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IMMUNOASSAY :ANTIBODY

Melisa Intan Barliana


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Introduction

Immunoassays are the methods of choice tomeasure biochemicals or analytical interestspecies that are enable to measure by enzymaticassay.

These analytes whether macromolecular or of relatively low molecular weight, may not even bespecies that are commonly found in living

systems.

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Many routine test procedures are antibody-based, such as:

1. Tests rely upon ability of antibodies to aggregate(agglutination) particulates antigens (e.g., blood typing) or toprecipitate soluble antigens (e.g., radial immunodiffusion,immunoelectrophoresis).

2. Assays rely upon chemically modified antibodies to quantitateantigens (e.g., radioimminoassays and immunosorbent assay)

with exquisite specificity and sensitivity.3. Additional assay (e.g., immunofluorescence and flow

cytometry) utilize fluorochrome-labeled antibodies to assessantigen expression within and on the cell surface.

4. Immune fuction also can be assessed in the laboratory (e.g.,complement fixation, proliferation and cytotoxic T lymphocyteassay) or in a clinical setting (assesment of hypersensitivity)


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Immunoassays rely on the selective binding propertiesof antibodies.

Antibody :• large glycoproteins of molecular weight 150 kDa,that possess two identical binding sites per moleculeand are thus called bivalent.

• an immunoglobulin molecule with specificity for anepotipe of the molecules that recognize antigen.

• produced in living organisms via the immuneresponse, in response to an immunogen (agentcapable of eliciting an immune response).

Introduction

Antibody

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Antibody

The N-terminal ends of the light polypeptide chains called Fabfragments. This fragments used in immunoassays based

on primary antigen–antibody interactions.

The heavy chains (H) in the so-called Fc fragments determinesthe antibody class: the most abundant classes are called IgG,IgM, IgA, IgE, and IgD.


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Antibodies isotypes

1. IgM

Most B cells display IgM on their cell surfaces.In general, IgM is thefirst immunoglobulin to be formed following antigenic stimulation.

2. IgDIgD has a monomeric structure and is almost exclusively displayed onB cell surfaces. Little is known of its function.

3. IgGMany IgG antibodies are effective in activating complement,opsonizing and neutralizing microorganisms and viruses, and initiatingantibody-dependent cell-mediated cytotoxicity, and they function in awide variety of hypersensitivity functions.

4. IgAIgA found in mucus, saliva, tears, breast milk, and gastrointestinalsecretions.

5. IgEIgE is present in relatively low serum concentration; most is adsorbedonto the surfaces of mast cells, monocytes, and eosinophils. Cross-linking of IgE on mast cell sur-faces by antigen triggers the release ofhistamine and other inflam-matory mediators, leading to immediatehypersensitivity (allergic)responses.


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Properties of Common Immunoglobulisn


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Once antibodies have been generated, their selective bindingproperties toward the high molecular weight immunogens

allow their interaction with other speciesthat possess certain structural similarities, which is:

Antigen is an organism, a molecule, or part of a moleculethat is recognize by the immune system.

Antigens are not necessarily capable of generating the immuneresponse themselves; thus, all antigens are not immunogens, whileall immunogens are also antigens.

Hapten is are low molecular weight compounds that can onlyelicit an immune response when they are chemically bound

to a high molecular weight compound, such as a carrierprotein.


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A schematic representation immune response


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Antigen-antibody reactions

The selectivity of antigen (Ag) – antibody (Ab) orhapten-antigen interactions is analogous to theselectivity of substrate–enzyme interactions,most specific noncovalent biochemical reactions.

The antigen-binding specific site of an antibody

called an epitope.

Ag + Ab AgAb

The strength of the binding of an antibody to an antigenreferred to its affinity.The reaction Ag-Ab is reversible depends on its affinity.


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Antigen-antibody Selectivity Interaction

The portion of antigen that interacts specifically withantigen-binding site on the antibody is called the antigenicdeterminant, or epitope.

The epitope size : ~0.7x1.2x3.5 nm equivalent to ~5-7amino acid residues

The complementary site on the antibody is called paratope,has the same size.



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Primary interactions :involve the specific recognition and combination of anantigenic determinant (epitope) with the binding site(paratope) of its corresponding antibody.

All Ab–Ag interactions begin with a primary interaction,

which occurs very rapidly (on a millisecond time scale),and is macroscopically invisible.


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Secondary antibody–antigen reactions :

occur as a result of antigen multivalency, and result inagglutination or precipitation of a polymeric antigen–antibody network.

