Immunology & Immunological Preparations

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By: Bijaya Kumar Uprety

Immunology Branch of biological science concerned with the study of immunity,

Or concerned with the structure and function of immune system.

ImmunityLatin term immunis meaning exempt.

Immunity means the state of protection from infectious disease.

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Year Name Event

430 B.C Earliest written

reference to the

phenomenon of immunity.

Thucydides (great

historian of the

Peloponnesian war).

In describing plague in Athens, he wrote during the war, only who had recovered from the

plague could nurse the sick because they would not get the disease for the second time.

15th century- First record

to induce immunity

deliberately.

Chinese and Turks Dried crust derived from small pox pustules were either inhaled into the nostrils or

inserted into small cuts in the skin (technique known as variolation).

1718 Lady Mary Wortley

(wife of british

ambassador to

Constantinople)

Performed variolation on her own children after realizing the technique was effective

among her native people.

1798 Edward Jenner

(physician)

Propounded an idea that introducing fluid from a cowpox pustule into people might

protect them from smallpox and he tested his idea on eight year old kid which was

successful.

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Louis Pasteur grew the fowl cholera causing bacterium in culture and when this was injected it into chicken they developed cholera but later on when he once again injected them with the same old culture they got ill but recovered later on.

He grew fresh culture and tried it on same chickens. They completely recovered.

Hence, Hypothesized and proved that aging had weakened the virulence of the pathogen and concluded that the attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine.

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Continue.. Later extended his findings to other diseases and

demonstrated it is possible to attenuate, or weaken apathogen and administer them to use it as a vaccine.

In 1881, Pasteur vaccinated one group of sheep withheat-attenuated anthrax bacillus (bacillus anthracis)and left another group of unvaccinated sheep . Allunvaccinated sheep died while other lived.

This was the beginnings of the discipline ofimmunology. In 1885, he first administered his firstvaccine to a human (a young boy) against rabies.

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Pasteur proved vaccination worked but didnt know how it worked.

In 1890, Emil von Behring and Shibasaburo Kitasato gave first insight into the mechanism of immunityGot nobel prize in 1901.

They demonstrated that serum from animals previously immunized to diptheria could transfer the immune state to unimmunized animals.

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During next decade, it was demonstrated by variousresearchers that an active component from immuneserum could neutralize toxins, precipitate toxins andagglutinate bacteria and active agent was named for itsactivity it exhibited: antitoxin, precipitin andagglutinin resp.

Initially different serum component was thought to beresponsible for each activity but during 1930, ElvinKabat (mainly him) found that gamma-globulin (nowimmunoglobulin, also a fraction of serum) wasresponsible for all these.

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The active molecules in the immunoglobulin fraction are called antibodies.

Because the immunity was mediated by antibodies contained in the fluids (known at that time as humors), it was called humoral immunity.

In 1883, Elie Metchnikoff demonstrated that cells also contribute to the immune state of an animal. He hypothesized that cells rather than serum components were major effector of immunity.(term phagocytes was coined and an idea of cell-mediated immunity dvpt).

Controversy developed between two concepts.

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But latter proved that both were correct.

Immunity requires both humoral and cellular responses.

In 1950, lymphocyte was identified as the cell responsible for both cellular and humoral immunity and experiments on chicken pioneered by Bruce Click at Mississippi State University indicated that there are two types of lymphocytes.

1. T- lymphocytes derived from thymus mediated cellular immunity.

2. B-lymphocytes from bursa of Fabricius were involved in humoral immunity.

Both these systems work hand in hand to protect our body against various foreign attack.

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Introduction to Immune system Remarkably versatile defense system that protect

animals against various invading micro-organisms and cancer.

Able to generate enormous variety of cells and molecules capable of specifically recognizing and eliminating large variety of foreign invaders.

Invaders human body immune system respond eliminate or destroys the invaders

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Body endowed with different defense system.

At first, external defense system comes into play whichincludes, skin, secretion of mucus, ciliary action,lavaging action of bactericidal fluids (e.g. tears),gastric acid and microbial antagonism.

If penetration occurs, bacteria are destroyed bysoluble factors such as lysozyme and by phagocytosiswith intracellular digestion.

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Functionally, immune response can be divided into two related activities-

1. Recognition Remarkable for its specificity.

2. Response.

Immune system is able to recognize subtle chemical differences that recognize one foreign pathogen from another.

Able to discriminate between foreign molecules and the bodys own cells and proteins.

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Once a foreign organism has been recognized, it recruits a variety of cells and molecules to mount an appropriate response, called effector response, to eliminate or neutralize the organism.

The immune response enables the elimination or neutralization of the cells/molecules (pathogens) from the body.

Convert initial recognition event variety of effectorresponses eliminate or neutralize particular pathogen.

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Later exposure to the same foreign organism induces a memory response, characterized by a more rapid and heightened immune reaction that serves to eliminate the pathogen and prevent disease.

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Types of immunity Two types of immunity:

1. Innate or nonspecific immunity.

2. Acquired or specific immunity.

Innate immunity:

It is the basic resistance to diseases that an individual has from the time of its birth.

Not specific to any one pathogen but rather constitutes a first line of defense.

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It consists of following four types of defensive barriers:

1. Anatomic barriers

2. Physiologic barriers

3. Endocytosis /phagocytosis barriers

4. Inflammatory barriers

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Endocytosis- Process of cellularingestion of macromolecules byinvagination of plasma membrane toproduce an intracellular vesicle whichencloses the ingested material.

3 types-

Phagocytosis (for particulates),

Pinocytosis (liquid),

Receptor mediated endocytosis(LDL) .

Most phagocytosis (most common) isdone by blood monocytes, neutrophils,and tissue macrophages.

Fig. Steps in phagocytosis of a bacterium.

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Fig 2 showing the major events in the inflammatory response.[ vasoactive and chemotactic factors i.e kinin and histamine. Additionally , bradykinins which is a type of kinin stimulate pain receptors and fibrin-clot] 19

Acquired Immunity Also known as adaptive immunity.

Capable of recognizing and selectively eliminating specific foreign microorganisms and molecules (i.e. foreign antigens).

Displays four characteristic attributes:

1. Antigenic specificity

2. Diversity

3. Immunologic memory

4. Self/nonself recognition.

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Components of Acquired Immunity Involves the following two major groups of cells1. Lymphocytes which includes B and T lymphocytes.

2. Antigen presenting cells (APCs)- Group of B-cells, dendritic cells and macrophages. They express class II MHC molecules on their

membranes & They are able to deliver a co-stimulatory signal that is

necessary for TH cell activation.

APC have Class II MHC (major histocompatibilitycomplex) molecules on their surfaceMHC molecules bind to antigen derived peptides present them to lymphocytes immune system activated.

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B lymphocytes B lymphocytes mature within the bone marrow; when they leave it, each expresses a

unique antigen-binding receptor on its membrane . This antigen-binding or B-cellreceptor is a membrane-bound antibody molecule.

