The document provides an overview of enzyme-linked immunosorbent assays (ELISA), including their principles, various assay formats, and development tips. It discusses the advantages and disadvantages, along with troubleshooting methods, and highlights the Lightning-Link technology for antibody labeling. Key information on data analysis and different detection methods is also included to aid in effective assay implementation.

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ELISA
An introduction to the basic principles
and assay formats

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Agenda
Agenda:
• Introduction to ELISA
• The different assay types
• Assay development tips
• Data analysis tips
• Troubleshooting tips
• Indirect vs. Direct antibody detection
• Using Lightning-Link for primary antibody labeling
• Immuno PCR

3. © Innova Biosciences ltd. 2014. All rights reserved
Helene Fransolet
International Distributor Manager and
Sales Executive at Innova Biosciences

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Introduction
ELISA:
• Enzyme-Linked Immunosorbent Assay
• Immunoassay utilising antibodies linked to enzymes for detection by colour
change
• Evolved from RIA in the 1960s
• Antibody or analyte being detected is absorbed to a solid surface, meaning
unbound materials can be washed away with ease
• With time, more techniques were developed and ELISA is now used to
describe any assay where a molecule is absorbed on a solid phase

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Introduction
Advantages:
• Qualitative and Quantitative assay
• Screen a large number of samples in one go
• Relatively easy to perform and to transfer to an automated format
Disadvantages:
• No information on the biochemical properties of the analyte being detected
• Other techniques such as Western Blotting or IHC need to be employed

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Introduction
Basic protocol:
• Coat solid phase with antibody or analyte
• Coat the remainder of the solid phase binding sites with blocking agent
• Add analyte (1) or anti-analyte antibody (2)
• Wash the plate
• Add anti-analyte enzyme linked antibody (1) or add anti-Ig enzyme linked
antibody (2)
• Wash the plate
• Add the substrate corresponding the to enzyme to induce a colour change

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Assay Formats
Capture (Sandwich) vs Direct:
Capture Direct
Antibody is immobilised Analyte/antigen is immobilised
The analyte/antigen is being detected Immune response is being detected

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Assay Formats
Choosing antibodies as sandwich pairs
Capture antibody Detection antibody Result
Monoclonal (epitope A) Monoclonal (epitope B) 
Monoclonal (epitope A) Monoclonal (epitope A)  (*)
Monoclonal Polyclonal 
Polyclonal Polyclonal 
Polyclonal Monoclonal 
* Antigen with repeated epitopes is an exception – e.g. CRP

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Assay Formats
Direct vs Indirect detection:
Indirect Direct
Labelled secondary antibody (or
Streptavidin) is used
Detection antibody is labelled
Additional wash steps required Reduced number of wash steps
Using secondary antibodies: Using primary antibodies
only:

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Assay Formats
Competitive vs non-competitive:
Competitive
Unknown amount of analyte from the
sample
Reference amount of analyte also present
Limited number of binding sites
Reference and sample analytes compete
Quantitation:

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Assay Formats
Competitive vs non-competitive:
Non- competitive
Unknown amount of analyte from the
sample
No reference amount of analyte
Antibody binding sites present in excess
Proportional assay
Activity
Concentration
Quantitation:

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Assay development
Tips:
• What is being detected?
• Is it a qualitative or quantitative assay?
• What is being measured?
• What level of sensitivity is required?

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Data Analysis
Tips:
• Controls are required
• A standard curve is required for quantitative assays
• Use replicates to generate robust data points
• Assay range must be established
• Determine the limit of detection

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Troubleshooting
• High background
• No signal
• Too much signal
• Poor standard curve
• Poor duplicates
• Poor reproducibility
• Edge effect

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Screening assay utilising ELISA

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What is Lightning-Link® technology?
The worlds fastest, easiest to use and most efficient conjugation technology!
• Only 30 seconds hands-on time!
• Over 50 labels available including:
Enzymes, fluorescent proteins / dyes, tandems, biotin & streptavidin
Antibodies – Proteins – Peptides
Fast – Easy-to-use – Reliable
Simple Antibody Labeling Kits

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What is Lightning-Link® technology?
• Chemistry expertise not required
• 100% antibody recovery
• Pack sizes range from 10ug, 100ug up to 5+mg
• Covalent conjugation ensures long-term stability
• Two ranges Lightning-Link® (3 hours incubation) and Lightning-Link® RAPID
(15 minutes)
Lightning-Link®

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Lightning-Link®

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 Oligonucleotide replaces the enzyme
 Same protocol as a normal ELISA
 Finish off with PCR to amplify the signal
 Increases sensitivity
 Use our Thunder-Link kit to easily conjugate your antibody to
your oligonucleotide of choice
Immuno PCR

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Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
facebook.com/InnovaBiosciences
@InnovaBioSci
Innova Biosciences

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Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries