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A Guide to Lateral Flow Immunoassay Development
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Our speaker today…
Emma Easthope
Technical Content Writer at Innova Biosciences
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Agenda
• Overview of Lateral Flow assays
• The components of a typical lateral flow test strip
• Detection methods
• Simplifying LFA development
• Q&A session
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Type in questions
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Q&A session
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What is an immunoassay?• An immunoassay is a bioanalytical method which relies on an
antibody: antigen interaction• Western blotting
• Immunocytochemistry (ICC)
• Immunohistochemistry (IHC)
• Flow cytometry
• ELISA
• Sensitive
• Selective
• Applicable to the detection of a wide range of analytes
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What is the readout of an immunoassay?• The first immunoassay, developed in 1959, detected radioactive
iodine131
• Handling and disposal issues are associated with the use ofradioactivity
• Alternative readouts have been developed• Colorimetric
• Gold nanoparticles, latex beads, enzymatic conversion of a chromogenic substrate
• Fluorometric• Fluorescent proteins, synthetic fluorescent dyes
• Chemiluminescent• Enzyme-driven, a wide range of chemiluminescent substrates is available
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What is a Lateral Flow Immunoassay?• A lateral flow immunoassay is unidirectional, and is used to detect the
presence (or absence) of a target analyte in a sample
• Applicable to point-of-care (POC) testing
• Minimal amount of sample preparation
• Widely used in a variety of settings
• Rapidly growing market
Unilever’s Clearblue home pregnancy test
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When might a lateral flow immunoassay be used?
Animal health
Agriculture
Aquaculture
Medicaldiagnostics
Foodsafety
Environmentalmonitoring
Drugs-of-abusetesting
Forensicscience
Pregnancy &fertility testing
Therapeuticmonitoring
Applications
Infectious diseasetesting
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Advantages and DisadvantagesAdvantages Disadvantages
Applicability to point-of-care testing Restriction on total test volume can impose a
limit on sensitivity
Wide range of applications Test-to-test reproducibility can be
problematic
Relatively short timeline for development,
low cost
Qualitative or semi-quantitative readout
High sensitivity and specificity Unclear patent situation in some instances
Low sample volume required
Long shelf-life, no need for refrigeration
Simple, user-friendly operation
One-step assay, no wash steps necessary,
short time to result
Easily scalable
High potential for commercialization
Amenable to multiplexing
Can be integrated with reader systems
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The basis of a lateral flow immunoassay
• Sample application pad• Conjugate release pad• Membrane• Wicking pad• Plastic cassette
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The sample application pad• An absorbent pad on to which the sample is
applied
• Typically a cellulose fiber or a woven mesh
• Promotes even, controlled sample transfer
• Cellulose fibers can be modified to allow pre-treatment of the sample• Reduce non-specific binding
• Increase sample viscosity
• Alter pH
• Remove red blood cells
• Pre-treatment is usually carried out byimmersion, followed by drying
Urine,water,
blood etc
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The conjugate release pad• An absorbent pad into which the detection
reagent has been dried
• Exhibits low non-specific binding
• A consistent bed volume is important toensure a constant amount of detectionreagent in each lateral flow test strip
• May require pre-treatment• Increase wettability
• Decrease non-specific interactions
• Control pH
• The conjugate can be added to the pad viadipping or dispensing methods
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The membrane• Typically composed of nitrocellulose
• Capillary flow time - time taken for liquid to movealong and fill a membrane of defined length
• Capture antibodies are immobilized across themembrane, usually in two distinct lines
• Instrumentation is required for precise applicationof the capture antibodies
• A number of parameters require optimization• Antibody concentration
• Antibody diluent
• Reagent dispensing rate
• Drying method
Capillary flow time
[analyte]
Effect of capillary flow time on assay sensitivity
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The wicking pad• Functions to increase the volume of sample
which enters the test strip
• Helps to reduce background
• Prevents backflow
