A guide to lateral flow immunoassay development

www.innovabiosciences.com
A Guide to Lateral Flow
Immunoassay Development

Our speaker today…
Emma Easthope
Technical Content Writer at Innova Biosciences

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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Q&A session

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

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 of
radioactivity
• 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

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

When might a lateral flow immunoassay be used?
Animal health
Agriculture
Aquaculture
Medical
diagnostics
Food
safety
Environmental
monitoring
Drugs-of-abuse
testing
Forensic
science
Pregnancy &
fertility testing
Therapeutic
monitoring
Applications
Infectious disease
testing

Advantages and Disadvantages
Advantages 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

The basis of a lateral flow immunoassay
• Sample application pad
• Conjugate release pad
• Membrane
• Wicking pad
• Plastic cassette

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 by
immersion, followed by drying
Urine,
water,
blood etc

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 to
ensure a constant amount of detection
reagent 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 via
dipping or dispensing methods

The membrane
• Typically composed of nitrocellulose
• Capillary flow time - time taken for liquid to move
along and fill a membrane of defined length
• Capture antibodies are immobilized across the
membrane, usually in two distinct lines
• Instrumentation is required for precise application
of 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

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 of
sample that is taken up by the test strip

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

Assay formats
• Direct (sandwich) assay
• Larger analytes with multiple antigenic
sites
• Competitive assay
• Smaller analytes with a single antigenic
determinant

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 magnetic
particles can be used
• The antibody and the detection moiety should be of high quality to
ensure a successful lateral flow immunoassay

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

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 flow
immunoassay
• Lateral flow evaluation should be introduced as early as possible during assay
development

The detection reagent – the detection moiety
• Gold nanoparticles or latex beads are the most commonly
used detection moieties in lateral flow immunoassays
• These should be of a uniform size and of a regular spherical
shape
• Ensures a consistent rate of transfer
• Conjugation of the antibody to the detection moiety
should be simple, scalable and reproducible

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

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 passive
adsorption of antibodies to ultra-stable gold nanoparticles

Colloidal gold from Innova Biosciences
• Uniform spherical shape
• Narrow size distribution
• Different nanoparticle sizes,
concentrations and pack
sizes available
• Fully scalable - available at
high 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 

Colloidal gold from Innova Biosciences
• Stringently QC tested
• Consistent high quality
• Batch-to-batch reproducibility
• Detailed Certificate of Analysis supplied
with every batch

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 antibodies
to gold nanoparticles
• Latex conjugation kits for covalent attachment of
antibodies to colored latex beads

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
• Available as easy-to-use conjugation kits, or
separately as carboxylated gold nanoparticles TEM image of a 40nm InnovaCoat®
GOLD coated nanoparticle

InnovaCoat® GOLD conjugation kits
• Quick and easy to use
• The conjugate is ready for use within 20
minutes
• Extensive pH optimization is not necessary
• Allow rapid screening of multiple
antibodies for assay development
• Freeze-dried
• Ship at ambient temperature
• Long shelf-life

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

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 antibody
labeling
• InnovaCoat® GOLD Carboxyl (-COOH) is optimized for
single-step EDC coupling

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 buffer
exchange

Latex bead conjugation kits
• Blue, red and black latex conjugation kits available
• Ideal for multiplexing

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

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

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 Targetgroup
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

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

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

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

Thank you!

Get in touch..
For more information please get in touch or visit our website
+44 (0) 1223 661000
www.linkedin.com/company/innova-biosciences@InnovaBioSciwww.facebook.com/InnovaBiosciences

A guide to lateral flow immunoassay development

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A guide to lateral flow immunoassay development