads, conjugate release pads, nitrocellulose membranes, and absorbent materials. We also offer diagnostic services to support your immunoassay development. Test line Nitrocellulose membrane Sample pad Blood separator Conjugate release Control line Absorption pad Diagnostic services Lateral-flow starter packs Lateral-flow starter packs from Cytiva Name Description Catalog number CF/GF starter pack A sample pack containing all our cellulose and glass fiber pads, including all blood separators, sample pads, conjugate pads, and absorbent pads 29363634 Nitrocellulose membrane starter pack A sample of all our nitrocellulose grades for LFA 13549210 Combo starter pack A pack containing both our CF/GF and nitrocellulose starter packs 29428668 Starter packs are a pack of materials, typically A4 sheets, that can be stored on the shelf in an R&D lab for quick access during development. They contain a variety of grades and speeds of membranes and pads, so you can test as many options as possible. Cytiva has three starter pack options, one contains cellulose and glass fiber pads, another contains a range of nitrocellulose membranes, and a third contains both pads and membranes. Choosing a sample pad with the properties that suit your sample will get your LFA off to a good start. Some key properties to consider when selecting a sample pad for your LFA are: Thickness: generally, the thicker the pad the higher the water absorbency Water absorption: measured in mg/cm2 , determines total amount of sample that can be applied Wicking rate: how quickly the sample will move through the sample pad onto the conjugate release pad, which ultimately contributes to speed and sensitivity of the test. Generally, the slower the wicking rate, the higher the sensitivity. Also called lateral-flow rate of the liquid. Beyond the basics: sample pad additives Additives in the sample wick can influence and mitigate some undesired properties in the sample. Some sample types show a broad sample-to-sample variation. For example, urine samples differ considerably in pH, ionic strength, and molecular composition. Impregnation of the sample pad with salts and buffers can make samples more uniform when they reach the conjugate release and test zones. In other cases, an addition of a substance to make the sample more viscous may reduce the wicking rate and enhance the sensitivity of detection. Blocking agents Blocking agents can be generic proteins such as bovine serum albumin (BSA) or detergents such as Tween 20. They help avoid nonspecific binding of the analyte or detection reagents to the reaction membrane. Efficient blocking will increase the signalto-noise ratio, enhancing visibility of the test lines by reducing background stain. The addition of blocking agents to the sample pad is often much easier and more cost efficient than adding them to the reaction membrane. This method is often called “blocking on the fly.” Guidance for sample pad selection Sample pads from Cytiva To ensure that your assay begins well, Cytiva offers a complete range of sample pads manufactured in controlled environments from high-quality materials, with your requirements in mind. Consistent absorbency and wicking rates help ensure test-to-test reproducibility. Low protein binding prevents loss of analyte, so test sensitivity is maintained. Naturally hydrophilic materials can be rewetted rapidly, even after prolonged storage. Formats are compatible with most styles of housing and can be easily customized upon request. The sample pad begins the LFA by receiving the sample from the point of application and allowing the flow across the other test components. Grade Properties Volume (per cm2) Thickness (μm @ 53 kPA) Wicking rate (s/4 cm) Water absorption (mg/cm2) CF1 A thin, smooth-surfaced cotton linter paper with a liner flow rate, suitable for small volumes. < 50 µL 176 187 16 CF3 A medium thick cotton linter paper, originally used for separation of inorganic compounds. Handles larger sample volumes than CF1. 50–100 µL 322 161 31 CF4 A medium thick cotton linter paper with acid treatment to improve wet strength and reduce trace impurity content. Similar weight and thickness to CF3 with faster wicking. 100–150 µL 482 65 46 GR470 Untreated bound cellulose suitable for whole blood or serum. Performs well with one or two drops of whole blood. > 150 µL 840 77 78 Standard 14 Untreated bound glass fiber for faster flow than cotton with lower sample retention. Higher absorption capacity than Standard 17. > 50 µL 355 23.1 50.9 Standard 17 Untreated bound glass fiber for faster flow than cotton with lower sample retention. Greater tensile strength than Standard 14. > 50 µL 370 34.5 44.9 GF/DVA Untreated bound glass fiber particularly suitable for saliva samples and raw milk samples. > 50 µL 785 28.2 93 VF2 Bound glass fiber used as single or multiple layers for separation. > 50 µL 785 23.8 86.2 Sample pad selection from Cytiva Request a sample: https://info.cytivalifesciences.com/diagnostics-product-enquiries.html For a rapid test, especially when intended for point-of-care use, the classical methods of serum or plasma preparation are not practical. There is an increasing demand for whole-blood assays, which require separation of blood cells within the test device. The separation of blood cells from plasma using a filter is a demanding task. The percentage of particulates (mainly erythrocytes; the contribution of leukocytes is negligible) in the hematocrit or packed cell volume (PCV) is between 36% and 48% for females and between 40% and 53% for males. Peak values can be up to 70% (typically found in blood of Tour de France participants). These percentages mean that a droplet of blood from a finger stick does not contain much liquid. It is common for lateral-flow immunoassays with whole-blood samples to add some reagent to the sample. The added reagent ensures that there is sufficient liquid volume to perform the assay. An anti-coagulant should also be added to this reagent to avoid clotting problems. With such an additive, the effective sample in the assay is plasma, not serum. A blood separator for immunochromatographic assays is typically a cellulosic or glass fiber depth filter. The separator must efficiently remove blood cells and yield a high recovery of plasma. It should not cause red blood cells to break down (act hemolytic) and not interact with the analyte in question. Blood separators from Cytiva Cytiva offers a family of blood separators to meet the increasing demands and strict requirements of the rapid diagnostics market. Their features include: Choice of separation times allows for test optimization. Separation in 30–120 s for rapid assays that save time. No appreciable red cell hemolysis for improved reproducibility of results. Consistency of materials provides reliable tests. Materials suitable for use in a range of tests, giving flexibility in test optimization. Separators appropriate for a range of blood volumes to enhance the separation rate according to the volume of blood available. Asymmetric polysuflone plasma separation membranes from Cytiva Cytiva also offers asymmetric polysuflone plasma separation membranes (PSM). The Vivid™ plasma separation membranes use a patented process where highly asymmetric membranes are specifically engineered Guidance for blood separation materials selection for the generation of plasma from whole blood, allowing the cellular components of the blood (red cells, white cells, and platelets) to be captured in the larger pores without lysis. The plasma flows down into smaller pores on the downstream side of the membranes. White blood cell isolation filtration media from Cytiva Leukosorb™ is a filtration medium for use in assays that require the reduction or isolation of heterologous and possibly activated leukocytes that can potentially interfere in the analysis. Leukosorb is a hydrophilic, fibrous matrix designed for use in procedures requiring the isolation of leukocytes from whole blood. Blood serum is one of the most interesting sources for important diagnostic markers. They wrap themselves around the fiber (x10,000) Red cell Red cell stick to glass fiber (x1,000) Blood separators selection Grade Materials and uses Volume (per cm2) Thickness (μm @ 53 kPA) Wicking rate (s/4 cm) Water absorption (mg/cm2) VF2 Vertical separator used as single or multiple layers for separation. < 50 µL 785 23.8 86.2 GF/DVA Untreated bound glass fiber. <