Product guide:
Lateral Flow Assay
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© Expedeon Ltd.
Index Introduction....................................... ............................................ 3 1. Particles, labeling kits and conjugates for lateral flow assays............. 3 1.1. Colloidal gold........................................................................... 3 1.2. InnovaCoat® GOLD nanoparticle conjugation kits........................ 4 1.3 Gold nanoparticle conjugates...................................................... 6 1.4. Latex conjugation kits .............................................................. 7 1.5. Europium conjugation kit........................................................... 8 1.6. Fluorescent dye antibody labeling kits......................................... 10 1.7. Conjugate Check&Go! Kit.......................................................... 11 1.8. Universal lateral flow assay kit................................................... 11 2. Custom services from Expedeon................................................... 13
Expedeon products are sold for research purposes only, and our terms and conditions of sale include a limited use license to our Intellectual Property for internal research applications. Commercial use, such as use within manufacturing, re-sale to third parties, or incorporation into kits, requires a separate written agreement, conferring relevant additional rights, with Expedeon. Lightning-Link®, InnovaCoat® and Thunder-Link® are registered trademarks of Expedeon. Cy and CyDye are trademarks of GE Healthcare. DyLight® is a registered trademark of Thermo FisherScientific Inc. and its subsidiaries. FluoProbes® is a registered trademark of interchim. Texas Red® is a registered trademark of Life Technologies Corporation. © EXPEDEON • All rights reserved.
Lateral Flow Assay Guide
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Introduction Lateral flow immunoassays rely on the use of antibodies which have been conjugated to colored detection moieties such as gold nanoparticles or latex beads, or to fluorescent labels such as fluorescent dyes, fluorescent proteins or europium particles. Expedeon offers a wide range of products to help you simplify the development of your own lateral flow immunoassay.
1. Particles, labeling kits and conjugates for lateral flow assays 1.1. Colloidal gold Gold nanoparticles are ideally suited for lateral flow immunoassays due to their intense ruby red color, which makes them easy to visualize on the test strip. Expedeon offers ultra high quality colloidal gold for use in traditional passive conjugation of antibodies. Our colloidal gold nanoparticles are extremely uniform spherical particles with a narrow size distribution, minimizing variability within your assay. They are available in various sizes (10 to 80 nm), concentrations and volumes for maximum flexibility. Uniform spherical shape Narrow size distribution Ultra-high quality Batch-to-batch consistency Available at high concentrations to simplify assay development Particles are buffer exchanged post manufacture to ensure consistent formulation Available in bulk quantities Our nanoparticle production process allows particularly tight control of shape, completely preventing the formation of irregular structures seen in many other commercial products (Fig 1).
Figure 1. Transmission electron microscopy of commercially available gold particles. Note that the Expedeon particles have the most regular shape.
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Our colloidal gold is superior when compared in a lateral flow assay against uncoated gold from a leading supplier (Fig 2). 500
Signal
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Competitor
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Ab C5
Ab C7
Figure 2. Lateral flow assay of two CRP antibodies (C5 and C7) as passive conjugates. Note that Expedeon’s gold nanoparticles give a greater signal than a leading competitor’s gold in each case.
1.2. InnovaCoatÂŽ GOLD nanoparticle conjugation kits The use of colloidal gold for lateral flow immunoassays requires passive adsorption of the antibody to the surface of the nanoparticle, a process which can be challenging and which necessitates specialist knowledge. To overcome this, we have developed our InnovaCoat GOLD nanoparticle conjugation kits. InnovaCoat GOLD is a one-step gold nanoparticle conjugation kit for antibodies, proteins and peptides. The nanoparticles within the kit have a proprietary surface coating which covalently binds antibodies to form highly stable conjugates. The kits facilitate standard, orientated, or site specific labeling of IgG antibodies for use in many applications such as lateral flow assays.
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InnovaCoat ÂŽ GOLD particles
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+ add
your antibodies
Gold conjugate in 20 minutes
Quick & easy to use - save time, no specialist knowledge required Generate antibody - gold conjugate in 20 minutes Proprietary surface coat forms ultra-stable conjugates Consistent high quality - excellent batch-to-batch reproducibility Different nanoparticle sizes, concentrations and chemistries available
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Figure 3. Antibody conjugation using InnovaCoat GOLD vs traditional passive conjugation techniques with uncoated gold nanoparticles, showing both enhanced signal intensity and improved specificity. 40 nm gold particles were labeled with anti-IgA antibody, and used to measure IgA concentration in a lateral flow inhibition assay, with IgA bound to a lateral flow strip.
