Lateral Flow Assay Development and the use of gold nanoparticles

© Innova Biosciences ltd. 2012. All rights reserved
in association with
For a full recording of the webinar please visit: www.innovabiosciences.com/videos.html

A Beginner’s Guide to
Lateral Flow Assay Development

Dr. Ahmed Jehanli, IPRO Interactive Ltd
Antibody and Antigen Selection for Lateral Flow Tests
Dr. Klaus Hochleitner, GE Healthcare Life Sciences
Lateral Flow Rapid Tests: Material Selection, Material Properties and
Basic Troubleshooting
Tom Speedy, Innova Biosciences Ltd
Covalent attachment of antibodies and ligands to gold nanoparticles

Antibody and Antigen Selection for
Lateral Flow Tests
Ahmed Jehanli
IPRO Interactive Ltd
Oxfordshire, UK
Ahmed.jehanli@iprointeractive.com

Introduction
• Lateral flow (LF) immunoassays point-of-
contact tests are simple to use, provide rapid
results with minimum amount of sample
preparation
• Lateral flow immunoassays underwent huge
expansion following the development of rapid
pregnancy tests in the 70s
• LF tests are widely available in the medical,
veterinary, environmental, and other fields.
• Global market in billions of dollars

Basic Components of Lateral flow
test
• Membrane strip/ test site
• Sample pad
• Conjugate (detector) pad
• Absorbent (sink) pad
• Antibody and antigen reagents

Lateral Flow test Types:
• Sandwich assay format: Used for
large analytes (proteins) with
multiple antigenic determinants
• Competitive assay format: Used
for small molecules (hormones,
drugs, etc

Sandwich LF Assay
Double antibody sandwich
for antigen detection
Sandwich assay for
antibody detection
Detector label
Detector
antibody
analyte

Competitive Inhibition LF Assay
Analyte negative sample Analyte positive sample
Carrier molecule
with covalently
attached
analyte, e.g.
BSA-cortisol

Antibodies
Polyclonal
• Advantages:
– High affinity
– Wide choice of species
• Disadvantages:
– Purity of the antigen is essential
to achieve high specificity
– Less than 5% of the
immunoglobulin fraction will be
the wanted antibody.
Immunoaffinity purification is
essential
– Supply is limited
Monoclonal
• Advantages:
– Unlimited supply
– High specificity
– Immunoaffinity purification is
not necessary.
• Disadvantages:
– High affinity antibodies can be
difficult to achieve
– Limited choice of species (murine
monoclonal antibodies)

Source of Antibodies & Antigens
• Commercial sources:
– Use antibody data bases for searching for reagents, e.g.,
Biocompare.com, Linscott’s Directory
– Antigen-antibody pairing are available for cardiac markers,
steroids, drugs of abuse, etc. Many already designed for
lateral flow assays
– Must ensure that continuous supply of reagents can be
provided

Source of Antibodies & Antigens
• In House:
– Several companies exist for carrying out contract immunisation and
antibody production both poly- and mono-clonal
– For polyclonal antibodies, rabbit, sheep, goat and chicken can be used
– For monoclonal antibodies, murine is the choice. Other species
monoclonal antibody production is available but can be costly and
royalty payments might be expected. For sheep monoclonal antibodies
see Bioventix PLC
– Recombinant and engineered antibodies are not widely commercially
available and tend to be costly

Immunisation
• Antigens:
– Purity of the antigen used for immunisation is crucial for raising polyclonal
antibodies but not so for monoclonal antibodies
– In general, molecules over 5000 molecular weight can be used for
immunisation with out further treatment
– If the antigen is not very immunogenic, treat as a “hapten” and conjugate it
with a “carrier” molecule, KLH for immunisation, example platelet derived
growth factor (PDGF)
– Small molecules (haptens) like hormones, drugs and small peptides must be
covalently attached to a large “carrier” protein prior to immunisation.
Substitution ratio of the hapten: protein has an impact on the affinity of the
raised antibody
– Type of spacer used to link the hapten to the carrier can have an effect on the
nature of antibody specificity
– Avoid using BSA or OVA as carriers as both are commonly used in LF assays as
blocking agents

Antibody Evaluation
• Prior to setting up the LF assay, evaluate reagents using
enzyme immunoassays (EIA) (sandwich or competitive
inhibition depending on the antigen). Validate, the specificity,
sensitivity and matrix suitability of the reagents.
• Although LF assays also use Sandwich and competitive
formats they are different from EIAs. The former format is an
“open” system while the latter is a “closed” system.
• It is very important that the analyte matrix is introduced to
the LF evaluation very early in assay development.
Don’t waste too much time on validation work in buffers.