A visible product is formed over a time scale of minutesto hours.


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The intrinsic association constant that characterizesantibody–epitope (hapten) binding is called the affinity:

Typical affinity values forantibody–hapten interactionsrange from 105 to 1012 M-1.

Affinity values are experimentally determined by maintaining aconstant total Ab concentration, and varying the H concentration.

[H]bound

[H]free [Ab]total


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Epitope Detection by Antibodies

A. Particulate AntigensParticulate antigens such as erythrocytes, bacteria, oreven antigen-coated latex beads are normally evenlydispersed in suspension.

Cross-linking between antigen-antibody causesclumping of the particles, also known as agglutination.

Agglutination has the effect of entrapping microbialinvaders within molecule, inhibiting their mobility, andrendering them more susceptible to destruction.


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Agglutination occurs only over a certain range of serum dilutions

because of either antigen or antibody excess.In the prozone, a large excess of antibody is present, so that eachantibody behaves univalently and cross-linking does not occur

Agglutination

At very high serum dilutions, a large excess of antigen exists, sothat not enough antibody is present to cross-link the antigen andcause agglutination. In this case, the antigen behaves in a

univalent manner


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1. Direct agglutinationThis reaction usually involves IgMantibodies that cross-link epitopeson cells or particles.


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Direct Agglutination


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2. Indirect or passive agglutinationThis techniques is often used to detect non-IgM (IgG)antibodies or antibodies in concentration too low to bedetected by direct agglutination.

Particulate antigen is incubated witha primary antibody. A secondaryantibody, also called an antiglobulin

or anti-immunoglobulin, is added toreact with the primary antibody tocause cross-linking or agglutination.


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The precipitin reaction

is the interaction of soluble antigenand soluble antibody that result information of Ag-Ab complexes(lattices) large enough toprecipitate from solution.

At a certain Ab/Ag ratio, the cross-linked polymeric network ofantibody and antigen loses itssolubility and precipitates.

B. Soluble Antigens


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1. Radial immunodiffusion (Mancini techniques)or Single radial Immunodiffusion (SRID)

This test is based upon the diffusion of soluble antigenwithin agar gel that contains a uniform concentrationof antibody.


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2. Double-diffusion(Ouchterlony technique)

This test is based upon thediffusion both antigen(loaded in one well) andantibody (loaded in anotherwell) through an agar gel.


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3. Immunoelectrophoresis (IEP)

This technique is a modificationof double diffusion.

Antigen are loaded into a wellwithin the agar, an electricalcurrent is applied, and antigensmigrate according both theirsize and their electrical charge.The electrical current isremoved, a trough is cut intothe agar, and antibody is placedin the trough.


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Quantitative precipitin curve.

Antibody-containing serum is

usually serially diluted, producing`half` concentrations of antibodies

Antigen in equal concentrationsis added to the antibody dilutions

resulting in differing degrees ofantigen-antibody complex formation

Antigen-antibody complexes areformed in three zones, zone of

antibody excess, equivalence zone,and zone of antigen excess.

Reaction with particulate antigens,caused agglutination

Reaction with soluble antigen

Reaction with particulate antigen


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Polyclonal and Monoclonal antibodies

Polyclonal antibodies (or antiserum)are antibodies that are obtained from different B cellresources.

The immunogen used to generate polyclonalantibodies may consist of a small molecule (a hapten)covalently bound to a carrier protein.

The resulting antiserum will contain a mixture of antibodiesthat bind to different epitopes of the hapten–carrierconjugate, as well as antibodies generated in response toother immunogens present in the organism.

This results in an antiserum possessing a wide range ofselectivities and affinities, and may result in significantcross reactivities or interferences, when employed inimmunoassays.


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Monoclonal antibodies

Monoclonal antibodies are a homogeneous population ofidentical antibody molecules because it derived from asingle B cell. It has identical paratopes and affinity for asingle antigenic epitope.

These antibodies are specific, is very restricted in antigen

to which it will bind, making it a very specific reagent.


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The generation of hybridoma cell lines formonoclonal antibody production

(Milstein and Kohler’s procedure)

HPRT : HipoxanthinePhosphoribosyltransferasefunction in purine nucleotidesproduction


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(1) Immunisation of a mouse(2) Isolation of B cells from the spleen(3) Cultivation of myeloma cells(4) Fusion of myeloma and B cells(5) Separation of cell lines(6) Screening of suitable cell lines(7) in vitro (a) or in vivo (b) multiplication(8) Harvesting


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