• Can be used to optimize the volume ofsample that is taken up by the test strip
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The plastic cassette• Ensures that the end user applies the sample only to the sample
application pad
• Protects the test strip
• Can be labeled
• Available as an off-the-shelf product
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Assay formats• Direct (sandwich) assay
• Larger analytes with multiple antigenicsites
• Competitive assay• Smaller analytes with a single antigenic
determinant
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The detection reagent• Typically antibodies which have been conjugated to gold
nanoparticles or latex beads
• Colorimetric readout, no development process required
• Fluorescent labels, enzymes, other colloidal metals and magneticparticles can be used
• The antibody and the detection moiety should be of high quality toensure a successful lateral flow immunoassay
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The detection reagent – the antibody• A consistent supply of good quality antibodies is essential
• Availability for the lifetime of the finished product
• Antigen specificity
• Antibody stability• Reactivity after adsorption to the conjugate release pad
• Tolerance of drying for a defined time period
• Instant reactivity following rehydration
• Monoclonal antibodies are preferred• Unlimited supply
• High specificity
• Immunogen affinity purification is not required
Antibody recognition of a common epitope results in binding to
multiple proteins
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The detection reagent – the antibody• The binding affinity of the antibody should be assessed
Ab + Ag⇌ Ab-Ag KD = [Ab] [Ag]
[Ab-Ag]
• A fast on-rate is critical to a successful lateral flow immunoassay
• ELISA testing is not predictive of antibody behaviour in a lateral flowimmunoassay• Lateral flow evaluation should be introduced as early as possible during assay
development
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The detection reagent – the detection moiety
• Gold nanoparticles or latex beads are the most commonlyused detection moieties in lateral flow immunoassays
• These should be of a uniform size and of a regular sphericalshape• Ensures a consistent rate of transfer
• Conjugation of the antibody to the detection moietyshould be simple, scalable and reproducible
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Preparation of the detection reagent• There are several methods of attaching an antibody to a detection
moiety• Passive adsorption
• Covalent attachment to surface-functionalized particles
• Attachment to pre-coated particles
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Passive adsorption• Relies on hydrophobic attractions and electrostatic interactions
between the antibody and the particle
• Requires specialist knowledge and lengthy optimization• Antibody titration
• Identification of a suitable buffer
• pH titration
• Innova Biosciences offers high quality colloidal gold for passiveadsorption of antibodies to ultra-stable gold nanoparticles
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Colloidal gold from Innova Biosciences
• Uniform spherical shape
• Narrow size distribution
• Different nanoparticle sizes,concentrations and packsizes available
• Fully scalable - available athigh concentration (>20 OD)and large volume (litres)
10nm colloidal gold available as 20ml and 100ml pack size
20nm, 40nm and 80nm gold available as 10ml and 100ml pack size
10nm 20nm 40nm 80nm
1 OD
10 OD
15 OD
20 OD
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Colloidal gold from Innova Biosciences
• Stringently QC tested• Consistent high quality
• Batch-to-batch reproducibility
• Detailed Certificate of Analysis suppliedwith every batch
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Covalent attachment to nanoparticles• Covalent attachment provides a number of advantages
over passive adsorption• Increased conjugate stability
• Tighter control over assay variability
• Uses up to 2.5x less antibody than passive adsorption
• InnovaCoat® GOLD for covalent attachment of antibodiesto gold nanoparticles
• Latex conjugation kits for covalent attachment ofantibodies to colored latex beads
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InnovaCoat® GOLD• Gold nanoparticles with a proprietary surface
coating• Covalent binding to form highly stable conjugates
• Metal-protein interactions are prevented
• Uniform spherical shape
• Narrow size distribution
• Stringently QC tested
• Available as easy-to-use conjugation kits, orseparately as carboxylated gold nanoparticles TEM image of a 40nm InnovaCoat®
GOLD coated nanoparticle
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InnovaCoat® GOLD conjugation kits• Quick and easy to use
• The conjugate is ready for use within 20minutes