Major Issues with LF Assays
Sensitivity & non-specific signal
Can be dealt with by changing antibody-gold particle
substitution ratio, signal amplification, amount of
reagents deposited on the membrane, and sample
buffer components

Conclusion
• The quality of the antibody and antigen used in LF is
very important
• Antibody affinity and specificity is very critical for a
successful LF assay.
• Purity and type of antigen used in the LF assay can
impact on assay sensitivity and specificity especially
for competitive inhibition assays

Lateral Flow Rapid Tests:
Material Selection,
Material Properties and
Basic Troubleshooting
Dr. Klaus Hochleitner
Global Lead Technical Product Specialist Diagnostics
GE Healthcare Life Sciences
Contact: Klaus.Hochleitner@ge.com

Sample
Self-Adhesive
Plastic Support
Sample Pad:
Sample
Preparation
Conjugate Pad:
Sample meets
Detection Reagents
NC Membrane:
Bind the target
See the result
Absorbent Paper:
Dispose remaining
sample liquid
Not shown: Tapes,
Housings, Packaging Materials
Reagents, Dispensing Equipment, Result Analysis
The Typical Rapid Test: A Lateral Flow Device

Sample pad selection: What do you
need to know about your sample before?
• Variability of target molecule concentration
(defines sample volume to be applied).
• Variability of sample composition, e.g. pH
(sample composition may have to be
adjusted by sample pad pretreatment).
• Sample viscosity (limits density of the pad
material).
• Unspecific interactions of your target with
the pad material (defines pad blocking
requirements).
• Unspecific interactions of the target with
test line reagents (may require additional
adjustments).
• Need of retention of particles contained in
the sample (e.g. red blood cells).

Sample Pad Selection
• Specify sample volume to be applied on test strip.
• GE provides material properties
(absorption capacity in µl/cm², paper raw materials, presence
of binders).
• Select high quality chromatography paper as sample pad, if
possible made of cotton linters (the most reproducible paper
raw material).
• If the sample pad is to retain particles, especially red blood
cells, or is to serve as a combined sample and conjugate pad,
select a glass fiber pad material.

Sample Pad Pretreatment
• Usually done by immersion.
• Dry material in a forced air convection oven.
• Store the coated material at 18 – 25°C and less than 20% rel. humidity.
Holds true for ALL coated materials in lateral flow tests!
Pretreatment buffer:
pH adjustment
(always necessary)
Salts
(try to avoid or
use low
concentrations)
Blockers (proteins,
polymers as e.g.
PVP, PVA, PEG)
Nonionic surfactants
(increase wettability
of pad material,
support blocking, may
help to reduce
unspecificities)
Beware of hemolytic reagents if blood is your sample

Blood Samples: Retention of RBCs
Cells are retained by mechanical
interaction
Wrap around fibers
General Recommendations:
• Define blood volume to be applied very carefully, and select appropriate pad
– too much volume leads to red blood cell breakthrough problems!
• Avoid hemolysis as this will release free hemoglobin to your membrane
which will result in a background color difficult to deal with.

The Conjugate pad:
Basic Considerations
• Typically, it is the physically smallest part in a lateral flow test.
• Fulfills a diversity of functions:
• Absorbs the volume in which the detector conjugate is added to the pad.
• Does not interact with the conjugate.
• Maintains the conjugate integrity upon drying.
• Maintains the conjugate integrity in the dry state
(can easily be more than a year at “room temperature“).
• Releases the conjugate easily and completely upon contact with the
sample liquid.
• Allows for interaction between the detector reagents in the conjugate
and the target in the sample.

Conjugate pad selection: What do you
need to know before?
• Type of conjugate do you want to use
(Metal colloids, latex beads, covalent or
non-covalent binding of the detector
molecules to the particle, no use of
particles but directly labeled
antibodies/antigens).
• Amount of detector molecules needed in a
test in order to obtain the required
sensitivity.
• Maximum concentration that can be
achieved with the conjugate in solution
without inducing aggregation of particles.
• As a result of these considerations: What is
the volume of conjugate solution that must
be applied to the conjugate pad per test?
Define the absorption capacity
required per cm² of pad.
Select the pad material.
Calculate the pad size needed
per test.

