ELISA Enzyme Linked Immunosorbent Assay

1. ELISA Enzyme Linked Immunosorbent Assay

2. What is an ELISA? • Enzyme-linked immunosorbent assay • Name suggests three components • Antibody • Allows for specific detection of analyte of interest • Solid phase (sorbent) • Allows one to wash away all the material that is not specifically captured • Enzymatic amplification • Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader

3. What is ELISA? Technique used to detect (assay) specific molecules (e.g. proteins & carbohydrates) in samples.  Immunological technique: uses antibodies.  Quantitative.  Very sensitive.  Commonly used in medicine and scientific research.

4.  Proteins secreted by B-lymphocytes (type of white blood cell), in vertebrates. Antibodies Recognise and bind to molecules (antigens) on foreign particles, marking them for destruction by T-lymphocytes.  Each antigen may generate several antibodies for different sites (epitopes) on antigen. Fab fragments Fc fragments IgG molecule

5. Basic steps of ELISAEnzyme Linked Immunosorbent Assay 1. Antigen of interest is absorbed on to plastic surface (‘sorbent’). 2.Antigen is recognised by specific antibody (‘immuno’). 3. This antibody is recognised by second antibody (‘immuno’) which has enzyme attached (‘enzyme-linked’). 4. Substrate reacts with enzyme to produce product, usually coloured. Coloured product = measure (assay) of antigen present

6. Secondary antibody Enzyme Coloured product Primary antibody Substrate Different antigens in sample

7. Monoclonal antibody production (hybridoma technology) Inject mouse with antigen Grow mouse myeloma (tumour) cells in culture Obtain Mouse spleen B-lymphocytes Fuse B-lymphocytes with myeloma cells Antibody-producing hybridoma cells

8. B-lymphocyte and myeloma mixture Unlimited supply of antibody specific for single epitope Keep clone producing antibody which best detects antigen Select fused and reproducing hybridoma cells via growth medium ? Make clones from individual antibody-producing cells Screen hybridomas for antibody production

9. Mouse serum injected into a different species, e.g. rabbit, goat. Polyclonal antibodies which can recognise any mouse antibody Animal makes various antibodies against the different antigens in serum Select anti-mouse antibodies from plasma Take blood from animal Secondary antibody production

10. Enzymes used in ELISA Peroxidase from horseradish Alkaline phosphatase from E. coli b-galactosidase from E. coli React with a colourless substrate to produce a coloured product. Must work fast at room temperature so the colour develops quickly.  Have minimal interference from factors in sample.

11. 8. Observe colour development 7. Add substrate for enzyme 1. Add antigen 2. Wash with PBST (detergent) 6. Wash with PBST 4. Wash with PBST 3. Add primary antibody 5. Add secondary antibody

12. Type of ELISA

13. Sandwich ELISA

14. Competitive ELISA • Less is more. More antigen in your sample will mean more antibody competed away, which will lead to less signal

15. Uses of ELISA outside the classroom Disease detection in people, animals and plants (e.g. HIV in humans).  Detection of allergens in food, e.g. peanuts.  Detection of illegal drugs in humans.  Detection of hormones, e.g. pregnancy testing kits.

16. ELISA in the curriculum Higher Biology, Biotechnology and Human Biology E.g. Biotechnology: production & use of monoclonal antibodies  Advanced Higher Biology: Biotechnology Unit Environmental Biology Unit Investigations

17. Advanced Higher Biology Investigation ideas Detection of Botrytis in fruit and vegetables from market or garden. Quantification of Botrytis as infection develops.  Detection of Botrytis in tissue before symptoms are observed.  Investigation on effect of temperature on rate of Botrytis development.

18.  Based in the Institute of Cell Biology at the University of Edinburgh. Role: to enhance engagement with biotechnology through interactions with the scientific community, school students, teachers and the general public.

19. Immunofluorescence

20. Introduction: • Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. • This technique is sometimes used to make viral plaques more readily visible to the human eye. • Immunofluorescent labeled tissue sections are studied using a fluorescence microscope. • Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.

21. There are two ways of doing IF staining • Direct immunofluorescence • Indirect immunofluorescence 1. Direct immunofluorescence • Ag is fixed on the slide • Fluorescein labeled Ab’s are layered over it • Slide is washed to remove unattached Ab’s • Examined under UV light in an fluorescent microscope • The site where the Ab attaches to its specific Ag will show apple green fluorescence • Use: Direct detection of Pathogens or their Ag’s in tissues or in pathological samples

22. Direct immunofluorescence

23. Indirect immunofluorescence: • Indirect test is a double-layer technique • The unlabelled antibody is applied directly to the tissue substrate • Treated with a fluorochrome-conjugated anti-immunoglobulin serum

24. Advantage over direct IF • Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test. • It is also more time-efficient since it is only one signal labelled reagent, the anti-immunoglobulin, is prepared during the lengthy conjugation process

26. Indirect immunofluorescence of iron-regulated cell wall mannoprotein FIT1of S. cerevisiae

28. Confocal image to detect phosphorylated AKT (green) in cardiomyocytes infected with adenovirus

29.  Immunofluorescence image of Cryptosporidium parvum oocysts       

32. Concentrated groundwater methanotrophic bacteria on 0.2 m m filter labeled with fluorescent antibodies.

33. Radioimmunoassay (RIA):A Remarkably Sensitive Bioassay

34. Biochemistry Principles

35. Radioimmunoassay Procedure

36. Standard Curve & Unknown Sample

37. Characteristics of Binder and Ligand • Availability • Synthetic • Natural – Produced From Animals • Monoclonal Antibodies • Purity – Competing Reactions with Impurities • Stability – Store in Albumin Serum • Specificity – Binding Constant

38. Characteristics of Tracer • Must Have Similar Binding Properties as Unlabeled Ligand • Internally Labeled Ligand • 14C and 3H • Externally Labeled Ligand • 131I and 125I • By-Products or Incomplete Synthesis • Purification by chromatography (gel filtration)

39. Separation of Bound and Free Ligand • Electrophoresis • Gel Filtration • Adsorption Chromatography • Fractional Precipitation • Centrifugation • Filtration • Partition Chromatography • Dialysis

40. Increasing Assay Precision

41. Sensitivity – Decrease [Ligand]

42. Sensitivity – Decrease [Binder]

43. Comparison of Assays • Enzymeimmunoassays (EIA) • millimolar • Fluoroimmunoassays (EIA) • micromolar • Radioimmunoassays (EIA) • nanomolar to picomolar

44. Applications of Radioimmunoassays • Endocrinology • Insulin, HCG, Vasopressin • Detects Endocrine Disorders • Physiology of Endocrine Function • Pharmacology • Morphine • Detect Drug Abuse or Drug Poisoning • Study Drug Kinetics

45. Applications of Radioimmunoassays • Epidemiology • Hepatitis B • Clinical Immunology • Antibodies for Inhalant Allergens • Allergy Diagnosis • Oncology • Carcinoembryonic Antigen • Early Cancer Detection and Diagnosis

46. Summary • Based on Simple Biochemistry Principles • Establish Ideal Binder and Ligand • Synthesize Tracer Ligand • Separation of Bound and Free Parts • High Precision and Sensitivity • Powerful Applications to a Wide Range of Medical Fields