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 and is useful & powerful method in estimating ng/mL to pg /mL ordered materials in the solution .

Why known as ......? Enzyme Linked Immunosorbent Assay

1. Antigen of interest is absorbed on to plastic surface ( sorbent ). 2. Antigen is recognized by specific antibody (immuno). 3.This antibody is recognized 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 developed in 1970 and became rapidly accepted.

Principle of ELISA
Substrate Secondary antibody

Coloured product Primary antibody

Different antigens in sample

ELISA Qualitative/Quantitative
Qualitative determines antigen or antibody is present or absent. Quantitative determines the quantity of the antibody the highest dilution of the specimen usually serum which gives a positive reaction in the test

ENZYME SUBSTRATE
Initially the substrate should be colorless. After degradation by the enzyme it should be strongly colored or fluorescent.

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.

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

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

Specimen Sample For ELISA

SERUM CSF SPUTUM URINE SEMEN SUPERNATANT OF CULUTRE STOOL

PRINCIPLE OF INSTRUMENT

TYPES OF ELISA
Competitive assay Non-competitive assay

Competitive Elisa
used to determine small molecule antigens.(T3,T4,progesterone etc.) antibody coated microwell serum antigen and labelled antigen added together in competition. antibody-antigen-enzyme complex bound is inversely related to the concentration of antigen present in the sample.

. Contd
the bound enzyme conjugate reacts with the chromogenic substrate added to produce a color reaction (blue to yellow color). . 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 added

Noncompetitive
Direct Assay
Antigen capture ELISA Antigen adsorbed directly detected by labeled enzyme Antibody capture ELISA Antibody adsorbed directly by labeled enzyme.

Indirect Assay
Antigen directly adsorbed onto the solid phase is first incubated with patient serum, and then with a labeled antibody specific for human immunoglobulin In In-direct ELISA color change is directly proportional to the concentration of specific antibodies in specimen

Sandwich Assay
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-antigenantibody/enzyme bound to the microwell. Enzyme reaction product is directly proportional to the concentration of standard or analytical antigen

Results

Enzymes Used in ELISA

Horseradish peroxidase (most commonly used) Alkaline Phosphatase - galactosidase Lactoperoxidase Tetra Methyl benzidine In case of peroxidase, the substrate hydrogen peroxide is converted into water and O2 in the presence of electron donors . (like diaminobenzidine or 4chloronaphthol 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

ELISA Plate
Microtitre wells Generally 96 wells Marked on one side

alphabetically
Numerically on the other side. Comes with the kit

TEST PERFORMANCE
Using a clean Pipette , add 100 L of diluted serum sample (Dilute the serum to be tested 1:100 in the sample diluents) to each well. Incubate 1 hour at 37C After incubation empty out contents of wells into waste container. Using pipette to fill the wells with washing buffer then drain out after sometime. Tap wells upside down on paper towel to remove all droplets. Wash the wells 5 times. At the end of the washing process, the wells must be entirely dry after the last wash. Distribute 100 L of anti-human immunoglobulin conjugate in each well. Incubate 30 minutes at 37C.

RESULT DETERMINATION
A) IgA and IgE Tests:-Plot the O.D. result of each reference , except for the negative reference on the vertical axis (Y-axis) in relation to the number of corresponding unit on the horizontal axis (X-axis). Using the absorbance value for each sample , determine the corresponding concentration of antibodies expressed in units/ml from the reference curve. (B) IgM Tests :-We can calculate the cut off value A450nm sample / A450nm Positive limit reference. The normalized value of the positive reference is 1 . All samples whose value is comprised between 0.8 and 1.0 are considered dubious and all samples whose normalized value is above 1.0 are considered positive for IgM antibodies.

Advantages of ELISA
Reagents are relatively cheap & have a long shelf life. ELISA is highly specific and sensitive No radiation hazards occur during labeling 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
Hormones

Proteins Infectious Agent ( Viral, Bacterial, Parasitic, Fungal ) Tumor Markers Serum Proteins Vaccine Quality Control For GMO IgG, IgM, IgA In New Born Screening In Clinical Research

FLUORESCENCE ACTIVATED CELL SORTER

 Discovery: The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg for cell sorting of viable cells at University of Stanford, USA. The term FACS was coined by Len Herzenberg who won the Kyoto Prize in 2006 for his work in flow cytometry. It is based on the Coulter principle which can be applied to count and size the various cells that make up whole blood.

