The document discusses various immunological techniques, particularly focusing on antigen-antibody interactions and their applications in immunologic assays for disease diagnosis and monitoring. Key interactions include noncovalent forces like hydrogen bonds and ionic bonds that influence specificity in binding. Notable techniques include agglutination, complement fixation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and western blotting among others, used for detecting antigens or antibodies in clinical samples.

1. Immunological techniques
Agglutinations,
Complement fixation,
Immune diffusion,
Immunoelectrophoresis,
RIA and ELISA,
Western blotting

2. Strength of Antigen-Antibody Interactions
• The noncovalent interactions that form the basis of antigen-antibody
(Ag-Ab) binding include (Figure 6-1).
• hydrogen bonds,
• ionic bonds,
• hydrophobic interactions, and
• van der Waals interactions
• These interactions are individually weak (compared with a covalent
bond), a large number of such interactions are required to form a
strong Ag-Ab interaction.
• Furthermore, each of these noncovalent interactions operates over a
very short distance, generally about 1X 10-7
mm (1 angstrom, Å);
consequently, a strong Ag- Ab interaction depends on a very close fit
between the antigen and antibody.
• Such fits require a high degree of complementarity between antigen
and antibody, a requirement that underlies the exquisite specificity
that characterizes antigen-antibody interactions.

3. The interaction between an
antibody and an antigen depends
on four types of noncovalent forces:
(1) hydrogen bonds, in which a
hydrogen atom is shared between
two electronegative atoms;
(2) ionic bonds between oppositely
charged residues;
(3) hydrophobic interactions, in
which water forces hydrophobic
groups together; and
(4) van der Waals interactions
between the outer electron clouds
of two or more atoms.
In an aqueous environment,
noncovalent interactions are
extremely weak and depend upon
close complementarity of the
shapes of antibody and antigen.

4. Antigen
An antigen is a foreign substance that enters your
body.
bacteria,
viruses,
fungi,
allergens,
Venom
Pollen grains and
other various toxins.

5. Antibodies are Immunoglobulins
• They are protective
glycoproteins produced by your
immune system.
• They attach to antigens (foreign
substances) — such as bacteria,
fungi, viruses and toxins and
remove them from your body.

6. • Antibodies are produced by B cells
specialized white blood cells.
• When an antigen comes into contact with a B
cell, it causes the B cell to divide and clone.
• These cloned B cells — or plasma cells — release
millions of antibodies into your bloodstream
and lymph system.

7. There are five types of antibodies with distinct functions
• IgM –
– Found in your blood and lymph system
– first line of defense against infections
– play a large role in immune regulation.
• IgG –
– Makes upto 70% to 75% of all immunoglobulins
– found mainly in blood and tissue fluids.
– protect the body from viral and bacterial infections.
• IgE-
– Found mainly in skin, lungs and mucus membranes,
– cause the mast cells -a type of white blood cell to release histamine into
the bloodstream.
– causing allergic reactions.
• IgD-
– IgD supports B cell maturation and activation.
• IgA-
– Found in saliva, tears, mucus, breast milk and intestinal fluid,
– protects against ingested and inhaled pathogens.

10. So
• The binding of Ag-Ab
interaction is due to high
degree of complementarity
between antigen and
antibody, offering the
exquisite specificity that is
their characteristic.
Antigen-Antibody Interactions
The basis of antigen-antibody (Ag-Ab) binding
are the weak noncovalent interactions
hydrogen bonds,
 ionic bonds,
 hydrophobic interactions,
 van der Waals interactions

11. • The exquisite specificity of antigen-antibody
interactions
has led
• to the development of a variety of immunologic
assays,
which can be used
• to detect the presence of either antibody or
antigen.
immunotechnique assays

12. • Immunoassays have played vital roles in
diagnosing diseases, monitoring the level of the
humoral immune response, and identifying
molecules of biological or medical interest.
• These assays differ in their;
–speed and sensitivity
–strictly qualitative and quantitative.

