ELISA Enzyme Linked Immunosorbent Assay

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

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.

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. Antibodies Proteins secreted by B-lymphocytes (type of white blood cell), in vertebrates. IgG molecule Fab fragments Fc fragments

Basic steps of ELISA Enzyme 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

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

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

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

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

Enzymes used in ELISA Peroxidase from horseradish Alkaline phosphatase from E. coli  -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.

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

Type of ELISA

Sandwich ELISA

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

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.

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

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.

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.

Immunofluorescence

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.

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

Direct immunofluorescence

2.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

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

Indirect immunofluorescence of iron-regulated cell wall mannoprotein FIT1 of S. cerevisiae

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

Immunofluorescence image of Cryptosporidium parvum oocysts

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

Radioimmunoassay (RIA): A Remarkably Sensitive Bioassay

Biochemistry Principles

Radioimmunoassay Procedure

Standard Curve & Unknown Sample

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

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

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

Increasing Assay Precision

Sensitivity – Decrease [Ligand]

Sensitivity – Decrease [Binder]

Comparison of Assays Enzymeimmunoassays (EIA) –millimolar Fluoroimmunoassays (EIA) –micromolar Radioimmunoassays (EIA) –nanomolar to picomolar

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

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

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