• Extensive pH optimization is not necessary
• Allow rapid screening of multipleantibodies for assay development
• Freeze-dried• Ship at ambient temperature
• Long shelf-life
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InnovaCoat® GOLD product range
• Different nanoparticle sizes, pack sizes and conjugation chemistries
Conjugation kits 10nm 20nm 40nm 60nm 80nm Target chemistries
InnovaCoat® GOLD Amine groups
InnovaCoat® GOLD Maleimide
Thiol groups (Fab’, oligo) ORIENTATED CONJUGATION
InnovaCoat® GOLD Hydrazide
Aldehyde groups (IgG, IgM) ORIENTATED CONJUGATION
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InnovaCoat® GOLD• InnovaCoat® GOLD targets primary amine (-NH2) groups
• AbPure™ kits for antibody concentration or buffer exchange
• InnovaCoat® GOLD Maleimide (-SH) and InnovaCoat®GOLD Hydrazide (-CHO) facilitate orientated antibodylabeling
• InnovaCoat® GOLD Carboxyl (-COOH) is optimized forsingle-step EDC coupling
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Latex bead conjugation kits• Quick and easy to use
• The conjugate is ready to use within 35 minutes
• Targets primary amine groups• Stable covalent bond
• Specially treated beads prevent aggregation
• Extensive pH optimization is unnecessary
• Accessory kits for antibody concentration or bufferexchange
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Latex bead conjugation kits• Blue, red and black latex conjugation kits available
• Ideal for multiplexing
• Stringently QC tested
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Other detection moieties• Enzymes and fluorescent labels are alternative detection moieties
• Enzymatic readouts require downstream processing
• Fluorescent assays are more sensitive than colorimetric assays, but require a specialized reader
• Lightning-Link® antibody labeling kits from Innova Biosciences• Enzymes – HRP, Alkaline Phosphatase, Glucose Oxidase
• Wide range of fluorescent proteins and fluorescent dyes
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Attachment to pre-coated particles• Occasionally it may be preferable to use pre-coated particles as the
detection reagent in a lateral flow immunoassay• The use of one detection method to label a number of different antibodies
during antibody screening can save time and money
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Pre-conjugated gold nanoparticles• Manufactured using InnovaCoat® GOLD nanoparticles
• Ultra high quality nanoparticles ensure optimal performance in lateral flow immunoassays
Conjugation kits 10nm 20nm 40nm 80nm Target group
InnovaCoat® GOLD Streptavidin Biotin
InnovaCoat® GOLD Biotin Streptavidin
InnovaCoat® GOLD Goat anti-mouse Mouse IgG
InnovaCoat® GOLD Goat anti-rabbit Rabbit IgG
InnovaCoat® GOLD Protein A IgG
InnovaCoat® GOLD Protein G IgG
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Basic troubleshooting
Issue Possible solution
Uneven lines Use membrane with different pore size
Reduce dispensing volume of reagent
Increase protein concentration of reagent
Check dispensing buffer composition
Check dispensing process
False positive signals Modify buffer in conjugate pad/solution
pH, salt concentration, surfactant concentration
Use a different conjugate
False negative signals See above, also:
Use membrane with smaller pore size
Increase sample volume
Uneven liquid fronts of migrating sample
Check membrane shelf life
Use membrane with different/more surfactant
Check relative humidity (very low?)
Contact membrane supplier (membrane surface properties?)
Increase surfactant conc. in conjugate pad
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Custom services• InnovaCoat® GOLD nanoparticle conjugate micro-optimization service
• Production of 8 different antibody-gold nanoparticle conjugates using your antibody, at your chosen scale
• InnovaCoat® GOLD custom conjugate formulation service• Production of bulk quantities of antibody-gold nanoparticle conjugate
• Lateral flow assay development services• Conjugation service
• Assay development
• Lateral flow immunoassay optimization
• Manufacturing strips for lateral flow immunoassays
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Summary• Rapidly growing market, wide range of applications
• Innova Biosciences’ products to facilitate colorimetric detection• Colloidal gold
• InnovaCoat® GOLD
• Latex conjugation kits
• Pre-conjugated gold nanoparticles
• Custom services available
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Type in questions
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Q&A session
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Thank you!
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Get in touch..For more information please get in touch or visit our website
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+44 (0) 1223 661000
www.linkedin.com/company/[email protected]/InnovaBiosciences