Antibodies are glycoproteins that consist of two identical heavy polypeptide chainsand two identical light polypeptide chains. Each heavy chain is joined with a lightchain by disulfide bonds, and additional disulfide bonds hold the two pairstogether.

The amino-terminal ends of the pairs of heavy and light chains form a cleft withinwhich antigen binds.

When a naive B cell (one that has not previously encountered antigen) firstencounters the antigen that matches its membrane bound antibody, the binding ofthe antigen to the antibody causes the cell to divide rapidly; its progeny differentiateinto memory B cells and effector B cells called plasma cells.

Memory B cells have a longer life span than naive cells, and they express the samemembrane-bound antibody as their parent B cell.

Although plasma cells live for only a few days, they secrete enormous amounts ofantibody during this time. It has been estimated that a single plasma cell can secretemore than 2000 molecules of antibody per second. Secreted antibodies are themajor effector molecules of humoral immunity.

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T lymphocytes T lymphocytes also arise in the bone marrow. Unlike B cells, which mature

within the bone marrow, T cells migrate to the thymus gland to mature.

During its maturation within the thymus, the T cell comes to express a uniqueantigen-binding molecule, called the T-cell receptor, on its membrane.

Unlike membrane-bound antibodies on B cells, which can recognize antigenalone, T-cell receptors can recognize only antigen that is bound to cell-membrane proteins called major histocompatibility complex (MHC)molecules.

MHC molecules that function in this recognition event, which is termedantigen presentation, are polymorphic (genetically diverse) glycoproteinsfound on cell membranes.

There are two major types of MHC molecules: Class I MHC molecules, which are expressed by nearly all nucleated cells ofvertebrate species, consist of a heavy chain linked to a small invariant proteincalled 2-microglobulin. Class II MHC molecules, which consist of an alpha and a beta glycoproteinchain, are expressed only by antigen-presenting cells.

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When a naive T cell encounters antigen combined with a MHC molecule on a cell,the T cell proliferates and differentiates into memory T cells and various effector Tcells.

There are two well-defined subpopulations of T cells: T helper (TH) and Tcytotoxic (TC) cells. Although a third type of T cell, called a T suppressor (TS) cell,has been postulated, recent evidence suggests that it may not be distinct from THand TC subpopulations.

T helper and T cytotoxic cells can be distinguished from one another by thepresence of either CD4 or CD8 membrane glycoproteins on their surfaces . Tcells displaying CD4 generally function as TH cells, whereas those displaying CD8generally function as TC cells. TH cells generally recognize antigen combined withclass II molecules, whereas TC cells generally recognize antigen combined with classI molecules.

After a TH cell recognizes and interacts with an antigen MHC class II moleculecomplex, the cell is activatedit becomes an effector cell that secretes variousgrowth factors known collectively as cytokines. The secreted cytokines play animportant role in activating B cells, TC cells, macrophages, and various other cellsthat participate in the immune response.

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Differences in the pattern of cytokines produced by activatedTH cells result in different types of immune response.

Under the influence of TH-derived cytokines, a TC cell thatrecognizes an antigenMHC class I molecule complexproliferates and differentiates into an effector cell called acytotoxic T lymphocyte (CTL).

In contrast to the TH cell, the CTL generally does notsecrete many cytokines and instead exhibits cell-killing orcytotoxic activity.

The CTL has a vital function in monitoring the cells of thebody and eliminating any that display antigen, such as virus-infected cells, tumor cells, and cells of a foreign tissue graft.Cells that display foreign antigen complexed with a class IMHC molecule are called altered self-cells; these are targets ofCTLs.

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ANTIGEN-PRESENTING CELLS Activation of both the humoral and cell-mediated branches of the

immune system requires cytokines produced by TH cells. It is essential that activation of TH cells themselves be carefully

regulated, because an inappropriate T-cell response to self-components can have fatal autoimmune consequences.

To ensure carefully regulated activation of TH cells, they can recognizeonly antigen that is displayed together with class MHC II molecules onthe surface of antigen-presenting cells (APCs).

These specialized cells, which include macrophages, B lymphocytes,and dendritic cells, are distinguished by two properties: (1) they expressclass II MHC molecules on their membranes, and (2) they are able todeliver a co-stimulatory signal that is necessary for TH-cell activation.

Antigen-presenting cells first internalize antigen, either byphagocytosis or by endocytosis, and then display a part of that antigenon their membrane bound to a class II MHC molecule. The TH cellrecognizes and interacts with the antigenclass II MHC moleculecomplex on the membrane of the antigen-presenting cell .Anadditional costimulatory signal is then produced by the antigen-presenting cell, leading to activation of the TH cell.

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Humoral Immune Responses It is based on antibodies.

It can be conferred on nonimmune individuals by administration of serum antibodies from an immune individual.

Antibodies act as an effector of humoral response.

They bind to the antigens and facilitate their elimination.

Elimination could be in various ways.

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Fig showing the structure of Antibody.

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1. By forming clusters through cross-linking of antigen molecules, which are readily ingested by phagocytic cells.

2. By binding of antibodies to a microorganism can activate the complement system, which lyses the mos.

3. Antibodies bind to toxins and viral particles, and prevent their subsequent binding to host cells.

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Cell-mediated Immune Responses Based on T cells, which are a type of lymphocyte.

T cells are of the following two types:

1. T helper (TH)

2. T cytotoxic (TC) cells.

TH cell interacts with an antigen MHC II molecule complex present on an APC cytokines secreted

cytokines activate B cells, Tc Cells, and various phagocytic cells.

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Activated phagocytic cells able to kill mos (bacteriaand protozoa).

When Tc cell interacts with an antigen-MHC Icomplex, the Tc cell proliferates under the influence ofcytokines produced by activated TH cells.

These Tc cells differentiate into cytotoxic Tlymphocytes (CTLs). The CTLs kill all such cells thatdisplay foreign antigens complexed with MHC Imolecules. Such cells are called altered self-cells, theyare usually virus-infected cells, tumor cells and foreigntissue cells.

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Thus TH cells and CTLs are effectors of the cell-mediated immune response.

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Fig 2. Overview of humoral and cell mediated immune responses.37

Passive Immunization It is the administration of preformed antibodies (usually

IgG) either intravenously or intramuscularly.

Used to provide rapid protection in certain infections suchas diptheria or tetanus or in the event of accidentalexposure to certain pathogens such as hepatitis B.

Also used to provide protection in immune compromisedindividuals who are unable to produce appropriateantibody response or in some instances incapable ofmaking any antibody at all (i.e. severe combinedimmunodeficiency).

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Antibodies given to immune deficient patients are usually IgG- derived from pooled normal plasma and are administered on a continuous basis (ideally every three weeks) as they are continuously catabolized and are effective only for short duration.

Preformed antibodies from animals, notably horse are also administered for some diseases but it presents a danger of immune complex formation and serum sickness (if repetitively injected).

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Active immunization Administration of vaccines containing microbial

products with or without adjuvants in order to obtain long term immunological protection against the offending microbe.