1.3 Gold nanoparticle conjugates We offer a range of gold nanoparticle conjugates produced using our InnovaCoat GOLD technology. All of our gold nanoparticle conjugates are stringently QC tested, minimizing assay variability which makes them ideal for lateral flow assays. Conjugates available are Goat anti-Mouse, Goat anti-Rabbit, streptavidin, biotin, protein A and protein G.
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1.4. Latex conjugation kits Latex beads are another popular choice for lateral flow immunoassays since they are available in a range of vibrant colors, making them easily visible and allowing for the simultaneous detection of different analytes. For use in a lateral flow immunoassay, latex beads require conjugation to the detection antibody. Traditional methods of latex conjugation are technically complex and can waste a lot of precious material; passive onjugation requires extensive optimization to identify the appropriate pH, while covalent conjugation is prone to aggregation of the latex. The Expedeon Latex Conjugation Kits have been designed to greatly simplify the process. Quick and easy to use - the conjugate is ready to use in just 35 minutes Resistant to aggregation - generate high quality data No extensive pH optimization required - save precious antibody / protein Different colors available - ideal for multiplexing
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Quench the reaction
Pellet particles
Resuspend into storage buffer
antibodies to latex beads
Figure 4. Multiplexed lateral flow immunoassay using Expedeon’s latex beads. Latex beads of three colors have each been conjugated to a different antibody or protein.
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1.5. Europium conjugation kit For lateral flow immunoassays that require higher levels of sensitivity, we offer our unique Europium Conjugation Kit which significantly simplifies the conjugation of antibodies and proteins to 200 nm Europium (Eu) particles. Our Europium Conjugation Kit is a unique product; the specially-treated 200 nm europium beads within the kit become covalently bound to the antibody or protein to form highly stable conjugates. The antibodyeuropium conjugates generated can be used in immunochromatographic lateral flow assay. Detection of europium conjugates in a lateral flow assay will require either a fluorescence strip reader or an UV transilluminator.
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Quench the reaction
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Resuspend into storage buffer
Pellet particles
add
antibodies to Europium particles
Data collected from the UV transilluminator: 2 ng/mL
Lateral Flow Assay Guide
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0.5 ng/mL
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0.063 ng/mL
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Data collected from the fluorescence strip reader: 1250
Signal (Arbitrary Units)
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Figure 5. Typical results obtained from a UV transilluminator (top) and a fluorescence reader (bottom), showing europium conjugated to a monoclonal anti-CRP antibody. The conjugate is easily able to detect as little as 63 pg/mL CRP, in part due to the low background and lack of aggregation.
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1.6. Fluorescent dye antibody labeling kits An alternative to particle-based detection is to use an antibody which has been labeled with a fluorescent dye or a fluorescent protein. Our Lightning-Link® antibody labeling kits enable the quick and easy conjugation of antibodies, proteins, peptides, or any other biomolecule which contains free amine groups, to a wide range of fluorescent detection moieties. Quick and easy to use – requires only 30 seconds hands-on time No separation steps, retain 100% of your antibody Wide range of labels available Reliable – cited in over 300 publications
Lightning-Link® Labeling Kits
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your antibodies
30 seconds hands-on time results in labeled antibodies
The use of fluorescently-labeled antibodies provides the opportunity for multiplexing in lateral flow immunoassays. A specialized strip reader allows the readout to be quantified, although it is possible to visualize many fluorescentlylabeled antibody conjugates by eye provided the antibody conjugate is present at a suitable concentration.
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1.7. Conjugate Check&Go! Kit The Expedeon Conjugate Check&Go! Kit is a dipstick lateral flow assay for confirming the successful conjugation of an IgG antibody to a colored label. This kit is compatible with IgG antibodies from multiple species, provided that they have affinity for either Protein A or G, which are immobilized on the T-line of the half strip. The Conjugate Check&Go! Kit can be used to confirm the successful conjugation of antibodies using Expedeon’s latex, gold nanoparticle and fluorescent labeling technologies, and are also compatible with any other conjugation technology on the market that uses colored labels. Quick and easy to use – just 10 minutes to confirm successful conjugation Requires only a small volume of diluted conjugate No specialist equipment required
Antibody conjugated to a coloured label
Protein A/G strip Protein A/G
Nitrocellulose membrane
Test line Control line
Wicking pad
Figure 6. 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.
1.8. Universal lateral flow assay kit The Expedeon Universal Lateral Flow Assay (LFA) Kit is designed to enable the easy development of customized sandwich lateral flow assays. The great advantage of this kit is its adaptability to any pair of capture and detection antibodies, which allows the detection of any type of analyte* without the need to stripe the capture antibody onto LFA strips using expensive equipment.