14. 1. Agglutination techniques
Agglutination, which refers to the clumping of
particles together, is an antigen-antibody
reaction that occurs when an antigen (i.e., a
molecule capable of triggering the adaptive
immune response) is mixed with its
corresponding antibody at a suitable pH and
temperature

15. In agglutination reactions, serial dilutions of
the antibody solution are made and a
constant amount of particulate antigen is
added to serially diluted antibody solutions.
After several hours of incubation at 37°C,
clumping is recorded by visual inspection

16. Agglutination
• In active agglutination, direct agglutination of
particulate antigen with specific antibody
occurs.
• Whole pathogens are used as a source of
antigen.
• It measures the antibody level produced by a
host infected with that pathogen.
• The binding of antibodies to surface antigens
on the bacteria results in visible clumps

17. Active agglutination can be of following types:
Slide/Tile agglutination:
• performed on a slide.
• Used for identification of bacterial types
• A suspension of unknown antigen on the slide is allowed to interact
with a drop of standardized antiserum is added or vice versa.
• A positive reaction results in formation of visible clumps. E.g. Widal
test,
Tube agglutination:
• It is performed in tube
• It is a standard quantitative technique for determination of antibody
titer.
• In this method serum is diluted in a series of tubes and standard
antigen suspensions (specific for the suspected disease) are added
to it.
• After incubation, antigen-antibody reaction is indicated by visible
clumps of agglutination.

18. Examples
• Agglutination reactions for blood grouping
• The 4 blood groups are A, B, AB and O.
• Each of these will be either Rh-positive or Rh-
negative.

19. • The blood groups refer to the presence on human red
blood cells of certain antigens, the blood group
factors.
• One very important group of factors present on the
red blood cells is the ABO system.
• The ABO group of a person depends on whether
his/her red blood cells contain one, both, or neither of
the 2 blood group antigens A and B.
• There are, therefore, 4 main ABO groups: A, B, AB and
O.

20. • Antibodies (agglutinins) for the antigens A and
B exist in the plasma and these are termed
anti-A and anti-B.
• The corresponding antigen and antibody are
never found in the same individual since,
when mixed, they form antigen-antibody
complexes, effectively agglutinating the blood.

21. • One end of a slide is labelled Anti-A, and the other
Anti-B. A drop of Anti-A test serum is added to the
end marked Anti-A, and a drop of Anti-B serum is
added to the end marked Anti-B.
Testing for ABO Group – Procedure
One drop of blood is added to each end
of the slide, and mixed well, using
separate wooden sticks.
The results are read directly from the slide. The subject
is blood group A if agglutination occurred with the
Anti-A test serum; group B if agglutination occurred
with the Anti-B test serum; group AB if agglutination
occurred with both test serums, and O if there was no
agglutination in either case.
In the sample to the right, we conclude the subject
has type A blood.

22. • When transfusing blood, it is important to
remember that the donor's blood must not
contain red blood cells that the recipient's
antibodies can agglutinate.
• Theoretically, then, individuals belonging to
blood group O are universal donors, while
those of blood group AB are universal
recipients.

23. Antiglobulin (Coombs) test:
• This’ test was devised by Coombs, Mourant, and
Race for detection of incomplete anti-Rh antibodies
that do not agglutinate Rh+ erythrocytes in saline.
• When serum containing incomplete anti-Rh
antibodies is mixed with Rh+ erythrocytes in saline,
incomplete antibody antiglobulin coats the surface
of erythrocytes but does not cause any
agglutination.
• When such erythrocytes are treated with
antiglobulin or Coombs serum (rabbit antiserum
against human gamma globulin), then the cells are
agglutinated.
• Coombs test can be direct as well as indirect.

24. Hemagglutination uses erythrocytes as the biological carriers
of bacterial antigens, and purified polysaccharides or proteins
for determining the presence of corresponding antibodies in a
specimen.

26. Complement fixation
The complement fixation test is
an immunological medical test that can be
used to detect the presence of either
specific antibody or specific antigen in a
patient's serum, based on
whether complement fixation occurs.

