Biochemistry clinical practice CLS 432 ENZYME LINKED IMMUNO SORBENT ASSAY (ELISA) & RADIO IMMUNO ASSAY(RIA) Dr. Samah Kotb Nasr El-deen Biochemistry clinical practice CLS 432 Lecturer of Biochemistry 2015

ENZYME LINKED IMMUNOSORBENT ASSAY

CONTENTS ELISA RIA Introduction to ELISA Principle & procedure Materials needed Types of ELISA Advantages & disadvantages of ELISA Applications RIA Introduction to RIA Principle & procedure Materials needed Advantages & disadvantages of RIA Instrumentation Applications

INTRODUCTION TO ELISA ELISA, or enzyme-linked immunosorbent assay, are quantitative immunological procedures in which the Ag-Ab reaction is monitored by enzyme measurements. The term ELISA was first used by Engvall & Perlma in 1971. The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.

Why known as ......? 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.

BASIC PRINCIPLE OF ELISA Use an enzyme to detect the binding of antigen (Ag) antibody (Ab). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag : Ab binding. An ELISA can be used to detect either the presence of Antigens or antibodies in a sample depending how the test is designed. ELISA was dveloped in 1970 and became rapidly accepted.

Materials Needed Testing sample. Antibody (1st, 2nd) / Antigen. Polystyrene microtiter plate. Blocking buffer. Washing buffer. Substrate. Enzyme.

ANTIGEN (Ag) Any molecule that induces production of antibodies when introduced in the body of an animal is called antigen. OR any “thing”, foreign to the immune system. e.g. bacteria, viruses, (or their parts), pollen, etc. Protein molecule. Carbohydrate molecule. Microorganisms. Allergens. Viruses Etc. SYMBOL FOR ANTIGEN

ANTIBODY ( Ab) Antibody: proteins produced by the immune system which help defend against antigens. SYMBOL FOR ANTIBODY Y

Antibodies (Immunoglobulins)

Specimen Sample For ELISA SERUM CSF SPUTUM URINE SEMEN SUPERNATANT OF CULUTRE STOOL

Enzymes Used in Elisa Horseradish peroxidase (most commonly used). Alkaline Phosphatase. β-galactosidase. Lactoperoxidase. Tetra Methyl benzidine is a chromogenic substrate used in staining procedures in immunohistochemistry .

In case of peroxidase, the substrate hydrogen peroxide is converted into water and o2 in the presence of electron donors(like diaminobenzidine or 4-chloronaphthol which themselves oxidized in the reaction). Oxidation of diaminobenzidine produces dark brown color while that of 4-chlorornaphthol yields purple color which is the basis of ELISA.

ENZYME SUBSTRATE Initially the substrate should be colorless After degradation by the enzyme it should be strongly colored or fluorescent. ENZYME SUBSTRATE CHROMOGEN STOPPING Alkaline Phosphatase p-NPP p-NPP+ diethandamine +MgCl2 1 M NaOH Horse radish Peroxidase H2O2 Tetramethylbenzidine + Phosphate – Citrate buffer 1 M H2SO4 O – Phenylenediamine + HCl 1 M HCl

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

Basic Steps Of Enzyme-Linked Immunosorbant Assay Review activity steps

TYPES OF ELISA Indirect Elisa Sandwich Elisa Competitive Elisa

Indirect Elisa

Sandwich ELISA Antigens such as tumor markers, hormones and serum proteins may be determined Antigen in the sample binds with the capture antibody on the microwell and becomes immobilized. The antibody of the enzyme conjugate binds with the immobilized antigen to form a sandwich of antibody-antigen-antibody/enzyme bound to the microwell. Enzyme reaction product is directly proportional to concentration of standard or analytical antigen

enzyme produces colour ELISA SANDWICH FORMAT Y Y Y Y Y Y enzyme produces colour Antibody 2nd antibody with enzyme Y Y Y Y Y Y Y Antibody/Antigen

Competitive Elisa Primary antibody (unlabeled) is incubated with sample antigen. Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the same antigen. Unbound antibody is removed by washing the plate. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.") The secondary antibody that is specific to the primary antibody  and conjugated with an enzyme is added. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

Competitive Elisa Increased serum antigen results in reduced binding of the antigen-enzyme conjugate with the capture antibody producing less enzyme activity and color (yellow) formation Substrate product concentration is inversely proportional to the concentration of standard or test antigen added

Comparison between Indirect Sandwich & Competitive ELISA to detect Ab (HIV, HCV) to detect Ag ( Tumor Markers, Hormones ) to detect Ag ( Free Testosterone)

Results

Importance of incubation step During the test performance incubation time and mentioned temperature is must required For the proper binding between antigen and antibody and also binding with conjugate and color development of substrate. Importance of Washing :- For the removal of any unbound Antibody/Antigen proper washing and taping is required other wise we get the incorrect result. So incubation & washing is much important for good results.

Elisa Plate Microtitre wells. Generally 96 wells. Marked on one side alphabetically. Numerically on the other side. Comes with the kit.

TEST PERFORMANCE  Incubate 1 hour at 37°C .  Using a clean Pipette , add 100 µL of diluted serum sample (Dilute the sera to be tested 1:100 in the sample diluents) to each well.  Incubate 1 hour at 37°C .

