lateral flow assay development and the use of gold nanoparticles

© Innova Biosciences ltd. 2012. All rights reserved

in association with

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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

[email protected]


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: [email protected]


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 7000

20

40

60

80

100

Wavelength

Ab

so

rban

ce

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 7000

20

40

60

80

1001M NaCl1M HCl1M NaOHWater

naked

InnovaCoat Gold(4 curves)

Wavelength

Ab

so

rban

ce

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 Ctrl Strip 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

% B

ind

ing

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


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Lightning-Link® is a registered trademark of Innova Biosciences