27. • The complement fixation test is a blood test
that can determine the presence of antigen-
specific antibodies by incubating patient
serum with antigen and complement.
• This assay takes advantage of the requirement
for complement to be activated by the
combination of antigen–antibody complexes

28. Steps of the method
•serum proteins react with antigen-antibody complexes.
•If this reaction occurs on a cell surface, it will result in the formation of
trans-membrane pores leading to the destruction of the cell.
•The basic steps of a complement fixation test are as follows:
1.A serum sample is taken.
2.It is then heated at about 56 °C to remove the complement proteins
already present in the sample.
3.The serum is then adsorbed with washed sheep RBC. It prevents
interference in the test by anti-RBC antibodies which are cross-
reactive.
4.Then the antigen and complement are added to the sample.
5.It is then subjected to incubation at a temperature of 37 °C for 30
minutes. It provides conditions and time for the formation of the Ag-
Ab complex.
6.And the indicator system is then added and the sample is observed
for change due to occurrence or non-occurrence of hemolysis.
7.If the color changes to pink then the test is negative

30. 3. Immunodiffusion Reactions

31. • Antibody and soluble antigen
interacting in aqueous solution form a
lattice that eventually develops into a
visible precipitate.
• Antibodies that aggregate soluble
antigens are called precipitins.
• Although formation of the soluble Ag-
Ab complex occurs within minutes,
formation of the visible precipitate
occurs more slowly and often takes a
day or two to reach completion

33. Examples
– radial immunodiffusion - the Mancini method
– double immunodiffusion- the Ouchterlony double
diffusion method
• a semisolid medium such as agarose or agar.

35. Ab
As equivalence is reached, a visible line of
precipitation, a precipitin line, forms

36. Radial immunodiffusion
• An antigen sample is placed in a well and allowed to
diffuse into agar containing a suitable dilution of an
antiserum.
• As the antigen diffuses into the agar, the region of
equivalence is established and a ring of precipitation,
a precipitin ring, forms around the well

39. • The area of the precipitin ring is proportional
to the concentration of antigen.
• By comparing the area of the precipitin ring
with a standard curve the concentration of
the antigen sample can be determined.

40. 4. Immunoelectrophoresis
Combines Electrophoresis and Double
Immunodiffusion

41. • In immunoelectrophoresis, the
antigen mixture is first
electrophorezed to separate
its components by charge.
• Troughs are then cut into the
agar gel parallel to the
direction of the electric field,
and antiserum is added to the
troughs.
• Antibody and antigen then
diffuse toward each other and
produce lines of precipitation
where they meet in
appropriate proportions

42. Application
• clinical laboratories to detect the
presence or absence of proteins -
antigens in the serum
• a patient produces abnormally low
amounts of one or more isotypes,
characteristic of certain
immunodeficiency diseases to diagnose
if a patient overproduces some serum
protein, such as albumin,
immunoglobulin, or transferrin.
Multiple myeloma
• serum from patients shows a heavy
distorted arc caused by the large
amount of myeloma protein-
monoclonal Ig

43. • A related quantitative technique,
rocket electrophoresis, does
permit measurement of antigen
levels.
• In rocket electrophoresis, a
negatively charged antigen is
electrophoresed in a gel
containing antibody.
• The precipitate formed between
antigen and antibody has the
shape of a rocket, the height of
which is proportional to the
concentration of antigen in the
well.

44. RIA – radioimmunoassay
Radioimmunoassay (RIA) is a competitive assay
technique in which the reagent, the antibody
(Ab), is used in a limited amount as compared
with the amount of analyte antigen (Ag).
To perform a RIA, it is essential to have a
radioactively labeled antigen (Ag ),
∗ which is
used as the tracer.