ELISA PLATE READY FOR READING Measures the absorbance at 450 nm With the help of ELISA READER. Calculate the absorbance for each sample and reference. We used Ascent Software for Calculation of the result

Advantages of ELISA Reagents have a long shelf life. ELISA is highly specific and sensitive. No radiation hazards occur during labelling or disposal of waste. Easy to perform and quick procedures. Equipment can be inexpensive and widely available. ELISA can be used to a variety of infections.

Disadvantages of ELISA Measurement of enzyme activity can be more complex than measurement of activity of some type of radioisotopes. Enzyme activity may be affected by plasma constituents. Kits are commercially available, but not cheap Very specific to a particular antigen. Won’t recognize any other antigen. False positives/negatives possible, especially with mutated/altered antigen.

Limitations Results may not be absolute. Antibody must be available. Concentration may be unclear. False positive possible. False negative possible.

APPLICATIONS OF ELISA 2) Detection of rotavirus in feces. 1) Detection of Mycobacterium antibodies in tuberculosis. 2) Detection of rotavirus in feces. 3) Detection of hepatitis B markers in serum. 4) Detection of HIV antibodies in blood samples 5) It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts , walnuts, almonds, and eggs.

APPLICATIONS OF ELISA 1- Hormones 7- Vaccine Quality Control 2- Proteins 8- FOR GMO (Genetically modified organism) 3- Infectious Agent ( Viral, Bacterial, Parasitic, Fungal ) 9- For Rapid Test 4- Drug Markers 10- IgG, IgM, IgA 5- Tumor Markers 11- In New Born Screening 6- Serum Proteins 12- In Clinical Research

Equipments for performing the ELISA test Pipettes Incubator ELISA reader

THERMOLAB SYSTEM (USA) ELISA READER THERMOLAB SYSTEM (USA)

Radioimmunoassay

INTRODUCTION Radioimmunoassay (RIA) is a very sensitive in vitro assay  technique used to measure concentrations of antigens  (for example, hormone levels in the blood) by use of antibodies. The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy. To perform a radioimmunoassay, a known quantity of an antigen  is made radioactive, frequently by labeling it with gamma-radioactive isotopes  of iodine  attached to tyrosine .

This radiolabeled antigen is then mixed with a known amount of antibody  for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum  from a patient containing an unknown quantity of that same antigen is added.

This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites. As the concentration  of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant  is measured using a gamma counter.

Principle of Radioimmunoassay Principle: Uses an immune reaction [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab  AgAb + Ag*Ab + Ag + Ab* Unbound Ag* and Ag washed out. Radioactivity of bound residue measured. Ligand conc. is inversely related to radioactivity. [Ag : ligand to be measured ; Ag* radiolabelled ligand]

Advantages & Disadvantages of RIA Highly specific: Immune reactions are specific. High sensitivity : Immune reactions are sensitive. Disadvantages Radiation hazards: Uses radiolabelled reagents. Requires specially trained persons. Labs require special license to handle radioactive material. Requires special arrangements for Requisition, storage of radioactive material. radioactive waste disposal.

Requirements for RIA Preparation & characterisation of the Antigen [Ligand to be analysed] . Radiolabelling of the Antigen. Preparation of the Specific Antibody. Development of Assay System.

Preparation & Radiolabelling of the Antigen Antigens prepared by.. Synthesis of the molecule. Isolation from natural sources. Radiolabelling [Tagging procedure] (3 H 14 C 125 I) are used as radioactive tags. Antigens are tagged to 3 H 14 C 125 Tagging should NOT affect Antigenic specificity & Antigenic activity !

Preparation of the Specific Antibody Antigen injected intradermally into rabbits or guinea pigs  antibody production. Antibodies recovered from the serum. Some ligands are not Antigenic Hormones, Steroids, Drugs  HAPTENS Eg: Gastrin, Morphine, Haptens conjugated to albumin  antigenic

Development of the Assay System A crucial step is separation of unbound antigens. This achieved by binding the antibodies to the microtitre well surface [Solid phase RIA]. Antigens bound to the fixed antibodies remain stuck to the inner surface. Decanting & washing the well removes unbound antigens. Other techniques of separation: Centrifugation.

Assay Procedure Add known amounts of the test sample + labelled antigen into the microtitre wells. Incubate  allow the reaction to reach completion. Decant & wash contents of the well  removes all unbound antigens. Radioactivity remaining in the Microtitre wells measured by a Counter [GM counter , Scintillation counter etc] Intensity of radioactivity is inversely correlated with the conc of antigens in the test sample. Sensitive to very low conc of antigens.

Antibodies: types of labelling -Radio-isotopes, -Enzymes Fluorescent Chemi-luminescent probes Metal tags

Radioimmunoassay (RIA) Advantages Flexibility Sensitivity Size Disadvantages Toxicity Shelf life Disposal costs

radiation (usually beta) INSTRUMENTATION Radiation will hit silver grains in emulsion and expose them Expose to film or emulsion Isotope will emit radiation (usually beta) Incubate tissue with radioactive ligand Autoradiography

INSTRUMENTATION