2 types:

1. Systemic Immunization.

2. Mucosal Immunization.

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Systemic Immunization This is the method of choice at present for most

vaccinations.

Carried out by injecting vaccine subcutaneously or intramuscularly into the deltoid muscle.

Ideally all vaccines given soon after birth but some deliberately delayed.

Common eg includes vaccines for measles, mumps, and rubella usually given at the age of 1. If given earlier maternal antibody would decrease their effectiveness.

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But carbohydrate vaccines for Pneumococcus,Meningococcus, and Haemophilus infections are givenat about 2 years as before this age they respond poorlyto polysaccharides unless they are associated withprotein components that can act to recruit T cell helpfor development of anti- polysaccharide antibody.

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Mucosal Immunization Most of the infectious agents gain entry to the systemic

system through mucosal route and the largest source oflymphoid tissue is also present at the mucosal surfaces.

Thus recent vaccination approaches have focussed on themucosal route as the site of choice for immunization eitherorally or through the nasal associated immune tissue(NALT).

Moreover, it eliminate the need for painful injection andallow for self-administration of certain vaccines such asthose for immunization against influenza.

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Adjuvant vaccines and live vector vaccines have been used to target mucosal immune system with some success.

Attenuated strains of salmonella can act as a powerful immune stimulus as well as acting as carriers of foreign antigens.

This approach has been used to immunize against mucosal surfaces against herpes simplex virus and human papilloma virus.

Bacterial toxins, eg those derived from cholera, E. coli etc which posses immunomodulatory properties are also being exploited in the dvpt of mucosally active adjuvants.

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Table 1. Passive immunization

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Vaccine Vaccine is a preparation containing a pathogen

(disease producing organism) either in attenuated orinactivated state.

This preparation is introduced into an individual toinduce adequate antibody production against thepathogen in question so that the individual becomesprotected against infection, at a later date, by thatpathogen.

The introduction of a vaccine in an individual is calledvaccination or immunization as it leads to thedevelopment of immunity in the vaccinatedindividuals to the concerned pathogen.

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The immunity is induced by the antigens of pathogen origin present in the vaccine.

Conventionally, various vaccines can be broadly classified into two groups:

1. Vaccines containing killed or inactivated pathogens, i.e. most bacteria vaccines and some virus vaccines (e.g. influenza virus inactivated by formalin, rabies virus inactivated by phenol and - priolactone).

2. Those containing live but attenuated pathogens, e.g., most virus vaccines.

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Attenuation means a drastic reduction in the virulence of a pathogen which is achieved as follows:

Several consecutive passages through an animal, which is not the usual host of the pathogen, e.g., small pox virus in calf.

Several passages through cultured cells of the host, e.g., rabies virus in human diploid cell culture, or of a different species, e.g., rabies virus, yellow fever virus in chick embryo cell culture.

Selection of less virulent strains of pathogens, e.g., a mutant strain of polio virus.

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Treatment of the pathogen with some chemicals, e.g., B.C.G. (Bacillus of Calmette Guerien) vaccine produced by culturing the bacteria on a medium containing bile.

Culturing pathogens under unfavourable conditions like high temp, e.g., anthrax vaccine obtained by cultivation of the bacterium (Bacillus anthracis) at 40-50 0C.

In general, inactivation of virus is always coupled with attenuation to minimize the accidental presence of active virulent particles which could cause disease in the vaccinated individuals.

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The different vaccines differ in their composition,efficacy and the duration of effective protection to thevaccinated individuals. These are one of the earliestexamples of biotechnological intervention in humanand animal health care.

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Types of various vaccines There are various types of vaccines,

1. Whole-Organism Vaccines (Conventional vaccines)

2. Purified Macromolecules as Vaccines (Conventional )

3. Subunit Vaccine

4. Recombinant- Vector Vaccines

5. DNA Vaccines

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Whole organism vaccines Many vaccines now available for humans, and animal

use are made using whole organisms (bacteria orvirus), either in the inactivated (killed) form orattenuated (live but avirulent) form.

Examples: Salmonella typhi (killed bacteria)againstTyphoid, Salmonella paratyphi (killed bacteria) againstparatyphoid, Vibrio cholerae (killed cells or cellextract) against cholera, Attenuated virus againstyellow fever, measles, mumps, rubella and polio.

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Preparation and storage of Typhoid-Paratyphoid A and B Vaccine [TAB-Vaccine]

Typhoid fever (enteric fever) is an acute generalized infection caused bySalmonella typhi ; whereas, paratyphoid fever is caused by Salmonella paratyphiA and Salmonella paratyphi B.

Preparation

(1) The vaccine is prepared by the general process and contains the following ineach millilitre : Typhoid bacilli (Salmonella typhi) : 1000 million Paratyphi Abacilli (S. paratyphi A) and Paratyphi B bacilli (S. paratyphi B) : 500 or 750million.

(2) The smooth strains of the three organisms known to produce the fullcomplement of O somatic antigens should be used. This specific strain of S.typhi must contain the virusassociated antigens (Vi-antigen).

(3) It has been duly established that when the organisms were killed with 75%ethanol and the resulting vaccine preserved with 22.5% ethanol, the potency ofthe alcohol treated vaccine was found to be almost double to that of theheat-treated vaccine, there by minimizing the possibility of both local andconstitutional reaction with the relatively smaller dose. Besides, alcohol treatedvaccines did possess definitely and predominantly longer life under theoptimal storage conditions [viz., storage between 2-4 C without allowing thevaccine to freeze].

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Purified macromolecules as vaccine The purified antigenic portions from the bacterial cell

wall, or viral coat protein are used as vaccines, and they can elicit immune reaction. Examples of macromolecule vaccines are:

1. Capsular polysaccharides

2. Surface antigens

3. And inactivated exotoxins called toxoids.

The macromolecule vaccines are generally safe since they dont contain live organism. Example is given in next slide.

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Preparation of Meningococcal Polysaccharide Vaccine

The Meningococcal Polysaccharide Vaccine consistsof one or more purified polysaccharides obtainedfrom appropriate strains of Neisseria meingitidis group A,group C, group Y and group W135 that have beenadequately proved to be capable of producingpolysaccharides that are absolutely safe and also capableof inducing the production of satisfactory levels ofspecific antibody in humans.

The vaccine is prepared immediately before use byreconstitution from the stabilized dried vaccine withan appropriate prescribed sterile liquid. It may eithercontain a single type of polysaccharide or any mixture ofthe types.

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The various Preparation steps adopted are as stated under :

(1) The preparation of the vacccine is based on a seed-lot system. Eachseed-lot is subjected to microbiological examination by culture in anappropriate media and microscopic examination of Gram-stainedsmears.

(2) The polysaccharide shown to be free from contaminating bacteria isprecipitated by the addition of cetrimonium bromide and thenpurified.

(3) Each polysaccharide is dissolved under aseptic conditions in a sterilesolution containing lactose or another suitable stabilizing medium forfreeze drying.