What is fluorescence???
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. A wide range of fluorophores can be used as labels in flow cytometry. These each have a characteristic peak excitation and emission wavelength. Also, the emission spectra of the labels often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available.

Introduction: Fluorescence-activated cell sorting is a specialized type of flow cytometry in which microscopic particles such as cells and chromosomes has been counted and examined by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. It is a powerful tool which enumerated lymphocytes by detecting and counting individual cells passing in a stream through a laser beam.

 Fluorescence-activated cell sorter provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.

Multiparametric analysis Total DNA content (cell cycle analysis, cell kinetics, proliferation etc. Total RNA content DNA copy number variation (by Flow-FISH) Protein expression and localization Protein modifications, phospho-proteins Cell surface antigens (CD markers) Intracellular antigens (cytokines, secondary mediators) Nuclear antigens Apoptosis (measurement of DNA degradation) Cell viability Membrane fluidity Protein expression, localization and Protein modifications Characterizing multidrug resistance (MDR) in cancer cells

 Principle: A beam of light, usually laser light, of a single wavelength is directed onto a hydrodynamically focused stream of fluid. The purpose of fluidics system is to transport particles in a fluid stream for laser beam interrogation, with only one cell or particle moving through the laser beam at any given moment. The flow of sheath fluid accelerates the particles and restricts them to the center of the sample core, in a process of hydrodynamic focusing.

 Two light beams, one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) is equipped with and one or more fluorescent detectors. Each suspended particle from 0.2 to 150 m passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source.

 This combination of scattered and fluorescent light is picked up by the detectors, and, by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak). Now it is possible to derive various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).

COMPONENTS
A flow cell - liquid stream (sheath fluid), which carries and aligns the cells so that they pass single file through the light beam for sensing. An optical system commonly used are measurement of impedance (or conductivity) and optical systems - lamps (mercury, xenon); high-power water-cooled lasers (argon, krypton, dye laser); lowpower air-cooled lasers (argon; 488 nm), red-HeNe (633 nm), green-HeNe, HeCd (UV); diode lasers (blue, green, red, violet) resulting in light signals.

 A detector and Analogue-to-Digital Conversion (ADC) system - which generates FSC (Forward Scatter) and SSC (Side Scatter) as well as fluorescence signals from light into electrical signals that can be processed by a computer An amplification system - linear or logarithmic A computer for analysis of the signal

Flow Cell
Injector Tip

Sheath fluid

Fluorescence signals Focused laser beam

Fluorescence Activated Cell Sorting

488 nm laser

FALS Sensor Fluorescence detector

Charged Plates
Single cells sorted into test tubes

Forward Angle Light Scatter

Laser
FALS Sensor

90 Degree Light Scatter

Laser
FALS Sensor

90LS Sensor

DATA ANALYSIS
Gating: The data generated by flow-cytometers can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates." Specific gating protocols exist for diagnostic and clinical purposes especially in relation to hematology.

Computational analysis: Recent progress on automated to population gating rare and identification strategies. hidden using computational methods has offered an alternative traditional of Automated populations. include identification systems could potentially help findings Representative Portal (ImmPort). automated methods

FLOCK in Immunology Database and Analysis

APPLICATIONS
It has broad application in medicine (especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting for sex preselection). Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. It can be used to sort out sperm that are most healthy. The resultant 'sex-sorted' spermatozoa are then able to be used in conjunction artificial insemination or in-vitro fertilization (IVF) to produce offspring of the desired sex.

 In protein engineering: Flow cytometry is used in to identify cell surface-displayed protein variants with desired properties by yeast display. In cell trafficking: Flow cytometry is widely utilized in lymphocyte trafficking by using intravital microscopy (IVM). IVM is a technique by which green fluorescent protein GFP expressed on a CD45 to see all cells with this receptor expressed, and we could compare levels between before and after counting antigen stimulation and experiments, extrapolating. through lymphocytes

 In Blood Cancers and AIDS: Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers and AIDS to count the population of lymphocytes. Purification of organelles: Since the organelle is surrounded by each type of organelle-specific proteins, FACS are very useful in purifying organelles and vesicles, particularly those that have a similar size and density.

Hybridoma technology mAb production

.and ..

HYBRIDOMA TECHNOLOGY
y A hybridoma is a hybrid cell obtained by fusion of B lymphocyte with usually a tumor cell of antibody forming system or b lymphocyte,(these are called myelomas). y The hybrid cell thus formed posses ability to produce antibodies due to B lymphocyte genome and the capability for the indefinte growth in vitro due to tumor(myeloma)cell involved in the fusion. y Therefore ,specific hybridomas are either cultured in vitro or passed through mouse peritoneal cavity to obtain monoclonal antibodies,this is called as hybridoma technology.

PRINCIPLE
y Antibodies are produced by B lymphocyte,each B

lymphocyte being pre programmed to respond to a single antigenic determinant. Antigenic determinant denotes the region of antigen molecule which reacts with an antibody that is specific to it. When an antigen reacts to the cell surface reactor of B lymphocyte,it proliferates rapidly to yield a population of B cells ,all of which produces antibodies of same specificity,this is called as clonal selection .

Thus a B lymphocyte produces antibodies of only one specificity. In addition , an antibody producing B lymphocyte cells called plasma cell is fully differentiated and does not divide when cultured in vitro,these features are critical to hybridoma technology. y B lymphocyte are isolated from spleen of an animal eg mouse wich had been immunized with antigen against which monoclonal antibodies are to be raised. Immunization is achived by injecting the antigen along with the suitable adjuvant either subcutaneously or in peritoneal cavity followed by the booster doses of antigen.

y Immunization enhances the population of B lymphocytes producing antibodies specific to antigen used(clonal selection),which greatly increases the chance of obtaining desired hybridoma clone. A large no. of these b lymphocytes are mixed with cells of selected myeloma and induce to fuse to form hybrid cell. y Myeloma cells are selected for mainly 2 features: 1:These cells must not produce antibodies themselves. 2:They must contain a genetic marker egHGPRT- trait,which permits an easy selection of resulting hybrid cells.

Improvements in hybridoma technology

y A continuous cell line was used as a fusion partner for the antibody producing B cell y Feeder layers consisting of extra cells to feed newly formed hybridomas were used for optimal growth and hybridoma production. y The most common feeder layers consisted of the following:
y Murine peritoneal cells y Macrophages derived from mouse,rat,or guinea pigs .. y Human fibroblast peripheral blood monocytes or thymus cells

Immunisation

Mice are immunised with Ag

Spleen cell,lymphocytes suspension is obtained .

Fusion

Lymphocytes are fused with myeloma cells

Myeloma cells are malignant lymphocytes . Cells are fused using PEG(polyethyleneglycol) to promote membrane fusion ..

Culture in HAT
T e e i i ic t e c lt re is

.
ec tai s . yp xa t i e,a i pteri ,t y i i e .

Myel

a cells are e gi eere t be eficie t i a e zy e yp xa t i e g a i e p tra sferase(HGPRT) ..

sp

rib syl

He ce t e

yel

a cells

t s rvive i c lt re

less t is e zy e is a

e t t e

e ia

O ly ybri cells s rvive

Splee are s

rt live i.e, t eir life spa is 7-10 ays,s t ey ie gra

HAT kills

yel

a cells ..

ally ..

..

The cells are cultured in HAT medium which is lacking the enzyme HGPRT

The powerful toxins of the media block the metabolic pathway Denovo pathway..essential for purine metabolismin DNA synthesis

Fused cells survive

Hybridoma cells consisting of immortalized plasma cells fused with B cells will survive in the absence of supplemented HGPRT in culture medium

Non fused, - HGPRT plasma cells and all non fused B cells die soon.

Screening
y ELISA y RIA y WESTERN BLOT

The application of monoclonal antibodies can be broadly categorized as:

a)

Diagnostic application b) Therapeutic application c) Drug targeting d) Miscellaneous application

Catalytic MAb (Abzymes) Auto antibody finger printing

MAbs are utilized in diagnostic kits for the diagnosis of various infectious diseases, detecting pregnancy, monitoring drug levels, matching histocompatibility antigen, detecting diabetes and cancer . Also used for diagnostic imaging (immunoscintigraphy) in cardiovascular diseases, bacterial infections, cancer. FDA licensed a new diagnostic imaging agent that can determine the extent of disease in patients diagnosed with small cell lung cancer (SCLC). Because these agents can detect tumor in different part of the body at one time, it can help physician to advice certain patients with advanced forms of the disease about treatment option without requiring further diagnostic tests. The new agent, Nofetumomab, is a fragment of a monoclonal antibody that when tagged with the radioisotope technique, can detect a protein found on the surface of most small lung cancer cells.

Improving the out come of bone marrow transplantation by using CD52 MAbs to prevent Graft-Versus-Host disease and Graft rejection. Graft-versus-Host Disease (GVHD) is a major cause of mortality and mobidity after allogenic bone marrow transplantation, but can be avoided by removing T-lymphocytes from the donor bone marrow. However, T-cell depletion increases the risk of graft rejection. This study examined the use of CD52 MAb to eliminate T- cells from both donor marrow and recipient to prevent both GVHD and rejection. Alemtuzumab is the monoclonal antibody against tumor B-cells. It imparts its action through cell lysis.

Catalytic MAb (Abzymes):

The antibodies are extremely efficient at binding ground states of the target molecule while enzymes obtained their catalytic efficiency from tight binding of the transition state for the reaction. Thus antibodies can be made efficient catalysts if they are made for reaction transition state. Lemer and his co-workers explored the probability of enzyme like action of antibodies by producing hapten-carrier complex where the hapten structurally resembled transition state and antihapten MAbs generated, gave catalytic activity. The hydrolysis of ester substrate increased thousand fold after incubation.

Anti-IgE MAb (omalizumab) IgE (Immunoglobulin E) is recognised as an important component of asthma pathophysiology, contributing to the early and latephase inflammatory cascade of the airways by its inhibition of allergen-induced activation of mast cells. Omalizumab has a dual effect; it binds free IgE and inhibits mastcell degranulation and, by reducing free IgE. It also downregulates Fc RI on basophils and mast cells (which prevents the need for almost complete removal of free IgE which would be otherwise necessary to elicit functional consequences on mast cells and basophils). It also decreases the overall inflammation of the airways in asthma. RCTs (randomised clinical trials) have demonstrated that omalizumab is effective in reducing asthma symptoms and improving quality of life while reducing the need for ICS (inhaled corticosteroids) in patients with allergic asthma.

 Anti-IL-2R-

MAb (daclizumab) IL-2 is intricately involved in T-cell activation. Daclizumab binds to the IL-2 receptor (CD25) and blocks IL-2 signalling and IL-2R- mediated T- and B-cell activation. It is efficacious in preventing renal transplant rejection and a clinical trial in moderatesevere asthma has also suggested efficacy on the basis that activated T-cells contribute to ongoing disease activity.

 Anti-IL-9

MAb Blocking the actions of IL-9 reduces allergeninduced airway inflammation and airway hyper-responsiveness in mouse models

Introduction
y Antibody and soluble antigen interacting in aq sol

form a lattice that eventually develops into a visible precpitate. y Antibody that aggregate soluble antigens are called precipitins. y Immnune precipitates can form not only in solution but also in agar matrix y When antigen and antibody diffuse towards one another in agar or when antibody is incorporated into agar and antigen diffuses into the antibody containing matrix ,a visible precipitate will form.

y Immunodiffusion is a technique for the identification

and quantification of any of the immunoglobulins. y It is based on the presence of a visible precipitate that results from an Ag-Ab combination under certain circumstances. y Gel diffusion is a technique that involves evaluation of the precipitin reaction in a clear gel. y Two commonly known immunodiffusion techniques are: double immunodiffusion(ouchterlony method) and radial immunodiffusion(mancini method).

Double immunodiffusion
y In this method both antigen and antibody diffuse

radially from wells towards each other,thereby establishing a concentration gradient. y Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). y Antibodies have at least two antigen binding sites, thus large aggregates or gel-like lattices of antigen and antibody are formed.

y Experimentally, an increasing amount of antigen is

added to a constant amount of antibody in solution, initially at low antigen concentration, all of the antigen is contained in the precipitate. y This is called the antibody-excess zone i.e. prozone phenomenon. y As more antigen is added, the amount protein precipitated increases until the antigen/antibody molecules are at an optimal ratio.

y This is known as the zone of equivalence or equivalence point. y The zone of equivalence lines may give a full identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a non-identity (i.e. the two lines cross completely). y Where the two diffusion fronts meet, if any of the antibodies recognize any of the antigens, they will bind to the antigens and form what is known as an immune complex. y This immune complex precipitates in the gel to give a thin white line, which is a visual signature of antigen recognition.

Radial immunodiffusion
y Radial immunodiffusion (or Mancini method)is a technique used in immunology to determine the quantity of an antigen by measuring the diameters of circles of precipitin complexes,surrounding samples of the antigen that mark the boundary between the antigen and an antibody suspended in a medium, such as an agar gel. y In radial immunodiffusion ,an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an anti-serum. y As the antigen diffuses into the agar the region of equivalence is establised and a ring of preciptation ,a precipitin ring,forms around the well.

y The area of precipitin ring is proportion to the concentration of antigen. y By comparing the area of precipitin ring with a standard curve,the concentration of the antigen sample can be determined. y The diameters of the circles increase with time as the antigen diffuses into the medium, reacts with the antibody, and forms insoluble precipitin complexes. y It can be applied when sensitivity is not required but specificity is, and antibody is plentiful. It is commonly used in clinically detecting levels of blood proteins in a patient.

Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity

y The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. y It represented the first time that hormone levels in the blood could be detected by an in vitro assay.

Technique of Radioimmunoassay
A mixture is prepared of y radioactive antigen y antibodies against that antigen. y Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. These compete for the binding sites of the antibodies.
y

y At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. y The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and y the radioactivity of each is measured.

y Precipitate the antigen-antibody

complexes by adding a "second" antibody directed against the first. y For example, if a rabbit IgG is used to bind the antigen, the complex can be precipitated by adding an antirabbitIgG antiserum (e.g., raised by immunizing a goat with rabbit IgG).

Advantages & Disadvantages of RIA

y Advantages y Highly specific: Immune reactions are specific y High sensitivity : Immune reactions are sensitive y Disadvantages y Radiation hazards: Uses radiolabelled reagents y Requires specially trained persons y Labs require special license to handle radioactive material y Requires special arrangements for
y y

Requisition, storage of radioactive material radioactive waste disposal.

Applications
y Epidemiology
y Hepatitis B

y Clinical Immunology
y Antibodies for Inhalant Allergens y Allergy Diagnosis

y Oncology
y Carcinoembryonic Antigen y Early Cancer Detection and Diagnosis

y plasma levels of: y most of our hormones; y digitoxin or digoxin in patients receiving these drugs; y certain abused drugs y for the presence of hepatitis B surface antigen (HBsAg) in donated blood; y anti-DNA antibodies in systemic lupus erythematosus (SLE).

Requirements for RIA

1. Preparation & characterisation of the Antigen [Ligand to be analysed] 2. Radiolabelling of the Antigen 3. Preparation of the Specific Antibody 4. Development of Assay System

Preparation & Radiolabelling of the Antigen by.. y Antigens prepared

y Synthesis of the molecule y Isolation from natural sources

y Radiolabelling [Tagging procedure] y 3 H 14 C 125 I are used as radioactive tags y Antigens are tagged to 3 H 14 C 125 y Tagging should NOT affect Antigenic specificity & Antigenic activity !

