ODD for human blood-M. Asif

2/25/2013
MUHAMMAD ASIF
Forensic Scientist (DNA/Serology)
Ouchterlony Double Immunodiffusion Assay
for Human Blood Detection
1

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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
optimal2/25/2013 29

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
lids.
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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 template
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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 7
100 x 15 mm petri
dish
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Filling Wells Procedure
5-7 µl
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Cross section of wells showing reagent levels
Correct
Over
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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ODD for human blood-M. Asif

ODD for human blood-M. Asif

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