(4) The solution is blended, if appropriate, with solution of thepolysaccharides of any or all of the other groups and passed through abacteria-retentive filter.

(5) Finally, the filtrate is freeze dried to a moisture content shown to befavourable to the stability of the vaccine

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Limitation of Conventional Vaccines Not all infectious agents can be grown in culture and no vaccines

have been developed for a number of diseases, where theinfectious agent is nonculturable.

Production of animal and human viruses requires animal cellculture, which is expensive.

Yield and rate of production of animal viruses is low. Extensive laboratory precautions are needed while dealing with

highly infectious agents. In spite of the best precautions, some batches of vaccines may

not be completely killed or attenuated. Attenuated strains may revert to pathogenic state, occasionally,

and may cause actual disease against which protection wassought.

Not all infectious disease are preventable by traditional vaccines(e.g. AIDS).

Have limited Shelf-life thus requires refrigeration.

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Vaccines made through recombinant DNA technology Recombinant DNA technology can be best used in the

following ways in vaccine development.1. Virulence genes can be deleted from the infectious agent

retaining the immunogenic properties.2. An organism (non-pathogenic) carrying antigenic

determinants can be created by insertion of the genescoding for the antigenic proteins.

3. For non-culturable agents, genes for the protein (criticalantigenic determinants) can be cloned and expressed inan expression vector (e.g. E. Coli or a mammalian cellline).

4. A targeted cell-specific killing system that kills only theinfected cells can be designed. In this technique, gene fora fusion protein is constructed. First, one part of thefusion protein binds to the infected cell. Then the otherpart kills the infected cell.

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Subunit Vaccines For viruses, it has been shown that specific protein from

the coat or envelope is enough to elicit the immuneresponse.

Vaccines with components of a pathogenic organism ratherthan the whole organism are called subunit vaccines. rDNAtechnology is best suited to develop subunit vaccines.

Purified proteins are more stable and are chemicallyprecise and safe from side effects. However, purification ofprotein can be expensive and sometimes purification canalter the configuration of protein and alter itsantigenicity!!!

These factors have to be assessed before making a proteinpreparation.

One of the example of subunit vaccines developed throughrDNA tech will be discussed in the upcoming slides.

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Subunit vaccine for foot and mouth disease virus (FMDV)

Formalin-killed FMDV was used as vaccine earlier. Thegenome of FMDV is single stranded RNA (ssRNA).

The cDNA complementary to this ssRNA, 8000nucleotide long is prepared . It is digested withrestriction enzymes, and the fragments are cloned inE. coli.

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Recombinant vector vaccines Genes that encode major antigens of especially virulent

pathogens can be introduced into attenuated viruses orbacteria.

The attenuated organism serves as a vector, replicating withinthe host and expressing the gene product of the pathogen.

A number of organisms have been used for vector vaccines,including vaccinia virus (it is most strong candidate as itis efficient in delivery and expression of cloned genes),the canarypox virus, attenuated poliovirus, adenovirus,attenuated strains of Salmonella, the BCG strain ofMycobacterium bovis and certain strains of streptococcus thatnormally exist in the oral cavity.

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Vaccinia virus has been widely employed as a vectorvaccine. This large, complex virus, with a genome ofabout 200 genes, can be engineered to carry severaldozen foreign genes without impairing its capacity toinfect host cells and replicate.

The process of producing a vaccinia vector that carriesa foreign gene from a pathogen is outlined in figurebelow.

The genetically engineered vaccinia expresses highlevel of the inserted gene product, which can thenserve as a potent immunogen in an inoculated host.

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Like the smallpox vaccine, genetically engineered vacciniavector vaccines can be administered simply by scratchingthe skin, causing localized infection in the host cells.

Antigen genes introduced into animal cells throughvaccinia virus genome inclues Rabies virus G protein,Hepatitis B surface antigen, Influenza virus NP and HAproteins, etc.

If the foreign gene product expressed by the vaccinia is aviral envelope protein, it is inserted into the membrane ofthe infected host cell, inducing development of cell-mediated immunity as well as antibody mediatedimmunity.

Similar to vaccinia vector vaccines other vector vaccineswhich have been recently tried include canarypox virus.

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DNA vaccines/ Gene vaccine (Genetic Immunization) Plasmid DNA encoding antigenic proteins is injected

directly into the muscle of the recipient. Muscle cells takeup the DNA and the encoded protein antigen is expressed,leading to both a humoral and cell-mediated response.

The DNA either integrate into the chromosomal DNA or tobe maintained for long periods in an episomal form.

It offers advantage over many of the existing vaccines few ofwhich are listed below:

1 . The encoded protein is expressed in the host in its naturalform- there is no denaturation or modification . Due to thisthe immune response is therefore directed to the antigenexactly as it is expressed by the pathogen.

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2. It induces both humoral and cell mediated immunity.

3. DNA vaccines cause prolonged expression of the antigen, which generates significant immunological memory.

4. Refrigeration is not required for handling and storage of the plasmid DNA (thus lowers cost and complexity of delivery).

5. The same plasmid vector could be custom tailored to make variety of proteins, so the same manufacturing techniques can be used for different DNA vaccines, each encoding an antigen from a different pathogen.

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An improved method of administering these vaccinesinvolves coating microscopic gold beads with theplasmid DNA and then delivering the coated particlesthrough the skin into the underlying muscle with anair gun (called gene gun). This will allow rapid deliveryof a vaccine to large populations without therequirement for huge supplies of needles and syringes.

Test of DNA vaccines in animal models have shownthese vaccines to be effective against various viraldiseases including influenza virus.

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Antigen-Antibody Interaction The antigen-antibody interaction is a biomolecular

association similar to an enzyme-substrate interaction.

However, it doesnt lead to an irreversible chemicalalteration in either the antibody or the antigen.

The association between an antigen and antibodyinvolves various noncovalent interactions between theantigenic determinant (epitope) of the antigen and thevariable-region (VH/VL ) domain of the antibodymolecule, particularly the hypervariable regions, orcomplementarity- determining regions (CDRs).

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The noncovalent interactions that form the basis ofantigen-antibody binding include hydrogen bonds,ionic bonds, hydrophobic interactions, and vanderWaals interactions.

Since these interactions are individually weak(compared with a covalent bond), a large number ofsuch interactions are required to form a strong Ag-Abinteraction.

Furthermore, each of these noncovalent interactionsoperates over a very short distance (1 angstrom or 1 x10-7 mm). Hence a strong Ag-Ab interaction dependson a very close fit between the antigen and antibody.Such fit require a high degree of complementaritybetween antigen and antibody.

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Cross-reactivity Although Ag-Ab reactions are highly specific, in some cases

antibody elicited by one antigen can cross- react with anunrelated antigen. Such cross-reactivity occurs if twodifferent antigens share an identical or very similar epitope.

Cross- reactivity is often observed among polysaccharideantigens that contains similar oligosaccharide residues.The ABO blood-group antigens, for example, areglycoproteins expressed on RBCs. Subtle differences in theterminal residues of the sugars attached to these surfaceproteins distinguish the A and B blood group antigens.