Preparation of the Specific Antibody

y Antigen injected intradermally into rabbits or guinea

pigs antibody production y Antibodies recovered from the serum y Some ligands are not Antigenic
y Hormones, Steroids, Drugs y Eg: Gastrin, Morphine, y Haptens conjugated to albumin

HAPTENS antigenic

Development of the Assay System

y A crucial step is separation of unbound antigens y This achieved by binding the antibodies to the

microtitre well surface [Solid phase RIA] y Antigens bound to the fixed antibodies remain stuck to the inner surface y Decanting & washing the well removes unbound antigens y Other techniques of separation: Centrifugation

Assay Procedure
y Add known amounts of the test sample +

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

VACCINES
A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "recognize" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

 Vaccination is also called active immunization because the immune system is stimulated to develop its own immunity against the pathogen. Passive immunity, in contrast, results from the injection of antibodies formed by another animal (e.g., horse, human) which provide immediate, but temporary, protection for the recipient.

HISTORY
The terms vaccination and vaccine derived from the work of Edward Jenner who, over 200 years ago, showed that inoculating people with material from skin lesions caused by cowpox (L. vaccinus, of cows) protected them from the highly contagious and frequently fatal disease smallpox.

 Since Jenner's time, the term has been retained for any preparation of dead or weakened pathogens, or their products, that when introduced into the body, stimulates the production of protective antibodies or T cells without causing the disease. In molecular terms, the goal is to introduce harmless antigen(s) with epitopes that are also found on the pathogen.

VACCINE PRODUCTION
Vaccine production has several stages. First, the antigen itself is generated.
Viruses are grown either on primary cells such as chicken eggs (e.g., for influenza), or on continuous cell lines such as cultured human cells (e.g., for hepatitis A) Bacteria are grown in bioreactors (e.g., Haemophilus influenzae type b)

VACCINE PRODUCTION
a recombinant protein derived from the viruses or bacteria can be generated in yeast, bacteria, or cell cultures.

After the antigen is generated, it is isolated from the cells used to generate it.
A virus may need to be inactivated, possibly with no further purification required Recombinant proteins need many operations involving ultrafiltration and column chromatography

VACCINE PRODUCTION
Finally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives as needed.
adjuvant enhances the immune response of the antigen stabilizers increase the storage life, and preservatives allow the use of multidose vials.

EXCIPIENTS
Beside the active vaccine itself, the following excipients are commonly present in vaccine preparations: Aluminum salts or gels are added as adjuvants. Adjuvants are added to promote an earlier, more potent response, and more persistent immune response to the vaccine; they allow for a lower vaccine dosage. Antibiotics are added to some vaccines to prevent the growth of bacteria during production and storage of the vaccine. Egg protein is present in influenza and yellow fever vaccines as they are prepared using chicken eggs. Other proteins may be present.

EXCIPIENTS
Formaldehyde is used to inactivate bacterial products for toxoid vaccines. Formaldehyde is also used to kill unwanted viruses and bacteria that might contaminate the vaccine during production. Monosodium glutamate (MSG) and 2-phenoxyethanol are used as stabilizers in a few vaccines to help the vaccine remain unchanged when the vaccine is exposed to heat, light, acidity, or humidity. Thimerosal is a mercury-containing preservative that is added to vials of vaccine that contain more than one dose to prevent contamination and growth of potentially harmful bacteria.

A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins. The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and "recognize" it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

Vaccination is also called active immunization because the immune system is stimulated to develop its own immunity against the pathogen. Passive immunity, in contrast, results from the injection of antibodies formed by another animal (e.g., horse, human) which provide immediate, but temporary, protection for the recipient.

The terms vaccination and vaccine derived from the work of Edward Jenner who, over 200 years ago, showed that inoculating people with material from skin lesions caused by cowpox (L. vaccinus, of cows) protected them from the highly contagious and frequently fatal disease smallpox.

Since Jenner's time, the term has been retained for any preparation of dead or weakened pathogens, or their products, that when introduced into the body, stimulates the production of protective antibodies or T cells without causing the disease. In molecular terms, the goal is to introduce harmless antigen(s) with epitopes that are also found on the pathogen.

Vaccine production has several stages. First, the antigen itself is generated.
 Viruses are grown either on primary cells such as

chicken eggs (e.g., for influenza), or on continuous cell lines such as cultured human cells (e.g., for hepatitis A)  Bacteria are grown in bioreactors (e.g., Haemophilus influenzae type b)

 a recombinant protein derived from the viruses or

bacteria can be generated in yeast, bacteria, or cell cultures.