Conjugate Pad Materials
• Options are glass fiber pads and non-wovens.
• Glass fibers are more versatile, especially when it comes to additional pad
functions as e.g. sample application or RBC retention.
• In general, glass fibers are recommended.
Pretreatment of Conjugate Pads
pH adjustment
(always necessary)
Do not use salts
(especially metal
colloids are not
compatible with high
salt concentrations)
Blockers (proteins,
polymers as e.g.
PVP, PVA, PEG)
Nonionic surfactants
(wettability, pad
blocking, membrane
blocking “on the fly“).

How To Get The Conjugate Into The Pad
Two options:
- Immersion/dipping of the pad in a conjugate solution
- Dispensing of defined conjugate volumes per conjugate pad length/area
Drawback of Immersion/Dipping:
- The pad material is variable in thickness. Soaking the pads with
conjugate will lead to variable amounts of conjugate in the tests strips
manufactured, and may lead to poor test reproducibility.
Drawback of Dispensing:
- Equipment needed
Strong recommendation: Dispense!

The Analytical Membrane
• Typically, this is a “large pore sized“ nitrocellulose (NC) membrane.
• The membranes are available in a very broad range of sample flow characteristics.
• All NC membranes contain a surfactant, usually an anionic surfactant, that makes
them hydrophilic.

The Structure of NC Membranes
• NC Membranes do not have “pores“.
• They are made of a meshwork of NC fibres:
drag and drop picture here
1000x
SEM FF 120 HP,
1000 x magnification

Characterization of NC Membranes: Capillary Flow Time
Describes the time a liquid (water) needs to migrate a defined distance (4 cm)
parallel to the membrane surface against gravity.
Test procedure:
Strip width: 10 mm
Water volume: 100µl
Typical Membrane Specifications:
Description Capillary Flow
FF80 HP 60 – 100 seconds
FF120HP 90 – 150 seconds
FF170HP 140 – 200 seconds

Membrane Selection
• Parameters to consider are sample type, test duration, and membrane
variability.
• The more viscous a sample the slower it will run through a membrane.
• The slower a membrane, the more NC it contains per cm² surface area which
means that it can bind more protein and generates more sensitive tests
• Especially for highly sensitive and/or quantitative tests, use membranes with
very low CVs on capillary flow times.
A general recommendation for test development:
Sample type Recommended Membrane Characteristics
Water Slow membrane as eg. FF 170 HP
Urine; low blood/serum volume with chase
buffer
Medium fast membrane as eg. FF 120 HP
Undiluted serum; saliva; resolubilized solids Fast membrane as eg. FF 80 HP
Please note that the reagent quality has a massive influence on the membrane
selection.

Dispensing Protein Lines
General recommendations:
• Use precision dispensing equipment as early as possible in test development.
• Typical dispensing rates are varying between 0.6 µl/cm and more than
1 µl/cm.
• Typical protein concentrations are varying between 0.75 and 1.25 µg/µl.
• Apply proteins to the membrane in a buffer close to the protein‘s pI.
• The buffer should not contain high salt concentrations.
• The buffer may contain a low concentration of methanol or ethanol
(up to 3 % v/v).
• Try to avoid the use of surfactants – they may lead to foaming problems
while being dispensed on the membrane.
• Low concentrations of Trehalose (recommendation: 0.5 – 1 % w/v) are
sometimes used to increase the stability of the protein of the membrane
surface.

The Wick
• Its task is to soak the sample liquid and all reagents that have not been
absorbed at the test and control lines.
• It must prevent the backflow of this liquid into the drying membrane as long
as possible.
• Select a cotton linters paper with an absorption capacity that is much higher
than the sample volume.

Some Basic Troubleshooting
Issue Possible Solution
Uneven Lines/Dots 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 conc.,
Change conjugated protein
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

“GE, imagination at work and GE monogram are trademarks of General Electric
Company”
“All goods and services are sold subject to the terms and conditions of sale of the
company within GE Healthcare which supplies them. A copy of these terms and
conditions is available on request. Contact your local GE Healthcare representative for
the most current information”
“© 2011 †General Electric Company – All rights reserved.
First published April. 2012‡”
“GE Healthcare UK Limited Amersham Place
Little Chalfont
Buckinghamshire. HP7 9NA
UK”
“Manufactured under a license to DE 10102744 and foreign equivalents thereof”
Regulatory Note: This is a technical report and the data contained within is not
intended to support any shelf life claims made for the product in the instructions for
use.