RBC glycoprotein sugars attached to the terminal endof it subtle difference in the terminal residues of thesesugars distinguish A and B blood group antigens.

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An individual lacking one or both of these antigens willhave serum antibodies to the missing antigen(s).

The antibodies are induced not by exposure to red bloodcell antigens but by exposure to cross-reacting microbialantigens present on common intestinal bacteria.

These microbial antigens induce formation of antibodiesin individuals lacking the similar blood-group antigens ontheir RBCs.

The blood-group antibodies, although elicited by microbialantigens, will cross-react with similar oligosaccharides onforeign RBCs. This provides the basis for blood typing testsand accounts for the necessity of compatible blood typesduring blood transfusions. Type A individual has anti- Bantibodies, type B has anti- A and type O has anti- A & B .

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Numerous viruses and bacteria have epitopes identicalor similar to normal host-cell components. In somecases, these microbial antigens have shown to elicitantibody that cross-reacts with the host-cellcomponents, resulting in a tissue- damagingautoimmune reaction.

For e.g. bacterium Streptococcus pyrogenes, expresscell wall proteins called M antigens . Abs producedagainst these antigens have shown to cross react withseveral myocardial and skeletal muscle proteinscausing kidney and heart damage followingstreptococcal infections.

Some vaccines also exhibit cross-reactivity.

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1. Precipitation Reactions Antibody and soluble antigen interacting in aqueous solution form a

lattice that eventually develops into a variable precipitate.

Antibodies that aggregate soluble antigens are called precipitins.

Although formation of the soluble Ag-Ab complex occurs withinminutes, formation of the visible precipitate occurs more slowly andoften takes a day or two to reach completion.

Formation of Ag-Ab lattice depends upon the valency of both:1. Ab should be bivalent; a precipitate will not form with monovalent

Fab fragments.

2. Ag must be either bivalent or polyvalent i.e. it must have at least twocopies of the same epitope or have different epitopes that react withdifferent antibodies present in polyclonal antisera.

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A. Precipitation reaction in fluids Precipitation reaction in fluids yields a precipitin

Curve.

A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amount of antigen to the tubes. At one time this method was used to measure the amount of antigen or antibody present in a sample of interest.

Once precipitate is formed each tube centrifuged to pellet the precipitate supernatant poured offamount of precipitate is measured.

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Plotting the amount ofprecipitate against increasingantigen concentrations yields aprecipitin curve.

The figure below shows that theexcess of either antigen orantibodies interferes withmaximal precipitation, whichoccurs at equivalence point.

Maximal precipitation occursat equivalence point.

As a large macromolecularlattice is formed at equivalence,complex increases in size andprecipitate out.

Follow figure and draw it!!!!it isimportant to draw figure.

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B. Precipitation Reaction in Gels Precipitation rxn in gels yields visible precipitin Lines.

Immune precipitates can form not only in solution but alsoin agar matrix.

When antigen and antibody diffuses towards one anotherin agar, or when Ab is incorporated into the agar andantigen diffuses into the antibody containing matrix, avisible line of precipitation will form.

As in precipitation reaction in fluid, visible precipitationoccurs in the region of antibody or antigen excess.

Two types of immunodiffusion reactions can be used todetermine relative concentration of antibodies or antigen ,to compare antigens, or to determine the relative purity ofan antigen preparation.

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Two types of immunodiffusion reactions both of whichare carried out semisolid medium such as agar.

1 Radial immunodiffusion(Mancini method): In thismethod, an Ag sample is placed in a well and allowedto diffuse into agar containing a suitable dilution ofantiserum. As antigen diffuses into the agar, the regionof equivalence is established and a ring ofprecipitation, a precipitin ring, forms around the well.The area of precipitin ring is proportional to theconcentration of antigen. By comparing the area of theprecipitin ring with a standard curve (obtained bymeasuring the precipitin areas of knownconcentrations of the antigen), the concentration of theantigen sample can be determined.

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2. Double immunodiffusion (the Ouchterlony method):

In this method both the antigen and antibody diffuse, radially from the wells towards each other , thereby establishing a concentration gradient. As equivalence is reached, a visible line of precipitation, a precipitin line is formed.

Please refer figure in next slide!!!!!!!!!!!!!!

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2. Agglutination Reaction The interaction between antibody and antigen results

in visible clumping called agglutination.

Antibodies that produce such reactions are called agglutinins.

Agglutination reaction are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens.

Just as an excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition is called prozone effect.

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Its applicationa. Hemagglutination is used in blood typing:Agglutination reactions are routinely performed to type

RBCs. In typing for the ABO antigens, RBCs are mixed on a slide

with antisera to the A or B blood-group antigens.If the antigen is present on the cells, they agglutinate,

forming a visible clump on the slide. Determination of which antigens are present on donor and

recipient RBCs is the basis for matching blood types fortransfusions.

b. Bacterial agglutination is used to diagnose infection.

c. Passive agglutination is useful with soluble antigens.

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Immuno-assay Techniques Radioimmunoassay

ELISA

Western Blotting

Immunofluorescence

Immunoelectron Microscopy.

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Immunoassays The exquisite specificity of antigen-antibody

interactions has led to the development of a variety ofimmunologic assays, which can be used to detect thepresence of either antibody or antigen.

Immunoassays have played vital roles in diagnosingdiseases, monitoring the level of the humoral immuneresponse, and identifying molecules of biological ormedical interest. These assays differ in their speed andsensitivity, some are strictly qualitative, other arequantitative.

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Radioimmunoassay One of the most sensitive techniques for detecting antigen or

antibody is radioimmunoassay (RIA).

Principle: The principle of RIA involves competitive binding ofradiolabeled antigen(usually labeled with gamma emittingisotope such as 125I but beta emitting isotopes such as tritium 3Hare also routinely used as labels) to a high- affinity antibody. Thelabeled antigen is mixed with antibody at a concentration thatsaturates the antigen-binding sites of the antibody.

Then test samples of unlabeled antigen of unknownconcentration are added in progressively larger amounts. Theantibody doesnt distinguish labeled from unlabeled antigen, sothe two kinds of antigen compete for available binding sites onthe antibody.

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As the concentration of unlabeled antigen increases, morelabeled antigen will be displaced from the binding sites.

The decrease in the amount of radiolabeled antigen bound tospecific antibody in the presence of the test sample is measuredin order to determine the amount of antigen present in the testsample.[Note: To determine the amount of labeled antigenbound, the Ag-Ab complex is precipitated to separate it fromfree antigen, and the radioactivity in the precipitate is measured.A standard curve can be generated using unlabeled antigensamples of known concentration (in place of test sample), andfrom this plot the amount of antigen in the test mixture may beprecisely determined.]

A microtiter RIA has been widely used to screen for the presenceof the hepatitis B virus.

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Enzyme-Linked Immunosorbent Assay Commonly known as ELISA (or EIA).

Its principle is similar to RIA but depends on an enzymerather than a radioactive label.

