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www.innovabiosciences.com
A Guide to Lateral Flow
Immunoassay Development
www.innovabiosciences.com
Our speaker today…
Emma Easthope
Technical Content Writer at Innova Biosciences
www.innovabiosciences.com
Agenda
• Overview of Lateral Flow assays
• The components of a typical lateral flow test strip
•...
www.innovabiosciences.com
Type in questions
Click to pop out the questions box
Q&A session
www.innovabiosciences.com
What is an immunoassay?
• An immunoassay is a bioanalytical method which relies on an
antibody: ...
www.innovabiosciences.com
What is the readout of an immunoassay?
• The first immunoassay, developed in 1959, detected radi...
www.innovabiosciences.com
What is a Lateral Flow Immunoassay?
• A lateral flow immunoassay is unidirectional, and is used ...
www.innovabiosciences.com
When might a lateral flow immunoassay be used?
Animal health
Agriculture
Aquaculture
Medical
dia...
www.innovabiosciences.com
Advantages and Disadvantages
Advantages Disadvantages
Applicability to point-of-care testing Res...
www.innovabiosciences.com
The basis of a lateral flow immunoassay
• Sample application pad
• Conjugate release pad
• Membr...
www.innovabiosciences.com
The sample application pad
• An absorbent pad on to which the sample is
applied
• Typically a ce...
www.innovabiosciences.com
The conjugate release pad
• An absorbent pad into which the detection
reagent has been dried
• E...
www.innovabiosciences.com
The membrane
• Typically composed of nitrocellulose
• Capillary flow time - time taken for liqui...
www.innovabiosciences.com
The wicking pad
• Functions to increase the volume of sample
which enters the test strip
• Helps...
www.innovabiosciences.com
The plastic cassette
• Ensures that the end user applies the sample only to the sample
applicati...
www.innovabiosciences.com
Assay formats
• Direct (sandwich) assay
• Larger analytes with multiple antigenic
sites
• Compet...
www.innovabiosciences.com
The detection reagent
• Typically antibodies which have been conjugated to gold
nanoparticles or...
www.innovabiosciences.com
The detection reagent – the antibody
• A consistent supply of good quality antibodies is essenti...
www.innovabiosciences.com
The detection reagent – the antibody
• The binding affinity of the antibody should be assessed
A...
www.innovabiosciences.com
The detection reagent – the detection moiety
• Gold nanoparticles or latex beads are the most co...
www.innovabiosciences.com
Preparation of the detection reagent
• There are several methods of attaching an antibody to a d...
www.innovabiosciences.com
Passive adsorption
• Relies on hydrophobic attractions and electrostatic interactions
between th...
www.innovabiosciences.com
Colloidal gold from Innova Biosciences
• Uniform spherical shape
• Narrow size distribution
• Di...
www.innovabiosciences.com
Colloidal gold from Innova Biosciences
• Stringently QC tested
• Consistent high quality
• Batch...
www.innovabiosciences.com
Covalent attachment to nanoparticles
• Covalent attachment provides a number of advantages
over ...
www.innovabiosciences.com
InnovaCoat® GOLD
• Gold nanoparticles with a proprietary surface
coating
• Covalent binding to f...
www.innovabiosciences.com
InnovaCoat® GOLD conjugation kits
• Quick and easy to use
• The conjugate is ready for use withi...
www.innovabiosciences.com
InnovaCoat® GOLD product range
• Different nanoparticle sizes, pack sizes and conjugation chemis...
www.innovabiosciences.com
InnovaCoat® GOLD
• InnovaCoat® GOLD targets primary amine (-NH2) groups
• AbPure™ kits for antib...
www.innovabiosciences.com
Latex bead conjugation kits
• Quick and easy to use
• The conjugate is ready to use within 35 mi...
www.innovabiosciences.com
Latex bead conjugation kits
• Blue, red and black latex conjugation kits available
• Ideal for m...
www.innovabiosciences.com
Other detection moieties
• Enzymes and fluorescent labels are alternative detection moieties
• E...
www.innovabiosciences.com
Attachment to pre-coated particles
• Occasionally it may be preferable to use pre-coated particl...
www.innovabiosciences.com
Pre-conjugated gold nanoparticles
• Manufactured using InnovaCoat® GOLD nanoparticles
• Ultra hi...
www.innovabiosciences.com
Basic troubleshooting
Issue Possible solution
Uneven lines  Use membrane with different pore si...
www.innovabiosciences.com
Custom services
• InnovaCoat® GOLD nanoparticle conjugate micro-optimization service
• Productio...
www.innovabiosciences.com
Summary
• Rapidly growing market, wide range of applications
• Innova Biosciences’ products to f...
www.innovabiosciences.com
Type in questions
Click to pop out the questions box
Q&A session
www.innovabiosciences.com
Thank you!
www.innovabiosciences.com
Get in touch..
For more information please get in touch or visit our website
www.innovabioscienc...
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A guide to lateral flow immunoassay development