After the antigen is generated, it is isolated from the cells used to generate it.
 A virus may need to be inactivated, possibly with

no further purification required  Recombinant proteins need many operations involving ultrafiltration and column chromatography.

Finally, the vaccine is formulated by adding adjuvant, stabilizers, and preservatives as needed.
 adjuvant enhances the immune response of the

antigen  stabilizers increase the storage life,  and preservatives allow the use of multidose vials.

Beside the active vaccine itself, the following excipients are commonly present in vaccine preparations:
 Aluminum salts or gels are added as adjuvants. Adjuvants

are added to promote an earlier, more potent response, and more persistent immune response to the vaccine; they allow for a lower vaccine dosage.  Antibiotics are added to some vaccines to prevent the growth of bacteria during production and storage of the vaccine.  Egg protein is present in influenza and yellow fever vaccines as they are prepared using chicken eggs. Other proteins may be present.

 Formaldehyde is used to inactivate bacterial products for

toxoid vaccines. Formaldehyde is also used to kill unwanted viruses and bacteria that might contaminate the vaccine during production.  Monosodium glutamate (MSG) and 2-phenoxyethanol are used as stabilizers in a few vaccines to help the vaccine remain unchanged when the vaccine is exposed to heat, light, acidity, or humidity.  Thimerosal is a mercury-containing preservative that is added to vials of vaccine that contain more than one dose to prevent contamination and growth of potentially harmful bacteria.

ATTENUATED VIRUSES AND BACTERIA:Attenuation often can be achieved by growing a pathogenic bacterium or virus for prolonged periods under abnormal culture conditions. This procedure selects mutants that are better suited to growth in abnormal cultre conditions and are therefore less capable of growth in normal host . For example, an attenuated strain of mycobacterium bovis called BCG was developed by growing M.bovis on a medium

Conatining increasing concentartion of Bile. ADVANTAGES prolonged exposure to the individual epitopes on the attenuated organisms, resulting in increased immunogencity. The ability to replicate within host cells make them particularly sutaible for inducing a cell mediated response. DISADVANTAGE They will revert to virulent form.

Another common approach in vaccine production is inactivation of the pathogen by heat or chemical means so that no longer capable of replication in the host. Its critically important to maintain the structure of epitopes on surface antigens during inactivation.Heat inactivation is generally unstisfactory because it causes extensive denaturation of proteins. Chemical inacativation with formaldehyde or various

alkylating agents has been successful.The salk poio vaccine is produced by formaldehyde inactivation. They require repeated boosters to maintain the immune status of the host.

Some of the risks associated with attenuated or killed whole organism vaccinces can be avoided with vaccines that consist of specific, purified macromolecules derived from pathogens. Inactivated exotoxins Capsular polysaccharides Recombinant microbial antigens

The current vaccine for streptococcus pneumoniae, which causes pneumococcal pneumonia, consists of 23 antigenically different capsular polysaccharides. The vaccines induces formation of opsonizing antibodies. Vaccine for Neisseria meningitidis, a common cause of bacterial meningitis, also consists of purified capsular polysaccharides.

Limitation:- Their inability to activate T helper cells. They activate B cells, resulting in IgM production. To solve this problem polysacchsride protein conjugate considerabily more immunogenic than the polysaccharide, because it activates T helper cells wich enables class switching from IgM to IgG.

Diptheria and tetanus vaccines can be made by purifiying the bacterial exotoxin and then inactivating the toxin with formaldehyde to form a toxoid. Vaccination with the toxoid induces antitoxoid antibodies. Which are also capable of binding to the toxin and neutralizing its effects.

This type of vaccines have been developed by cloning the gene for the major surface antigen of hepatitis B virus(HBsAg) and expressing in yeast cells. The recombinant yeast cells are grown in the large fermenters, and HBsAg accumulates in the cells. The yeast cells are harvested and disrupted by high pressure, releasing the recombinant HBsAg which is then purified by conventional biochemical techniques.This recombinant hepatits B vaccine has been shown to induce the production of protective antibodies.