An enzyme conjugated with an antibody reacts with acolorless substrate to generate a colored reaction product.Such a substrate is called a chromogenic substrate.

A number of enzymes have been employed for ELISA,including alkaline phosphatase, horseradish peroxidase,and - galactosidase. These assays approach the sensitivityof RIAs and have the advantage of being safe and lesscostly.

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Types: 3 types.

Indirect ELISA: used in determination of serum Absagainst HIV.

Sandwich ELISA

Competitive ELISA

In one of the version of ELISA usingchemiluminescence, a luxogenic (light-generating)substrate is used in place of chromogenic substrate(used in conventional ELISA). Its advantage isincreased sensitivity.

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Indirect ELISA Antibody can be detected or quantitatively determined

with an indirect ELISA (Figure 6-10a). Serum or some othersample containing primary antibody (Ab1) is added to anantigen-coated microtiter well and allowed to react withthe antigen attached to the well.

After any free Ab1 is washed away, the presence of antibodybound to the antigen is detected by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), whichbinds to the primary antibody.

Any free Ab2 then is washed away, and a substrate for theenzyme is added. The amount of colored reaction productthat forms is measured by specialized spectrophotometricplate readers, which can measure the absorbance of all ofthe wells of a 96-well plate in seconds.

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Indirect ELISA is the method of choice to detect the presence of serumantibodies against human immunodeficiency virus (HIV), thecausative agent of AIDS. In this assay, recombinant envelope and coreproteins of HIV are adsorbed as solid-phase antigens to microtiterwells.

Individuals infected with HIV will produce serum antibodies toepitopes on these viral proteins. Generally, serum antibodies to HIVcan be detected by indirect ELISA within 6 weeks of infection.

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SANDWICH ELISA

Antigen can be detected or measured by a sandwich ELISA(Figure 6-10b). In this technique, the antibody (rather thanthe antigen) is immobilized on a microtiter well.

A sample containing antigen is added and allowed to reactwith the immobilized antibody.

After the well is washed, a second enzyme- linked antibodyspecific for a different epitope on the antigen is added andallowed to react with the bound antigen. After any freesecond antibody is removed by washing, substrate is added,and the colored reaction product is measured.

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COMPETITIVE ELISA

Another variation for measuring amounts of antigen is competitiveELISA (Figure 6-10c). In this technique, antibody is first incubated insolution with a sample containing antigen.

The antigen-antibody mixture is then added to an antigen coatedmicrotiter well. The more antigen present in the sample, the less freeantibody will be available to bind to the antigen-coated well.

Addition of an enzyme-conjugated secondary antibody (Ab2) specificfor the isotype of the primary antibody can be used to determine theamount of primary antibody bound to the well as in an indirect ELISA.

In the competitive assay, however, the higher the concentration ofantigen in the original sample, the lower the absorbance.

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Western blotting Indentification of a specific protein in a complex mixture of proteins can be

accomplished by a technique known as Western blotting. In western blotting, a protein mixture is electrophoretically separated on an SDS-

polyacrylamide gel (SDS-PAGE), a slab gel infused with sodium dodecyl sulfate(SDS), a dissociating agent.

The protein bands are transferred to a nylon membrane by electrophoresis and theindividual protein bands are identified by flooding the nitrocellulose membranewith radiolabeled or enzyme linked polyclonal or monoclonal antibody specific forthe protein of interest.

The Ag-Ab complexes that is formed on the band ,containing the proteinrecognized by the antibody, can be visualized in a variety of ways. If the protein ofinterest was bound by a radioactive antibody, its position on the blot can bedetermined by exposing the membrane to a sheet of x-ray film, a procedure calledautoradiography.

However, the most generally used detection procedures employ enzyme-linkedantibodies against the protein. After binding of the enzyme antibody conjugate,addition of a chromogenic substrate that produces a highly colored and insolubleproduct causes the appearance of a colored band at the site of the target antigen.

The site of the protein of interest can be determined with much higher sensitivity ifa chemiluminescent compound along with suitable enhancing agents is used toproduce light at the antigen site.

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Western blotting can also identify a specific antibody in a mixture. Inthis case, known antigens of well-defined molecular weight areseparated by SDS-PAGE and blotted onto nitrocellulose.

The separated bands of known antigens are then probed with thesample suspected of containing antibody specific for one or more ofthese antigens.

Reaction of an antibody with a band is detected by using eitherradiolabeled or enzyme-linked secondary antibody that is specific forthe species of the antibodies in the test sample.

The most widely used application of this procedure is in confirmatorytesting for HIV, where Western blotting is used to determine whetherthe patient has antibodies that react with one or more viral proteins.

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Immunoprecipitation The immunoprecipitation technique has the advantage of

allowing the isolation of the antigen of interest for furtheranalysis.

It also provides a sensitive assay for the presence of a particularantigen in a given cell or tissue type.

An extract produced by disruption of cells or tissues is mixedwith an antibody against the antigen of interest in order to forman antigen-antibody complex that will precipitate.

However, if the antigen concentration is low (often the case incell and tissue extracts), the assembly of antigen-antibodycomplexes into precipitates can take hours, even days, and it isdifficult to isolate the small amount of immunoprecipitate thatforms.

When used in conjugation with biosynthetic radioisotopelabelling, immunoprecipitation can also be used to determinewhether a particular antigen is actually synthesized by a cell ortissue.

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Immunofluorescence In 1944, Albert Coons showed that antibodies could be

labeled with molecules that have the property tofluorescence.

If antibody molecules are tagged with fluorescent dye,or fluorochrome, immune complexes containing thesefluorescently labeled antibodies can be detected bycolored light emission when excited by light ofappropriate wavelength.

Antibody molecules bound to antigens in cells ortissue sections can similarly be visualized.

The emitted light could be viewed with fluorescencemicroscope, which is equipped with UV light source.

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In this technique (immunofluorescence), various fluorescent compounds in use include Fluorescein, Rhodamine, Phycoerythrin.

Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect.

In direct staining, the specific antibody (the primary Ab) is directly conjugated with fluorescein.

In indirect staining, the primary Ab is unlabeled and is detected with an additional flurochrome-labeled reagent.

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Immunoelectron microscopy Specificity of antibody has made them powerful tools

for visualizing specific intracellular tissue componentsby immunoelectron microscopy.

In this technique,

Electron-dense label conjugated to Fc portion of aspecific antibody for direct staining or conjugated toan anti-immunoglobulin reagent for indirect staining Electron dense label(commonly used are ferritinand colloidal gold) absorbs electrons it can bevisualised with electron microscope as small blackdots.

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Monoclonal Antibodies Monoclonal Antibodies are usually produced from

hybridoma clones.

Each hybridoma clone is derived by the fusion of amyeloma cell and an antibody producing lymphocyte,and the hybridoma clone producing the desiredantibody is identified and isolated.

Hybridoma cells are mass-cultured for the productionof monoclonal antibodies either (1) in vivo in theperitoneal cavity of mice or (2) in vitro in large scaleculture vessels.

