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MUHAMMAD ASIF

Forensic Scientist (DNA/Serology)

Ouchterlony Double Immunodiffusion Assay

for Human Blood Detection

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PART-1

IMMUNODIFFUSION

BASICS

Introduction

Definition: The passive diffusion of soluble antigens and/or antibodies toward

each other leading to their precipitation in a gel matrix.

The Ouchterlony double immunodiffusion (ODD) technique is one of the

simplest techniques extensively used to check antisera for the presence of

antibodies for a particular Ag and to determine its titre (concentration). This

method has been widely used for detection and qualitative diagnostic

procedures. The method is called "double" referring to the fact that in this

procedure, antigen and antibody are allowed to migrate towards each other in

a gel and a line of precipitation is formed where the two reactants meet. This

precipitation reaction is highly specific. The method is even today widespread

and used by people working with diagnosis or protein detection or comparing

antigens or antisera. The method is not very sensitive.

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An antigen reacts with a specific antibody to form a large macromolecular

immune complex.

The immune complex composition depends on:

1. Nature,

2. Concentration

3. Proportion of the initial reactants

Each antibody can associate and bind with more than one antigen and each

antigen can be bound by more than one antibody molecule. The noncovalent

interactions form the basis of antigen-antibody (Ag-Ab) binding:

1. Hydrogen bonds,

2. Ionic bonds,

3. Hydrophobic interactions,

4. Vander Waals interactions.

Principle

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Immunodiffusion in gels is used for the analysis of antigens and antibodies.

Types of Immunodiffusion

1. Single Immunodiffusion

2. Double Immunodiffusion

Immunodiffusion in Gels

Single (simple) diffusion in one dimension:

The process of diffusion of an antigen in an antibody-containing gel

The process of diffusion of an antibody in an antigen-containing gel.

Immunoprecipitin line is formed at the point of equivalence.

Single (simple) Immunodiffusion

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Both antigen and antibody are diffuse into the gel; diffusion in two dimension

(Ouchterlony method).

Immunoprecipitin line is formed at the point of equivalence.

Utilized as a rough estimation of antigen or antibody purity.

Used for semiquantitative analysis in human serological system.

Antigens from different species are loaded into two wells and the known

antibody is loaded in a third well located between the antigen wells to form a

triangle. Depending on the similarity between the antigens, different

geometrical patterns are produced between the antigen and antiserum wells.

The pattern of lines that form can be interpreted to determine whether the

antigens are same or different.

Double Immunodiffusion-1

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Double Immunoiffusion-2

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Antigen

A substance that when introduced into the body stimulates the production of an

antibody. Antigens include toxins, bacteria, foreign blood cells, and the cells of

transplanted organs.

In general, an antigen is defined as a substance that binds to specific antibodies,

which in the human body are used to find and neutralize any potentially harmful

foreign substances in the bloodstream. The specific binding between antigen and

antibody is similar to that of the lock-and-key binding model.

Antigen receptor, a surface protein located on B cells and T cells, binds to antigens

and initiates acquired immune responses. The antigen receptors on B cells are

called B cell receptors (or membrane immunoglobulins) and the antigen receptors

on T cells are called T cell receptors. The N-terminal region of the light and heavy

chains each have a domain of approximately 110 amino acids known as the

variable region and serve as the antigen binding site of the molecule.

In human blood, the different lettering of different blood types is designated by the

specific antigen present in the individual's blood cells. While all types contain the

oligosaccharide (O) antigen, the A and B blood types are defined by having N-

acetylgalactose (A) or galactose (B) monosaccharide. Likewise, the AB blood

group has both A and B antigens. Additional antigens are bound to define the

positive or negative state of the ABO blood groups. The structures of the enzymes

that bind to the antigen are similar and very slightly different, demonstrating

antigen specificity.

Antigen Receptor

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Characteristics of Antigens

Immunogenicity – property of substance (immunogens or antigens) to

induce a detectable immune response

Antigenic specificity – property of antigen molecule (or its part) to react

with the specific antibody.

Antigenicity – given by a surface structure of immunogen - antigenic

determinants. The organism responds only to those that are foreign to him.