Covalent attachment of antibodies
and ligands to gold nanoparticles
Tom Speedy – Corporate Business Manager

1.Innova Biosciences and bioconjugation
2.Traditional (passive) gold conjugation methods
3.Overview of covalent chemistries
4.Functionalisation of gold nanoparticles
5.Ultra-stable InnovaCoatTM GOLD nanoparticles

What is Lightning-Link® technology?
The worlds fastest, simplest and most efficient conjugation technology!
• Just 30 seconds hands-on time to set up the reaction
• Over 50 labels available including:
Enzymes, fluorescent proteins, fluorescent dyes, tandems, biotin & streptavidin
• 100% antibody recovery
• Fully scalable from R&D to Production / Manufacture
• Virtually eliminates batch to batch variability
• Covalent conjugation ensures long term stability
• Available as traditional Lightning-Link® (2 hour incubation) or new Lightning-
Link® RAPID (15 minute incubation)

The World’s fastest and easiest to use antibody labelling system

• 20 – 80nm gold used in diagnostic tests
• Antibody-gold conjugates made by a non-covalent (passive) adsorption technique
• Colloidal instability when attaching ligands or biomolecules to naked gold (or
nanoparticles in general)
• Need to optimise conditions for each antibody (pH, salt conc. etc.), centrifuge..

Ratio 650:530 is an aggregation parameter
400 500 600 700
0
20
40
60
80
100
Wavelength
Absorbance
aggregated
dispersed

Self assembly on planar gold surfaces:
thiol
alkanethiol
Au-S dative bond
carboxyl analogue

- -
-
-
-
--
-
-
-
-
-
Citrate ions
destabilised
successful
coating
dissociation
X+
X = COOH or NH2
COOH
or NH2

Gold
Latex
InnovaCoat
surface
Colloidal stabilisation
Surface functionalisation
Control the number of reactive groups

COOH
amine
hydrazide
maleimide
epoxide
aldehyde
Amine,
hydroxyl
Amines (lys)COOH
(glu, asp)
aldehyde
Thiol (cys)
NHS esters
hydrazide

400 500 600 700
0
20
40
60
80
100
1M NaCl
1M HCl
1M NaOH
Water
naked
InnovaCoat Gold
(4 curves)
Wavelength
Absorbance
Enhanced colloidal stability

Conjugate Min (to aggregate)
Biotin-Gold (competitor 1) <0.1
Biotin-Gold (competitor 2) <0.1
Carboxyl gold (competitor 3) (self assembly?) <0.1
Lipoid acid (self assembly) <0.1
Mercaptopropionic acid <0.1
Antibody/Naked gold (passive) ~5
Antibody-InnovaCoat™ (covalent) 170
InnovaCoat™ intermediates (e.g. carboxyl, amine) 90-150
InnovaCoat™ Biotin 150
InnovaCoat™ Streptavidin 120
Stability in 2.5M NaOH at 70°C

biotin biotin
Strep CtrlStrip type: Strep Ctrl

A B C D
A - InnovaCoat GOLD 10 OD
B – Washed
C – Naked gold 10 OD
D – Naked gold washed
IgG spotted strips,
BSA blocked
Integrity of the surface coat

What is InnovaCoat® technology?
A revolutionary method for conjugating nanoparticles to biomolecules
• Proprietary surface coat
• Ultra stable
• Covalent linking of antibodies, analytes and other biomolecules
• Increased assay sensitivity
• NO pH titrations – NO centrifugations – NO aggregation or instability!

Increase your limits of detection with InnovaCoat GOLD
Figure 1.
A comparison between the percentage binding of anti-cortisol antibodies conjugated to 40nm gold particles by a traditional passive method
against those conjugated covalently using InnovaCoat technology. All components of this competitive lateral flow assay are identical with the
exception of the method used to conjugate the antibodies to gold colloid. External data source.
0
10
20
30
40
50
60
70
80
90
100
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4
%Binding
Cortisol Concentration [ng/mL]
InnovaCoat GOLD
Passive Gold Conjugation

Innovative Cambridge Company Innova Biosciences
Secures Prestigious Development of Prototype Grant
from the Technology Strategy Board.
Innova Biosciences (Cambridge, UK), inventor of 'Lightning-Link®', the world’s easiest to
use antibody labelling technology, is pleased to announce it has been awarded a
development of prototype grant circa £210,000 by the Technology Strategy Board, with
matched company funding of approximately £250,000, to develop novel nanoparticle
products for diagnostics applications.
Innova Biosciences Press Release: May 2012

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences

Lateral Flow Assay Development and the use of gold nanoparticles

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