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Application When Mabs are used to detect the presence of a

specific antigen or of antibodies specific to an antigen in a sample or samples, this constitutes a diagnosic application.

Antibodies specific to a cell type, say, tumor cells, can be linked with a toxin polypeptide to yield a conjugate molecule called immunotoxin. This immunotoxin will bind to tumor cell and kills it.

Immunopurification.

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ANTIGENS AND HAPTENS The two terminologies viz., antigens and haptens are intimately

associated with immunology ; and, hence one may understand and have aclear concept about them as far as possible.

Antigens An antigen is either a cell or molecule which will bind with preexiting

antibody but will not definitely cause induction of antibody production. Antigen may also be defined as a macromolecular entity that

essentially elicits an immune response via the formation of specificantibodies in the body of the host.

In a broader perspective the antigen (or immunogen) is invariably regardedas the afferent branch of the prevailing immune system, and is any cell ormolecule which would provoke an immune response very much in animmunologically viable and competent individual. Generally, immunogens(antigens) must fulfill the following two characteristic features, namely:

(a) should be larger than 2000 in molecular weight, e.g., protein, glycoprotein andcarbohydrates, and

(b) must be absolutely foreign to the individual into whom they have beenintroduced appropriately.

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Example : The best example of an antigen is ones ownerythrocytes. Because, they will not induce antibodyformation in oneself but will definitely react with anantibody essentially contained in an improperly matchedblood transfusion.

Quite often an antigen is a protein, but it could also be apolysaccharide or nucleic acid or any other substance.

Importantly, it may also be possible that a foreignsubstance (e.g., protein)-not necessarily belonging to apathogenic microorganism, may act as an antigen sothat on being injected into a host, it may induce antibodyformation.

Besides, they may turn out to be antigenic and therebycause stimulation of antibody production, incase they areintimately and lightly get bound to certainmacromolecules, for instance : proteins, carbohydrates andnucleic acids.

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Haptens In usual practice, the relatively smaller, less rigid or rather less

complex molecules usually are not immunogenetic in their purestform, but may be made so by simply linking them strategically toeither larger or more complex structures. Consequently, the smallermolecules are invariably termed as haptens ; whereas, the largermolecules or cells are known as carriers.

Hapten may also be definedas a substance that normally doesnot act as an antigen or stimulate an immune response but that canbe combined with an antigen and, at a later time, initiate a specificantibody response on its own.

Furthermore, small molecules (micromolecular), such as : drugsubstances, that may serve as haptens and can normally be madeantigenic by coupling them chemically to a macromolecularsubstance e.g., protein, polysaccharide, carbohydrate etc. The haptenis obtained from a non-antigenic compound (micromolecule) e.g.,morphine, carteolol etc., which is ultimately conjugated,covalently to a carrier macromolecule to render it antigenic.

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One of the good example is of gastrin (hapten) which is dulycoupled to albumin (i.e., protein carrier) by treatment withcarbodiimides (CCD), which couple functional carboxyl,amino, alcohol, phosphate or thiol moieties.

Importantly, the hapten-conjugate thus obtained is normallysubjected to emulsification in a highly refined mineral oilpreparation containing-killed Mycobacterium (CompleteFreunds Adjuvant), and subsequently injected intradermallyeither in healthy rabbits or guinea pigs on several occasionsat intervals.

Evidently, the serum antibody should have not only highdegree of specificity but also a reasonably strong affinity forthe prevailing antigens.

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Hypersensitivity Immune system mobilizes variety of effector molecules that act to

remove antigen by various mechanisms.

Generally, these effector molecules induce a localized inflammatoryresponse that eliminates antigen without extensively damaging thehosts tissue. Under certain circumstances, however, this inflammatoryresponse can have deleterious effects, resulting in significant tissuedamage or even death. This inappropriate immune response is termedhypersensitivity or allergy.

Although the word hypersensitivity implies an increased response, theresponse is not always heightened but may, instead, be an inappropriateimmune response to an antigen. Hypersensitive reactions may developin the course of either humoral or cell-mediated responses.

Hypersensitivity may be defined as an abnormal sensitivity to astimulus of any kind.

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There are four types of hypersensitivity reaction:

1. Type I hypersensitivity (IgE Mediated Hypersensitivity)

2. Type II (IgG Mediated Hypersensitivity)

3. Type III (Immune complex mediated hypersensitivity)

4. Type IV (Cell Mediated Hypersensitivity)

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1. IgE mediated Hypersensitivity A type I hypersensitive reaction is induced by certain types of antigens (such as foreign

serum, vaccine, penicillin, rye grass, ant venom, bee venom, etc) referred to as allergens,and has all the characteristics of a normal humoral response.

That is, an allergen induces a humoral antibody response by the same mechanisms as forother soluble antigens, resulting in the generation of antibody-secreting plasma cells andmemory cells.

What distinguishes a type I hypersensitive response from a normal humoral response isthat the plasma cells secrete IgE. This class of antibody binds with high affinity to Fcreceptors on the surface of tissue mast cells and blood basophils.

Mast cells and basophils coated by IgE are said to be sensitized. A later exposure to thesame allergen cross-links the membrane-bound IgE on sensitized mast cells andbasophils, causing degranulation of these cells.

The pharmacologically active mediators released from the granules act on thesurrounding tissues. The principal effectsvasodilation and smooth-musclecontractionmay be either systemic or localized, depending on the extent of mediatorrelease.

The clinical manifestations of type I reactions can range from life-threatening conditions,such as systemic anaphylaxis and asthma, to hay fever and eczema, which are merelyannoying

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General mechanism underlying a type I hypersensitive reaction. Exposure to an allergen activates B cells to form IgEsecreting plasma cells. The secreted IgE molecules bind to IgE specific Fc receptors on mast cells and blood basophils.(Many molecules of IgE with various specificities can bind to the IgE-Fc receptor.) Second exposure to the allergen leadsto crosslinking of the bound IgE, triggering the release of pharmacologically active mediators, vasoactive amines, frommast cells and basophils. The mediators cause smooth-muscle contraction, increased vascular permeability, andvasodilation. 114

2. Antibody Mediated cytotoxic Hypersensitivity Type II hypersensitive reactions involve antibody-mediated destruction of cells.

Antibody can activate the complement system, creating pores in the membraneof a foreign cell, or it can mediate cell destruction by antibody dependent cell-mediated cytotoxicity (ADCC). In this process, cytotoxic cells with Fc receptorsbind to the Fc region of antibodies on target cells and promote killing of thecells .Antibody bound to a foreign cell also can serve as an opsonin, enablingphagocytic cells with Fc or C3b receptors to bind and phagocytose theantibody-coated cell.

Examples : The various examples are as stated below :

(i) Transfusion reactions i.e., when blood groups are not matched properly,

(ii) Haemolytic disease concerning the newly born babies via Rhesus incompatibility,

(iii) Graft destruction or rejection i.e., antibody-mediated graft destruction or rejection.

