Lateral Flow Webinar - Final - PDF PDF

in association with
For a full recording of the webinar please visit: www.innovabiosciences.com/videos.html

Innova Biosciences ltd. 2012. All rights reserved
A Beginners 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-ofcontact 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
Detector label
Detector
antibody
analyte
Double antibody sandwich

for antigen detection
Sandwich assay for

antibody detection
Competitive Inhibition LF Assay
Carrier molecule
with covalently
attached
analyte, e.g.
BSA-cortisol
Analyte negative sample
Analyte positive sample
Antibodies
Monoclonal
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
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, Linscotts 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
Antigens:
Immunisation
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.
Dont 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
The Typical Rapid Test: A Lateral Flow Device

Sample
Reagents, Dispensing Equipment, Result Analysis
Not shown: Tapes,

Housings, Packaging Materials
Self-Adhesive
Plastic Support
Sample Pad:
Sample
Preparation
NC Membrane:
Conjugate Pad:
Bind the target
Sample meets
See the result
Detection Reagents
Absorbent Paper:
Dispose remaining
sample liquid
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 l/cm, 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 25C 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 Innova Biosciences ltd. 2012. All rights reserved
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?
Define the absorption capacity

required per cm of pad.
Select the pad material.
Calculate the pad size needed
per test.
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?
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)
Blockers (proteins,
polymers as e.g.
PVP, PVA, PEG)
Do not use salts

(especially metal
colloids are not
compatible with high
salt concentrations)
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
SEM FF 120 HP,

1000 x magnification
1000x
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:
Typical Membrane Specifications:
Strip width: 10 mm
Description
Capillary Flow
FF80 HP
60 100 seconds
FF120HP
90 150 seconds
FF170HP
140 200 seconds
Water volume: 100l
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 cm 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 l/cm and more than
1 l/cm.
Typical protein concentrations are varying between 0.75 and 1.25 g/l.
Apply proteins to the membrane in a buffer close to the proteins 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 LightningLink RAPID (15 minute incubation)
The Worlds 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..
Absorbance
100
80
aggregated
60
40
20
0
400
dispersed
500
600
700
Wavelength
Ratio 650:530 is an aggregation parameter
Self assembly on planar gold surfaces:

carboxyl analogue
alkanethiol
thiol
Au-S dative bond
Citrate ions
- -- - -- - destabilised
COOH
or NH2
successful
coating
X
X = COOH or NH2
dissociation
Gold
InnovaCoat
surface
Latex
Colloidal stabilisation
Surface functionalisation
Control the number of reactive groups
COOH
(glu, asp)
Amines (lys)
COOH
amine
hydrazide
aldehyde
NHS esters
epoxide
hydrazide
maleimide
Amine,
hydroxyl
aldehyde
Thiol (cys)
Enhanced colloidal stability
Absorbance
100
1M NaCl
1M HCl
1M NaOH
Water
80
60
40
20
0
400
InnovaCoat Gold
(4 curves)
naked
500
600
700
Wavelength
Stability in 2.5M NaOH at 70C

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)

Antibody-InnovaCoat (covalent)
InnovaCoat intermediates (e.g. carboxyl, amine)
InnovaCoat Biotin
InnovaCoat Streptavidin
~5
170
90-150
150
120
biotin
Strip type:
Strep Ctrl
biotin
Strep Ctrl
Integrity of the surface coat

A
D
IgG spotted strips,
BSA blocked
A - InnovaCoat GOLD 10 OD
B Washed
C Naked gold 10 OD
D Naked gold washed
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!
% Binding
Increase your limits of detection with InnovaCoat GOLD

100
90
80
70
60
50
40
30
20
10
0
InnovaCoat GOLD
Passive Gold Conjugation
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
Cortisol Concentration [ng/mL]

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.
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 worlds 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.

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

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Загружено:

Авторское право:

Доступные форматы

in association with

For a full recording of the webinar please visit: www.innovabiosciences.com/videos.html

Innova Biosciences ltd. 2012. All rights reserved

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

Tom Speedy, Innova Biosciences Ltd

Covalent attachment of antibodies and ligands to gold nanoparticles

Antibody and Antigen Selection for

Basic Components of Lateral flow

Membrane strip/ test site

Lateral Flow test Types:

Innova Biosciences ltd. 2012. All rights reserved

Double antibody sandwich

Sandwich assay for

Competitive Inhibition LF Assay

Analyte positive sample

Innova Biosciences ltd. 2012. All rights reserved

Innova Biosciences ltd. 2012. All rights reserved

Source of Antibodies & Antigens

Innova Biosciences ltd. 2012. All rights reserved

Source of Antibodies & Antigens

Innova Biosciences ltd. 2012. All rights reserved

Major Issues with LF Assays

Innova Biosciences ltd. 2012. All rights reserved

Purity and type of antigen used in the LF assay can

Lateral Flow Rapid Tests:

Innova Biosciences ltd. 2012. All rights reserved

The Typical Rapid Test: A Lateral Flow Device

Not shown: Tapes,

Innova Biosciences ltd. 2012. All rights reserved

Sample pad selection: What do you

Variability of target molecule concentration

Innova Biosciences ltd. 2012. All rights reserved

Sample Pad Selection

Sample Pad Pretreatment

Blood Samples: Retention of RBCs

Cells are retained by mechanical

Wrap around fibers

The Conjugate pad:

Conjugate pad selection: What do you

Define the absorption capacity

Type of conjugate do you want to use

Innova Biosciences ltd. 2012. All rights reserved

Conjugate Pad Materials

Pretreatment of Conjugate Pads

Do not use salts

How To Get The Conjugate Into The Pad

The Analytical Membrane

Typically, this is a large pore sized nitrocellulose (NC) membrane.

Innova Biosciences ltd. 2012. All rights reserved

The Structure of NC Membranes

SEM FF 120 HP,

Innova Biosciences ltd. 2012. All rights reserved

Characterization of NC Membranes: Capillary Flow Time

Typical Membrane Specifications:

140 200 seconds

Water volume: 100l

Innova Biosciences ltd. 2012. All rights reserved

Recommended Membrane Characteristics

Slow membrane as eg. FF 170 HP

Urine; low blood/serum volume with chase