45. The principle and procedure :
•competitive binding of radiolabeled antigen and unlabeled antigen
to a high-affinity antibody.
•The labeled antigen is mixed with antibody at a concentration that
saturates the antigen-binding sites of the antibody.
•Then test samples of unlabeled antigen of unknown concentration
are added in progressively larger amounts.
•The antibody does not distinguish labeled from unlabeled antigen,
so the two kinds of antigen compete for available binding sites on
the antibody.
•As the concentration of unlabeled antigen increases, more labeled
antigen will be displaced from the binding sites.
•The decrease in the amount of radiolabeled antigen bound to
specific antibody in the presence of the test sample is the measure
of the amount of antigen present in the test sample.
•The antigen is generally labeled with a gamma-emitting isotope
such as 125
I, beta-emitting isotopes such as tritium 3
H as labels.

47. • A binding curve is then be generated which
allows the amount of antigen in the patient’s
serum to be derived.
• That means as the concentration of unlabeled
antigen is increased, more of it binds to the
antibody, displacing the labeled variant.
• The bound antigens are then separated from
the unbound ones, and the radioactivity of the
free antigens remaining in the supernatant is
measured.

48. • Counting radioactivity in the precipitates
allows the determination of the amount of
radiolabeled antigen precipitated with the
antibody.
• A standard curve is constructed by plotting the
percentage of antibody-bound radiolabeled
antigen against known concentrations of a
standardized unlabeled antigen, and
• the concentrations of antigen in patient
samples are extrapolated from that curve.

50. Radioimmunoassay (RIA) Applications
• It was first used for the detection of peptide
hormones.
• Detection of different viral antigens
• Detection of many hormones and drugs
• Detection of Hepatitis B surface antigens
• Detection of mycotoxins
• Detection of the early stage of cancer

51. 4. Enzyme-Linked Immunosorbent Assay (ELISA)
• An enzyme conjugated with an antibody
reacts with a colorless substrate to generate a
colored reaction product.
• A number of enzymes have been employed for
ELISA, including
– alkaline phosphatase,
– horseradish peroxidase, and
– beta-galactosidase

53. Types of ELISA include
– Indirect ELISA
– Sandwich ELISA
– Competitive ELISA

55. INDIRECT ELISA
• Antibody can be detected or quantitatively determined with an
indirect ELISA
• Serum or some other sample containing primary antibody (Ab1) is
added to an antigen-coated microtiter well and allowed to react with
the antigen attached to the well.
• After any free Ab1 is washed away, the presence of antibody bound to
the antigen is detected by adding an enzyme-conjugated secondary
anti-isotype antibody (Ab2), which binds to the primary antibody.
• Any free Ab2 then is washed away, and a substrate for the enzyme is
added.
• The amount of colored reaction product that forms is measured by
specialized spectrophotometric ELISA plate readers, which can
measure the absorbance of all of the wells of a 96-well plate in
seconds.
• Indirect ELISA is the method of choice to detect the presence of serum
antibodies against human immunodeficiency virus (HIV), the causative
agent of AIDS.

57. Sandwish ELISA
• Antigen can be detected or measured by a sandwich ELISA
• In this technique, the antibody is immobilized on a microtiter
well.
• A sample containing antigen is added and allowed to react with
the immobilized antibody.
• After the well is washed, a second enzyme-linked antibody
specific for a different epitope on the antigen is added and
allowed to react with the bound antigen.
• After any free second antibody is removed by washing, substrate
is added, and the colored reaction product is measured.

58. Competitive ELISA
• Antibody is first incubated in solution with a sample
containing antigen.
• The antigen-antibody mixture is then added to an antigen
coated microtiter well.
• The more antigen present in the sample, the less free antibody
will be available to bind to the antigen-coated well.
• Addition of an enzyme-conjugated secondary antibody (Ab2)
specific for the isotype of the primary antibody can be used to
determine the amount of primary antibody bound to the well
as in an indirect ELISA.

60. 5. Western Blotting
• Identification of a specific protein in a complex
mixture of proteins can be accomplished by a
technique known as Western blotting

61. • A protein mixture is electrophoretically separated
on an SDS-polyacrylamide gel (SDS-PAGE) as bands
• The protein bands are transferred to a nylon
membrane by electrophoresis and
• the individual protein bands are identified by
flooding the nitrocellulose membrane with
enzyme linked polyclonal or monoclonal antibody
specific for the protein of interest.
• After binding a chromogenic substrate that
produces a highly colored and insoluble product
causes the appearance of a colored band at the site
of the target antigen.

