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Fluorescent detection in lateral flow

Lateral flow assays, also known as immunochromatographic assays or strip tests, are immunoassays which are used to detect the presence, or absence, of a target analyte in a sample. Since they are suitable for point-of-care testing, provide a result extremely quickly, and offer simple, user-friendly operation they are rapidly growing in popularity. To find out more about the features and benefits of lateral flow assays, why not download our free guide, or watch our webinar?

Lateral flow assays have traditionally relied on the use of antibodies which have been conjugated to colored detection moieties such as gold nanoparticles or latex beads. Colorimetric readouts allow rapid visual assessment of the assay result however, despite the extensive and well-documented use of colorimetric detection methods, many researchers are instead turning to fluorescent labels such as fluorescent dyes, fluorescent proteins or Europium particles, since these offer several advantages:

  • Greater assay sensitivity
  • Quantitative readout
  • Possibility of multiplexing

Expedeon has created a wide range of products to help you simplify the development of your lateral flow assay, including kits to produce fluorescently-labeled reagents. Why not visit our application page to discover more?

 

Lightning-Link® for production of fluorescently-labeled antibodies

Our Lightning-Link® technology enables the direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups. The product range includes kits for labeling biomolecules with a wide range of fluorophores, covering the spectrum from UV to far infra-red, and is therefore ideally suited to the production of reagents for use in lateral flow assays which require an immunofluorescent readout.

The Lightning-Link® kits are extremely quick and easy to use. The conjugation reaction is initiated simply by adding the biomolecule to a vial of the lyophilized mixture which contains the label of interest and incubating. With no separation steps, 100% of the material is recovered; and since the bond which is formed is covalent, the resulting conjugates are highly stable.

 

Lightning-Link Conjugation Process

 

Although it is possible to visualize many fluorescently-labeled antibody conjugates by eye (provided the antibody conjugate is present at a suitable concentration), it is more common to use a specialized strip reader for fluorometric detection. This should be fitted with the relevant filters for detection of the fluorophore and, if a multiplexed lateral flow assay is performed, the properties of each fluorophore should be carefully considered.
To view the properties of each of our fluorophores, including maximal absorption and maximal emission wavelengths, extinction coefficient and Stokes shift data, why not download our fluorescence table?

Conjugate Check&Go! to confirm successful antibody conjugation

Our Conjugate Check&Go! kit is a dipstick lateral flow assay for confirming the successful conjugation of an IgG antibody to a colored label. It is compatible with antibody conjugates produced using many of the fluorescent Lightning-Link® antibody labeling kits, as well as with conjugates generated using colloidal gold, InnovaCoat® GOLD and Latex Conjugation Kits. The kit is compatible with IgG antibodies from multiple species, provided that they have an affinity for either Protein A or G, which are immobilized at the Test line.

 

Schematic representation of the Conjugate Check&Go! process

Schematic representation of the Conjugate Check&Go! process. The antibody conjugate flows along the nitrocellulose membrane and binds to the Protein A and Protein G which are concentrated at the test line. When the antibody is successfully conjugated to a colored label, a visible line appears on the strip.

 

We have used Conjugate Check&Go! to test antibody conjugates produced using a wide range of our Lightning-Link® antibody labeling kits.

Conjugate Check&Go! evaluation of antibody conjugates generated using Lightning-Link®

Conjugate Check&Go! evaluation of antibody conjugates generated using Lightning-Link®. Conjugate Check&Go! was used to confirm successful conjugation of an IgG to a fluorescent protein (RPE), tandem dye (APC/Cy5.5), or fluorescent dye labels (Fluorescein, Cy5.5). The line intensity varies according to the choice of label, with fluorescent dyes requiring a higher concentration of the antibody conjugate to produce a visible line compared to fluorescent proteins and tandem dyes. The intensity of the line is also dependent on the color of the label.

 

Europium conjugation kits

For lateral flow assays that require an even higher degree of sensitivity, we offer our Europium Conjugation Kit. These allow direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups to 200nm Europium (Eu) chelate microspheres. Our Europium particles have a specially treated surface, allowing the generation of highly stable, covalent conjugates which are resistant to aggregation and require no extensive pH optimization.

 

Europium Conjugation Kit

The Europium conjugation process.

 

Labeling with Europium particles provides up to 15-fold higher sensitivity compared with other particle-based assays, in part due to Europium’s large Stokes shift. Europium has a maximal absorbance of 365nm and a maximal emission of 610nm, with a wide excitation spectrum and a narrow emission spectrum. Since the emitted fluorescence has a long lifetime (µseconds), Europium can be used for time resolved fluorescence.
We have tested antibody conjugates produced using our Europium Conjugation Kit in both dipstick and strip test lateral flow assay formats, and evaluated the assay readouts to produce a qualitative result with a UV transilluminator, and a quantitative result with a lateral flow strip reader.

 

Dipstick lateral flow assay

During a dipstick assay, the conjugated antibody is added directly to a solution containing the target analyte, to which it binds. The lateral flow test strip is then dipped in to the solution, and the analyte and the detection reagent flow along the nitrocellulose membrane, towards the wicking pad. The analyte becomes bound at a Test line, which typically consists of a second specific antibody that has been immobilized on the membrane. A Control line, usually composed of an anti-species antibody (against the host species of the antibody conjugate), is included further along the strip.

The schematic represents the assay format

Dipstick lateral flow assay for evaluation of an antibody-Europium particle conjugate. The schematic represents the assay format: an anti-CRP antibody was conjugated to Europium particles using Innova’s Europium Conjugation Kit, and the conjugate was added to a solution containing a known concentration of CRP. A dipstick lateral flow test strip, consisting of a nitrocellulose membrane and a wicking pad, was dipped in to the solution. A positive result at the Test line indicates the presence of CRP. The image of the lateral flow strips shows data obtained from the UV transilluminator, demonstrating low background, no signs of aggregation, and a sensitivity of 0.25-0.5ng/ml. The graph shows the same data obtained from the lateral flow strip reader, with a sensitivity of 0.063ng/ml. Please note that a Control line was not included in this instance.

 

Strip test lateral flow assay

The main difference between a dipstick assay and a strip test assay is that a strip test includes a conjugate release pad, which is a portion of the lateral flow assay in to which the conjugated antibody has been dried. Download our free guide, or watch our webinar to find out more. The sample is added to the sample application pad, from which it flows in to the conjugate release pad. The analyte and the detection reagent then move along the nitrocellulose membrane, towards the wicking pad. The analyte becomes bound at a Test line and, as in the strip test, a Control line is usually included further along the strip.

Strip test lateral flow assay for evaluation of an antibody-Europium particle conjugate. The schematic represents the assay format: an anti-CRP antibody was conjugated to Europium particles using Expedeon’s Europium Conjugation Kit, and dried in to the conjugate release pad. A solution containing a known concentration of CRP, spiked in to CRP-depleted 100% serum, was then added to the sample application pad. A positive result at the Test line indicates the presence of CRP in the serum. The graph shows data obtained from the lateral flow strip reader, demonstrating low background and high sensitivity. Please note that a Control line was not included in this instance.

 

We offer a wide range of products which are suitable for fluorescent detection in lateral flow, including kits for labeling antibodies with fluorescent dyes, fluorescent proteins or Europium particles, as well as our Conjugate Check&Go! Kit to confirm successful antibody conjugation. If you have any questions, please contact us.