This latest presentation on lateral flow immunoassay development will provide a general overview of lateral flow assays, take you through the components of a typical lateral flow test strip, and will provide you with detail on the different detection methods which are employed. We will also describe how our products and custom services can greatly simplify the development of your lateral flow assay.

To find out more about Innova Biosciences' lateral flow assay development services visit our website:
https://www.innovabiosciences.com/b2b/lateral-flow-assay-development-service.html

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A guide to lateral flow immunoassay development

  1. 1. www.innovabiosciences.com A Guide to Lateral Flow Immunoassay Development
  2. 2. www.innovabiosciences.com Our speaker today… Emma Easthope Technical Content Writer at Innova Biosciences
  3. 3. www.innovabiosciences.com Agenda • Overview of Lateral Flow assays • The components of a typical lateral flow test strip • Detection methods • Simplifying LFA development • Q&A session
  4. 4. www.innovabiosciences.com Type in questions Click to pop out the questions box Q&A session
  5. 5. www.innovabiosciences.com 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
  6. 6. www.innovabiosciences.com 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
  7. 7. www.innovabiosciences.com 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
  8. 8. www.innovabiosciences.com 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
  9. 9. www.innovabiosciences.com 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
  10. 10. www.innovabiosciences.com The basis of a lateral flow immunoassay • Sample application pad • Conjugate release pad • Membrane • Wicking pad • Plastic cassette
  11. 11. www.innovabiosciences.com 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
  12. 12. www.innovabiosciences.com 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
  13. 13. www.innovabiosciences.com 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
  14. 14. www.innovabiosciences.com 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
  15. 15. www.innovabiosciences.com 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
  16. 16. www.innovabiosciences.com Assay formats • Direct (sandwich) assay • Larger analytes with multiple antigenic sites • Competitive assay • Smaller analytes with a single antigenic determinant
  17. 17. www.innovabiosciences.com 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
  18. 18. www.innovabiosciences.com 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
  19. 19. www.innovabiosciences.com 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
  20. 20. www.innovabiosciences.com 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
  21. 21. www.innovabiosciences.com 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
  22. 22. www.innovabiosciences.com 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
  23. 23. www.innovabiosciences.com 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 
  24. 24. www.innovabiosciences.com Colloidal gold from Innova Biosciences • Stringently QC tested • Consistent high quality • Batch-to-batch reproducibility • Detailed Certificate of Analysis supplied with every batch
  25. 25. www.innovabiosciences.com 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
  26. 26. www.innovabiosciences.com 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, or separately as carboxylated gold nanoparticles TEM image of a 40nm InnovaCoat® GOLD coated nanoparticle
  27. 27. www.innovabiosciences.com 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
  28. 28. www.innovabiosciences.com 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
  29. 29. www.innovabiosciences.com 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
  30. 30. www.innovabiosciences.com 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
  31. 31. www.innovabiosciences.com Latex bead conjugation kits • Blue, red and black latex conjugation kits available • Ideal for multiplexing • Stringently QC tested
  32. 32. www.innovabiosciences.com 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
  33. 33. www.innovabiosciences.com 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
  34. 34. www.innovabiosciences.com 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
  35. 35. www.innovabiosciences.com 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
  36. 36. www.innovabiosciences.com 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
  37. 37. www.innovabiosciences.com 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 www.innovabiosciences.com
  38. 38. www.innovabiosciences.com Type in questions Click to pop out the questions box Q&A session
  39. 39. www.innovabiosciences.com Thank you!
  40. 40. www.innovabiosciences.com Get in touch.. For more information please get in touch or visit our website www.innovabiosciences.com +44 (0) 1223 661000 www.linkedin.com/company/innova-biosciences@InnovaBioSciwww.facebook.com/InnovaBiosciences

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