A guide to lateral flow immunoassay development

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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 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


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

Medicaldiagnostics

Foodsafety

Environmentalmonitoring

Drugs-of-abusetesting

Forensicscience

Pregnancy &fertility testing

Therapeuticmonitoring

Applications

Infectious diseasetesting


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


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 byimmersion, 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 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


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


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


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 antigenicsites

• 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 magneticparticles can be used

• The antibody and the detection moiety should be of high quality toensure 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 flowimmunoassay• 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 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


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 passiveadsorption of antibodies to ultra-stable gold nanoparticles


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


Colloidal gold from Innova Biosciences

• Stringently QC tested• Consistent high quality

• Batch-to-batch reproducibility

• Detailed Certificate of Analysis suppliedwith 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 antibodiesto gold nanoparticles

• Latex conjugation kits for covalent attachment ofantibodies 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

• Stringently QC tested

• Available as easy-to-use conjugation kits, orseparately 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 20minutes

• Extensive pH optimization is not necessary

• Allow rapid screening of multipleantibodies 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 antibodylabeling

• InnovaCoat® GOLD Carboxyl (-COOH) is optimized forsingle-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 bufferexchange


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

• Ideal for multiplexing

• Stringently QC tested


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 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


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



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Q&A session


Thank you!


Get in touch..For more information please get in touch or visit our website


+44 (0) 1223 661000

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A guide to lateral flow immunoassay development

Science

Transcript of A guide to lateral flow immunoassay development