The number of antigenic determinants – usually varies with the size and

chemical complexity of macromolecule (egg ovalbumin, MW 42 000, has 5

antigenic determinants and thyroglobulin, MW 700 000, has many as 40).

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Antigens may either be proteins or polysaccharides.

Chemical nature of antigens:

proteins

polysacchrides

lipopolysaccharides

nucleoproteins

glycoproteins

steroid hormones

bacterial cells, viruses

synthetic polypeptides

synthetic polymers

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Characteristics of Antigens

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Antibodies (also called immunoglobulins or Ig's) are complex, Y-shaped secreted

protein on the surface of B cells/ lymphocytes that circulate through the blood

stream or lymph in response to an antigenic stimulus such as a bacterium, virus,

parasite, or transplanted organ, and bind to specific antigens, thereby attacking

microbes during an immune response. The body contains millions of different B

cells, each able to respond to one specific antigen.

Each antibody is made of four polypeptide (protein) chains: 2 heavy chains and

2 light chains. Both heavy chains are identical to each other and both light chains

are identical to each other. Each contains a constant region and a variable region.

The constant region forms the main part of the molecule while the variable regions

forms the antigen-binding site. Each antibody has 2 antigen-binding sites.

The portion that recognizes and binds to the foreign protein is the "Fab" portion,

which is identical within all species; its chemical structure does not differ between

species. However, the second portion ("Fc") is specific to each species.

Antibodies

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Polyclonal antibody – a sample that contains a mixture of antibodies active

against a specific antigen, each recognizing a different epitope or region of the

antigen.

1. Recognise multiple epitopes on any one antigen. Serum obtained will contain

a heterogeneous complex mixture of antibodies of different affinity

2. Polyclonals are made up mainly of IgG subclass

3. Peptide immunogens are often used to generate polyclonal antibodies that

target unique epitopes, especially for protein families of high homology

Monoclonal antibody – a quantity or culture of a single antibody type,

produced in vivo (in the body) or in vitro (in the lab), directed against a

specific epitope, and produced by a single clone of B cells, or a myeloma (cancer)

cell line.

1. Detect only one epitope on the antigen.

2. They will consist of only one antibody subtype. Where a secondary antibody

is required for detection, an antibody against the correct subclass should be

chosen.

Polyclonal/ Monoclonal Antibodies-1

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Polyclonal antibodies are obtained from serum of animals immunized with a

particular antigen. The antibody mixture obtained from serum is the result of

many B-cell clones, each producing one specific antibody. This mixture of

antibodies often can recognize multiple epitopes (region of an antigen that binds

to an antibody), making them more tolerant to small changes in the structure of

the antigen. It is often a better choice for detection of denatured proteins. Also,

polyclonal antibodies may be generated in a variety of species such as rabbit,

sheep, and donkey. This provides a greater number of experimental options to

researchers.

Monoclonal antibodies are produced by fusing myeloma cells with antibody

secreting B-cells. The resulting hybridoma cells produce large quantities of

homogenous antibodies that recognize a single epitope. Because of their

specificity, monoclonal antibodies are excellent as the primary antibody in an

assay, or detecting antigen in tissue, and will often give significantly less

background staining than polyclonal antibodies. However, due to its specificity to

only one type of epitope, a monoclonal antibody will not bind to its specific

antigen if it is degraded. In contrast, a polyclonal antibodies mixture may still

retain some of its binding ability even if certain species within the mixture are

structurally compromised.

Polyclonal/ Monoclonal Antibodies-2

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Function of antibodies includes:

1. Recognition and binding to antigens

2. Inactivation of the antigen

Function of Antibodies

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The antibodies are made of two identical polypeptide "heavy" chains (55 kD)

and two identical polypeptide "light" chains (25 kD). The carboxy-terminal

regions of the heavy chains fold together to form the Fc domain. The opposite

end of each heavy chain is called the amino-terminal region. These ends each

combine with light chains to form the Fab domain. The light and heavy chains

both have variable regions, attached by covalent bonds. Covalent disulfide

bridges as well as non-covalent bonds hold the four chains together. The

disulfide bridges between the heavy chains form flexible hinge-like structures

between the Fab and Fc domains and are composed mostly of proline, serine

and threonine. The hinges allow the antibody to conform to different sized

structures that contain the appropriate epitope. This is the region most

susceptible to damage .