(iv) Autoimmune reactions usually directed against the formed elements of the blood, and the kidney glomerular basement membrances, etc.

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Example 1: Transfusion Reactions Are Type II Reactions

A large number of proteins and glycoproteins on the membrane of red blood cellsare encoded by different genes, each of which has a number of alternative alleles. Anindividual possessing one allelic form of a blood-group antigen can recognize otherallelic forms on transfused blood as foreign and mount an antibody response. Insome cases, the antibodies have already been induced by natural exposure to similarantigenic determinants on a variety of microorganisms present in the normal floraof the gut. This is the case with the ABO blood-group antigens.

Antibodies to the A, B, and O antigens, called isohemagglutinins, are usually of theIgM class. An individual with blood type A, for example, recognizes B-like epitopeson intestinal microorganisms and produces isohemagglutinins to the B-likeepitopes.

If a type A individual is transfused with blood containing type B cells, a transfusionreaction occurs in which the anti-B iso-hemagglutinins bind to the B blood cellsand mediate their destruction by means of complement-mediated lysis. Antibodiesto other blood-group antigens may result from repeated blood transfusions becauseminor allelic differences in these antigens can stimulate antibody production. Theseantibodies are usually of the IgG class.

The clinical manifestations of transfusion reactions result from massiveintravascular hemolysis of the transfused red blood cells by antibody pluscomplement. These manifestations may be either immediate or delayed.

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Hemolytic Disease of the Newborn Is Caused by Type II Reactions Hemolytic disease of the newborn develops when maternal IgG antibodies

specific for fetal blood-group antigens cross the placenta and destroy fetal redblood cells. The consequences of such transfer can be minor, serious, or lethal.

Severe hemolytic disease of the newborn, called erythroblastosis fetalis, mostcommonly develops when an Rh+ fetus expresses an Rh antigen on its bloodcells that the Rh mother does not express.

This most commonly happens when a woman with Rh negative blood becomes pregnant by a man with Rh positive blood and conceives a baby with Rhpositive blood.

Red blood cells from the baby can leak across the placenta into the woman's bloodstream during pregnancy or delivery. This causes the mother's body to make antibodies against the Rh factor.

If the mother becomes pregnant again with an Rh-positive baby, it is possible for her antibodies to cross the placenta and attack the baby's red blood cells.

After birth, an affected newborn may develop kernicterus. This happens when bile pigments are deposited in the cells of the brain and spinal cord and nerve cells are degenerated.

Incompatibilities between ABO blood types can also cause this condition. These are less common than those of the Rh factor and tend to be less severe.

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3. Immune ComplexMediated (Type III) Hypersensitivity The reaction of antibody with antigen generates immune

complexes. Generally this complexing of antigen with antibodyfacilitates the clearance of antigen by phagocytic cells.

In some cases, however, large amounts of immune complexes canlead to tissue-damaging type III hypersensitive reactions.

The magnitude of the reaction depends on the quantity ofimmune complexes as well as their distribution within the body.When the complexes are deposited in tissue very near the site ofantigen entry, a localized reaction develops.

When the complexes are formed in the blood, a reaction candevelop wherever the complexes are deposited.

In particular, complex deposition is frequently observed onblood-vessel walls, in the synovial membrane of joints, on theglomerular basement membrane of the kidney, and on thechoroid plexus of the brain. The deposition of these complexesinitiates a reaction that results in the recruitment of neutrophilsto the site. The tissue there is injured as a consequence ofgranular release of lytic enzymes from the neutrophil.

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Type III hypersensitive reactions develop when immune complexes activate thecomplement systems array of immune effector molecules. The C3a, C4a, andC5a complement split products are anaphylatoxins that cause localized mast-cell degranulation and consequent increase in local vascular permeability. C3a,C5a, and C5b67 are also chemotactic factors for neutrophils, which canaccumulate in large numbers at the site of immune-complex deposition. Largerimmune complexes are deposited on the basement membrane of blood vesselwalls or kidney glomeruli, whereas smaller complexes may pass through thebasement membrane and be deposited in the subepithelium. The type oflesion that results depends on the site of deposition of the complexes.

Much of the tissue damage in type III reactions stems from release of lyticenzymes by neutrophils as they attempt to phagocytose immune complexes.The C3b complement component acts as an opsonin, coating immunecomplexes.

A neutrophil binds to a C3b-coated immune complex by means of the type Icomplement receptor, which is specific for C3b. Because the complex isdeposited on the basement- membrane surface, phagocytosis is impeded, sothat lytic enzymes are released during the unsuccessful attempts of theneutrophil to ingest the adhering immune complex. Further activation of themembrane-attack mechanism of the complement system can also contribute tothe destruction of tissue. In addition, the activation of complement can induceaggregation of platelets, and the resulting release of clotting factors can lead toformation of microthrombi.

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Type IV or Delayed-Type Hypersensitivity (DTH) When some subpopulations of activated TH cells encounter certain types

of antigens, they secrete cytokines that induce a localized inflammatoryreaction called delayed-type hyper- sensitivity (DTH).

The reaction is characterized by large influxes of nonspecific inflammatorycells, in particular, macrophages.

This type of reaction was first described in 1890 by Robert Koch, whoobserved that individuals infected with Mycobacterium tuberculosisdeveloped a localized inflammatory response when injected intradermallywith a filtrate derived from a mycobacterial culture.

He called this localized skin reaction a tuberculin reaction. The characteristic of a type IV reaction are the delay in time required for

the reaction to develop and the recruitment of macrophages as opposed toneutrophils, as found in a type III reaction.

In this type, sensitized TH1 cells release cytokines that activatemacrophages or TC cells which mediate direct cellular damage.

Macrophages are the major component of the infiltrate that surrounds thesite of inflammation.

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Antibody Production (Immunogen Preparation)

The production of specific antibody probes is arelatively straightforward process involvingimmunization of animals and reliance upon theirimmune systems to raise responses that result inbiosynthesis of antibodies against the injectedmolecule. Even so, several factors affect the probabilityof inducing an immunized animal to produce usefulamounts of target-specific antibodies. Antigens mustbe prepared and delivered in a form and manner thatmaximizes production of a specific immune responseby the animal. This is called immunogen preparation.

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Antibody production is conceptually simple. However, because itdepends upon such a complex biological system (immunity of a livingorganism), results are not entirely predictable. Individual animals even those that are genetically identical will respond uniquely to thesame immunization scheme, generating different suites of specificantibodies against an injected antigen. Even so, equipped with a basicunderstanding of how the immune system responds to injection of aforeign substance and a knowledge of available tools for preparing asample for injection, researchers can greatly increase the probability ofobtaining a useful antibody product.

For example, small compounds (drugs or peptides) are not sufficientlycomplex by themselves to induce an immune response or be processedin a manner that elicits production of specific antibodies. For antibodyproduction to be successful with small antigens, they must bechemically conjugated with immunogenic carrier proteins such askeyhole limpet hemocyanin (KLH). Adjuvants can be mixed andinjected with an immunogen to increase the intensity of the immuneresponse.

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