ELISA. An introduction to the basic principles and assay formats. Innova Biosciences ltd All rights reserved

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1 ELISA An introduction to the basic principles and assay formats

2 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 Helene Fransolet International Distributor Manager and Sales Executive at Innova Biosciences

4 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

5 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

6 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

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

8 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

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

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

11 Activity 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 Quantitation: Concentration

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

13 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

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

15 Screening assay utilising ELISA

16 Simple Antibody Labeling Kits 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

17 Lightning-Link 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)

18 Lightning-Link

19 Immuno PCR 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

20 Contact If you would like any more information, please contact us at Innova Biosciences

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