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Fully customizable – adaptable to any pair of capture and detection antibodies and detect any type of analyte Quick & easy to use – both conjugations can be set up in only 30 seconds No false negative results – assay is compatible with biological samples No specialized or costly equipment Can be qualitatively and quantitatively analyzed by using supplied scoring card or a LFA reader respectively * The antigen must contain at least two antigenic sites for the binding of the capture and detection antibodies
Capture antibodies
Gold-conjugated detection antibodies
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Gold-biotin conjugate
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Sample
Insert strip
Sample application pad
Test line Control line
Wicking pad
Mix antibodies
The kit combines Expedeon’s easy to use Lightning-Link antibody labeling and InnovaCoat GOLD nanoparticle conjugation technologies with an immunochromatography test performed on Universal-LFA strips. The capture antibody is conjugated to Lightning-Link Ulfa-Tag, while the detection antibody is conjugated to 40 nm InnovaCoat GOLD, both of which require only 30 seconds to set up. The capture and detection antibodies are diluted and incubated with the analyte and 40 nm InnovaCoat GOLDBiotin and then run on Universal LFA strips. Universal LFA strips consist of a nitrocellulose membrane containing a ‘Test line’ (T-line) of immobilized anti- Ulfa-Tag antibody, that binds the Ulfa-Tag-conjugated capture antibody which further binds the analyte in complex with the InnovaCoat GOLD-detection antibody. A red T-line appears when the analyte is present and the line intensity varies depending on the analyte concentration (see Figure 1). Universal LFA strips also contain a ‘Control-line’ (C-line) striped with streptavidin, which confirms that the test is valid, and an absorbent pad to promote and control the flow of sample through the membrane.
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2. Custom services from Expedeon Although lateral flow assay development can be relatively straightforward, we recognize that sometimes you might need some assistance. You may, for example, be struggling to optimize your antibody conjugation, time restraints could mean that you wish to outsource your bulk conjugations, or you might be a relative newcomer to lateral flow. To help you streamline your workload, Expedeon offers a wide range of custom services, including our lateral flow assay development service. Our lateral flow assay development service is designed to take your lateral flow immunoassay from an initial idea, through to R&D, and then on to trusted partners for bulk manufacturing. The full service is a multi-step project providing you with different options, from a proof-of-principle lateral flow immunoassay using a dipstick, to full strips with sample application pad, conjugate release pad and wicking pad, and small scale manufacture. Module 1 – conjugation services Module 1 consists of our conjugate micro-optimization services, and our custom conjugate formulation services. Our conjugate micro-optimization services enable you to find the best performing conjugate for your assay, using very small quantities of your antibody or protein; our experienced conjugation scientists will produce a range of different antibody-gold nanoparticle conjugates for you to test in your application. Our custom conjugate formulation service can be used as a follow-up service from our conjugate microoptimization service, or as an independent option; our team of expert conjugation scientists will quickly provide you with bulk quantities of your bespoke antibody-gold nanoparticle conjugate, in your chosen buffer and at your preferred concentration. Module 2 – assay development Our lateral flow assay development service is provided as a dipstick assay format, or as a full lateral flow test strip. The dipstick format consists of a wicking pad and a nitrocellulose membrane, and is designed to prove that the conjugation has been successful. The antibody-gold nanoparticle conjugate is mixed with the sample containing the antigen, and is then applied to the dipstick; a capture antibody on the nitrocellulose membrane is used to show that the assay set up is correct. The full lateral flow test strip format provides the same materials as the dipstick assay, with the addition of a conjugate release pad containing the dried antibodygold nanoparticle conjugate, and a sample application pad. Whichever format is chosen, the capture antibody is dispensed on the nitrocellulose membrane as either a spot or as a test line; greater amounts of antibody are required for the latter to allow for priming of the dispenser. If spots are dispensed on the nitrocellulose we will provide images (qualitative method), whereas if lines are dispensed the signal will be read and the sensitivity calculated (quantitative method).
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Module 3 – lateral flow assay optimization Within module 3, our experienced in-house team will determine the optimal amount of capture antibody and will test different pads and membrane treatments to provide you with your optimized lateral flow assay. Assay optimization will provide you with the best possible performance at the lowest manufacturing cost. Module 4 – strip manufacturing for LFA This service allows you to purchase assembled strips for your lateral flow assay, either as dipsticks or as full lateral flow test strips. For further information on our custom services please visit our website, www.expedeon.com or contact the team: solutions@expedeon.com
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Lateral Flow Assay Guide
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