67. An electron micrograph showing a cell
with magnetic beads attached to its
surface via antibodies.

68. 6. Immunofluorescence
• In 1944, Albert Coons showed that antibodies could be
labeled with molecules that have the property of
fluorescence.
• Fluorescent molecules absorb light of one wavelength
(excitation) and emit light of another wavelength (emission).
• If antibody molecules are tagged with a fluorescent dye, or
fluorochrome, immune complexes containing these
fluorescently labeled antibodies (FA) can be detected by
colored light emission when excited by light of the
appropriate wavelength.
• Antibody molecules bound to antigens in cells or tissue
sections can similarly be visualized.
• The emitted light can be viewed with a fluorescence
microscope, having a UV light source.

69. Dyes used
• Fluorescein, an organic dye absorbs blue light
(490 nm) and emits an intense yellow-green
fluorescence (517 nm).
• Rhodamine, absorbs in the yellow-green
range (515 nm) and emits a deep red
fluorescence (546 nm).
• Phycoerythrin a brilliant emitter of red
fluorescence,

70. In direct staining, the primary antibody is directly conjugated with
fluorescein;
In indirect staining, the primary antibody is unlabeled and is
detected with an additional fluorochrome-labeled reagent

71. • Indirect immunofluorescence staining has two
advantages over direct staining.
• First, the primary antibody does not need to be
conjugated with a fluorochrome.
• Because the supply of primary antibody is often a
limiting factor,
• indirect methods avoid the loss of antibody that usually
occurs during the conjugation reaction.
• Second, the sensitivity of staining increases because
multiple molecules of the fluorochrome reagent bind to
each primary antibody molecule,
• increases the amount of light emitted at the location of
each primary antibody molecule.

72. In this micrograph, antibody molecules
bearing heavy chains are detected by indirect
staining of cells with rhodamine-conjugated
second antibody.

73. 7. Immunoelectron Microscopy
• specific intracellular tissue components can be visualized by
immunoelectron microscopy
• In this technique,
– an electron-dense label is conjugated to the Fc portion of a specific
antibody for direct staining or
– conjugated to an anti-immunoglobulin reagent for indirect staining.
• A number of electron-dense labels have been employed,
Ferritin and colloidal gold.
• The electron-dense label absorbs electrons, it can be visualized
with the electron microscope (Transmission Electron
Mciroscope) as small black dots.
• In the case of immunogold labeling, different antibodies can be
conjugated with gold particles of different sizes, allowing
identification of several antigens within a cell by the different
sizes of the electron-dense gold particles attached to the
antibodies (Figure 6-16).

74. The electron-dense label absorbs
electrons, it can be visualized with
the electron microscope
(Transmission Electron Mciroscope)
as small black dots.

75. 8. Flow Cytometry – Fluorescence activated cell sorter
(FACS)
• Gives quantitative data
• The flow cytometer uses a laser beam and light
detector to count single intact cells in suspension
• Every time a cell passes the laser beam, light is
deflected from the detector, and this interruption of
the laser signal is recorded.
• Those cells having a fluorescently tagged antibody
bound to their cell surface antigens are excited by the
laser and emit light that is recorded by a second
detector system located at a right angle to the laser
beam.

76. FITC- fluorescein isothiocyanate
PE-phycoerythrin

77. lower left-hand panel not
to have reacted with
either antibody anti-A or
anti-B.
upper left panel
reacted with anti-
B but not anti-A,
those in the lower
right panel reacted
with anti-A but not
anti-B
upper right panel
contains cells that
react with both
anti-A
and anti-B
Four subpopulations
can be distinguished:

78. • Leukemia is the unchecked proliferation of an
abnormal clone of hematopoietic cells
• The diagnosis of leukemia is made on the basis of
two findings.
– One is the detection of abnormal cells in the
bloodstream, and the other is observation of abnormal
cells in the bone marrow.
• An abnormal cell displays surface immunoglobulin
would be assigned to the B-cell lineage and its
maturational stage would be that of a mature B cell.
• On the other hand, a cell that had cytoplasmic
heavy chains, but no surface immunoglobulin,
would be a B-lineage leukemic cell but at the
maturational stage of a pre-B cell.