Composition of Antibodies

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Different functions of different parts of the Antibody-1

The two structural portions of the antibody, i.e. the variable (Fab) and the constant (Fc) fragments, impart

distinct biological functions.

Fab-mediated functions:

1. Antigen recognition – One of the major functions of the Fab region is antigen recognition. The

immune system generates large number of antibodies that can recognize virtually all possible antigens

present in pathogens and their products. These may be on invading microbes such as bacteria, viruses,

and parasites as well as environmental antigens. Antibodies can be produced against all types of

molecules including carbohydrates, nucleic acids and phospholipids but are best suited to bind against

a protein.

2. Neutralization of pathogens – Once the antibodies recognize the antigens the binding occurs outside

the cell. This is where most of the bacteria and bacterial toxins are found. The binding prevents the

access of the pathogen into the cells and prevents infection or destruction of host cells. Antibodies

also block the binding of the bacteria to host cells by binding to cell-surface proteins. Antibodies

protect similarly from viral infections as well.

3. Antibodies are the first line of defence - IgM antibodies have a pentameric structure and are rapidly

generated in blood. They can bind to multivalent antigens, such as bacterial cell wall polysaccharides.

This is because each IgM pentamer has 10 antigen-binding sites. This enhances its strength and ability

to bind to antigens. IgM antibodies are also elective in complement activation. IgG also helps in

opsonization and complement activation. IgG diffuses into the tissues and binds to toxins rapidly. IgG

can thus neutralize foreign antigens and protect epithelial cells from infectious agents acting as first

line of defence.

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Fc-mediated effector functions:

1. Activation of effector cells – Through their Fc fragments, antibodies can activate

accessory elector cells. These include phagocytic cells like macrophages and

neutrophils, T cells like natural killer cells, and eosinophils and mast cells. Each of

these cells has a receptor for the Fc fragment. Thus these cells can identify an Fc

fragment and eliminate the pathogens.

2. Complement binding – Once bound to the antigen there is formation of antigen–

antibody complexes. This further activates a complex set of reactions called the

complement cascade. Complements are series of plasma proteins that help in release of

chemical mediators from mast cells (mast cell degranulation), phagocytosis (eating up

of bacterial and microbial cells by macrophages) and cell lysis (breaking down or

bursting of the invading cells). Complement activation begins when the C1q molecule

binds to antibody molecules attached to the surface of a pathogen and triggers the

classical pathway of complement activation. The main functions of the complements

are to enable phagocytes to destroy bacteria that they would otherwise not recognize.

Neither complement nor phagocytes are specific for the pathogen but the antibodies

are.

3. Opsonization – Those microbes that replicate outside cells are removed by an

interaction of the Fc part with specific receptors on the surface of elector cells. The

antibodies coat the surface of the pathogen and allow binding of their Fc domains to Fc

receptors present on elector cells. The macrophages and neutrophils then engulf the

pathogen and internalize the microbe causing its destruction.

Different functions of different parts of the Antibody-2

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There are 5 classes of antibodies (listed from most common to least common):

•IgG

•IgM

•IgA

•IgE

•IgD

Types of Antibodies

Variability of antibodies is subject to 5-classes of Ig: G, A, M, D, E

Heavy chains – g, a, m, d, e

Light chains – k, l

Subclasses of immunoglobulins:

IgG – g1, g2, g3, g4

IgA – a1, a2

IgM - m1, m2

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Types of Antibodies-2

Characteristics of Antibodies (Immunoglobulins)

Proteins with the property

of specific combination

with antigen (or one

antigenic determinant)

which elicited their

formation.

Immunoglobulins account

for ~ 20% of the total

plasma proteins.