79. • Immunophenotyping uses flow cytometry and
monoclonal antibodies
• The availability of monoclonal antibodies specific
for each of the scores of antigens found on
various types and subtypes of hematopoietic
cells has made it possible to identify patterns of
antigen expression that are typical of
– cell lineages,
– maturational stages, and
– a number of different types of leukemia

81. Application
• Most cancer centers are equipped with flow cytometers
that are capable of performing and interpreting the
multiparameter analyses necessary to provide useful
profiles of surface markers on tumor cell populations.
• Flow cytometric determination of immuno-phenotypes
allows:
– Confirmation of diagnosis
– Diagnosis when no clear judgment can be made based on
morphology or patterns of cytochemical staining
– Identification of aberrant antigen profiles that can help
identify the return of leukemia during remission
– Improved prediction of the course of the disease

83. SUMMARY
• Antigen-antibody interactions depend on four types of
noncovalent interactions: hydrogen bonds, ionic bonds,
hydrophobic interactions, and van der Waals interactions.
• The affinity constant, which can be determined by Scatchard
analysis, provides a quantitative measure of the strength of
the interaction between an epitope of the antigen and a single
binding site of an antibody.
• The avidity reflects the overall strength of the interactions
between a multivalent antibody molecule and a multivalent
antigen molecule at multiple sites.
• The interaction of a soluble antigen and precipitating
antibody in a liquid or gel medium forms an Ag-Ab precipitate.
• Electrophoresis can be combined with precipitation in gels in a
technique called immunoelectrophoresis.

84. • The interaction between a particulate antigen and agglutinating
antibody (agglutinin) produces visible clumping, or agglutination
that forms the basis of simple, rapid, and sensitive immunoassays.
• The enzyme-linked immunosorbent assay (ELISA) depends on an
enzyme-substrate reaction that generates a colored reaction
product.
• ELISA assays that employ chemiluminescence are the most
sensitive immunoassays available.
• In Western blotting, a protein mixture is separated by
electrophoresis; then the protein bands are electrophoretically
transferred onto nitrocellulose and identified with labeled antibody
or labeled antigen.
• Fluorescence microscopy using antibodies labeled with fluorescent
molecules can be used to visualize antigen on or within cells.
• Flow cytometry provides an unusually powerful technology for the
quantitative analysis and sorting of cell populations labeled with
one or more fluorescent antibodies.

85. Hemeagglutination
• Agglutination reactions are routinely performed to type
red blood cells
• 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 for transfusions.

86. 1. Cross-Reactivity
• In some cases antibody elicited by one antigen can
cross-react with an unrelated antigen.
• Cross-reactivity occurs if two different antigens
share an identical or very similar epitope
• Cross-reactivity is often observed among
polysaccharide antigens that contain similar
oligosaccharide residues.

87. Example:
 The ABO blood-group antigens, for example, are
glycoproteins expressed on red blood cells.
 Cross-reactivity is often observed among
polysaccharide antigens that contain similar
oligosaccharide residues.

88. • Type A individual has anti-B antibodies;
• Type B individual has anti-A;
• Type O individual has anti-A and anti-B

89. • Antibodies are produced to these antigens
• The antibodies are induced not by exposure to red blood
cell antigens but by exposure to cross-reacting microbial
antigens present on common intestinal bacteria.
• These microbial antigens induce the formation of
antibodies in individuals lacking the similar blood-group
antigens on their red blood cells
• These blood-group antibodies, although elicited by
microbial antigens, will cross-react with similar
oligosaccharides on foreign red blood cells,
• This provides the basis for blood typing tests and
accounting for the necessity of compatible blood types
during blood transfusions.

90. Thank you