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A precipitin band is formed from the integral binding of an antibody to a soluble

antigen, when the diffusing stain contains proteins that are recognized by IgG

molecules in the diffusing antiserum. An immune complex is formed in the region

of equivalence in an agar matrix due to antigen and antibody diffusion toward one

another. However, no visible precipitate is formed in regions of antibody or

antigen excess. Two types of immunodiffusion reactions can be used to determine

relative concentrations of antibodies or antigens, to compare antigens, or to

determine the relative purity of an antigen preparation. They are radial

immunodiffusion (the Mancini method) and double immunodiffusion (the

Ouchterlony method); both are carried out in a semisolid medium such as agar.

Immune Complex

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Precipitation occurs because the antigen is multivalent i.e., has several antigenic

determinants per molecule to which antibodies can bind. Antibodies have at least two

antigen binding sites, thus large aggregates or lattices of antigen and antibody are formed.

Precipitation will not occur if excess antigen is present or if excess antibody is present.

Cross-linking and lattice formation will only occur when antigen and antibody

concentrations are optimal. An increasing amount of antigen is added to a constant amount

of antibody in solution. This is called the antibody-excess zone (Prozone phenomenon). The

Ag and Ab concentrations are relatively higher near their respective wells. As they diffuse

farther from the wells, their concentration decreases. An antigen will react with its specific

antibody to form an Ag-Ab complex. As more antigens are added, the amount of protein

precipitated increases until the antigen/antibody molecules are at an optimal ratio. This is

known as the equivalence zone or equivalence point. When the amount of antigen in

solution exceeds the amount of antibody, the amount of precipitation will decrease. This is

known as the antigen excess zone.

Immune Complex

Imunoprecipitation reaction

Used for qualitative and quantitative detection of

antigens and antibodies:

phase one – formation of primary complexes

with low MW

Phase two – interconnection of Ag and Ab to

the three dimensional network (formation of

insoluble aggregates )

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Diffusion of Reagents

Ag As

Seen as a precipitin line

when concentrations are

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Diffusion of Reagents

Ag As

At 24 hours a

precipitin line is visible2/25/2013 30

The forces binding antigen to antibody

Electrostatic : between attraction oppositely charged ionic group – (-NH3-) of

lysine and (-COO-) of aspartate.

Hydrogen bonding – relatively weak and reversible hydrogen bridges between

hydrophilic group (-OH, -NH2, COOH).

Hydrophobic– non-polar, hydrophobic side chains of Val, Leu, Ile (hydrophobic

groups come close together and exclude water molecules between them. The force

of attraction increases.

Van der Waals – forces which depend upon interaction between the external

„electron clouds“. Non-specific attractive forces.

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Prozone : Ab excess,

precipitate does not

form ( (soluble

immune complexes)

Zone of

equivalence- optimal

ratio of Ag/Ab –

insoluble precipitate

Post-zone – excess of Ag

(soluble immune

complexes)

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The ratio of antigen / antibody

Precipitation and immunodiffusion in gels

The development of immune-complex is based on different rates of diffusion of Ag and

Ab into the gel, depending on their :

concentration

physicochemical properties

gel structure

Distance between wells (very important step)

Most widely used gels – agar a agarose

Tests are performed by pouring molten agar (agarose) onto glass slides

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Advantages:

•Group-specific test (detects antibodies to all subtypes, determine

antigenic relationships)

•Easy, requires few reagents/equip

Disadvantages:

•Semi quantitative

•Moderate sensitivity

•Subjective interpretation

•Requires 24 hr

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PART-2

Ouchterlony Double Immunodiffusion Assay

for Human Blood Detection

The following materials are necessary:

Agar

Sodium azide (NaN3)

Sodium chloride (NaCl)

Distilled water

Plastic petri dishes (100 x 15 mm)

Cork borer punch (5 mm diameter)

Rubber bulb

Pasteur pipettes

Flasks (125 ml and 250 ml)

Vacuum pump

Materials and Methods

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Preparation of Agar Gel Gel mixture

0.5 g agar

0.01 g NaN3 +

100 ml distilled water

Put agar and water in a flask stopped with

cotton/or aluminum foil. Heat the mixture

in an oven or water bath until the agarose

dissolves. Cool to approximately 40o C.

Add sodium azide. Mix carefully to

prevent the formation of air bubbles.

Dispense 8-10 ml of the solution on each

petri dish and partially cover until gel

solidifies. Remove condensed water from

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a) Use 1 ml tip and cut it to have 2-3 mm diameter of tip. Fine the tip by

rubbing on paper towel to make it even. Using 1 ml pipet and this

formed tip make wells in the gel according to a desired pattern i.e., 2-3

mm thick; 2-3 mm in diameter and 5 mm apart. The agar disks are

removed by using a vacuum system of pipette.

b) First, place the samples in the peripheral wells. Next, place the

antiserum in the center well. Use a clean pipette tip for each sample and

antisera.

c) Load sample/ antibody volume per well 5-7μl.

Preparation of the Test Plate & loading of Sample/ antibody

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Agar 2-3 mm thick

Wells 2-3 mm in diameter

(adjust distance as per

requirements)

Wells 5 mm apart (adjust

distance as per requirements)

Preparation of the test plate

Sample template2/25/2013 39

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Placement of Antibody/Samples

-Ve

+Ve

S S

Ag

Sample #1

Sample #4

Sample #2

S

S

Sample #3

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Numbering Patterns

1 2

3 4 5

6 7100 x 15 mm petri

dish

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Filling Wells Procedure

5-7 µl2/25/2013 43

Cross section of wells showing reagent levels

CorrectOver

filled

Under

filled

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Hold Tip Vertical When Filling Wells

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Method Not Recommended

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Place plates in a wet chamber. Use a hermetically sealed plastic container with a

piece of damp paper inside and a support to prevent the place from getting wet.

Readings and Interpretations of Results

The first reading is made after 2 hours and the second reading at 18 hours. For

easier reading, use a dark box consisting of a wooden support with an opening at

the top big enough to support the dish firmly in place.

Place a fluorescent light inside the box.

IMPORTANT SOPs:

1. The results will be accepted if standards (both negative and positive controls

are as expected as per QAS).

2. The precipitin band is sometimes clearly visible to the naked eye, but staining

and destaining is must to enhance sensitivity and clarity of casework results.

3. The results will be reviewed by two independent competent analysts/ lab techs.

4. The negative results will be reported and confirmation by re-testing and both

results will be reported in the data sheets.

Incubation of the test plate

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Chemical Tests

Chemically the blood is confirmed by the detection of hemoglobin or its

derivatives by the formation of specific crystals. In Takayama or hemochromogen

test, ferrous iron from hemoglobin reacts with pyridine to produce red feathery

crystals of pyridine ferroprotoporphyrin. Teichman reagent, consisting of a

solution of potassium bromide, potassium chloride and potassium iodide in glacial

acetic acid, and is heated to react with hemoglobin. The reaction first converts the

hemoglobin to hemin, and then the halides react with the hemin to form

characteristic brownish-yellow rhomboid crystals.

Immunoprecipitation (IP)Tests

Blood can be identified as being of human origin by precipitin reactions with

antisera specific for components of human blood. Usually this is an anti-human

serum serum - that is, an antiserum to human serum. Strictly speaking, this is a test

for human origin not for human blood, as serum constituents such as albumin and

some globulins are found in the extra-vascular space.

Confirmatory Tests for human blood

http://www.nfstc.org/pdi/Subject02/pdi_s02_m02_02_b.htm

There are some possible errors that could be encounter ed during this experiment:

1. Contamination of antibodies which may develop a precipitin lines

between antiserum wells on double diffusion.

2. Sample that contains lipemic(excessive amounts of fat and fatty substances in t

he blood; hyperlipemia) which produces non-specific precipitin of protein gel

3. Hemolysed sample were also given similar characteristic to lipemic sample.

4. Others non-antibody such as C-Reactive protein (a protein found in the blood,

the levels of which rise in response to inflammation (i.e. C-reactive protein is an

acute-phase protein), polysaccharides, immunoglobulin, DNA and others.

5. The sensitivity of the test depends on the distance and the reactants

concentrations i.e., the more closer the wells, the smaller amount reactant will

be needed. Furthermore the concentration, thickness and viscosity of the gel also

affect the sensitivity of the test. These problems can be prevented using serial

dilutions.

Possible Errors

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Thank you

for your attention

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