This document discusses antigen-antibody reactions including factors that affect their measurement and techniques used to measure them in the lab. Key points covered include: affinity and avidity being measures of strength of antigen-antibody binding; specificity and cross-reactivity relating to reaction with single or multiple antigens; and techniques including precipitation tests, agglutination, ELISA, radioimmunoassay, immunofluorescence, and complement fixation. Both qualitative and quantitative applications are discussed.

1. Antigen and antibody reactions

2. Affinity =  attractive and repulsive forces
Ab
Ag
High Affinity
Ab
Ag
Low Affinity
Affinity
• Strength of the reaction between a single antigenic
determinant and a single Ab combining site

3. Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq = 104
Affinity
106
Avidity
1010
Avidity

4. Specificity
• The ability of an individual antibody combining
site to react with only one antigenic determinant.
• The ability of a population of antibody molecules
to react with only one antigen.

5. Cross Reactivity
• The ability of an individual Ab combining site to
react with more than one antigenic determinant.
• The ability of a population of Ab molecules to
react with more than one Ag
Anti-A
Ab
Ag A
Anti-A
Ab
Ag B
Shared epitope
Anti-A
Ab
Ag C
Similar epitope
Cross reactions

6. Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation

7. Antigen antibody tests
• Used in both directions
• Qualitative
• Quantitative

8. Antigen and antibody reactions in
the lab
• Precipitation tests
• Agglutination
• ELISA
• Radioimmunoassay
• Immunofluorescence
• Complement Fixation

9. Qualitative/quantitative
• Qualitative
– determines antigen or antibody is present or
absent
• Quantitative
– determines the quantity of the antibody
– Titer
– The highest dilution of the specimen usually
serum which gives a positive reaction in the
test

10. Precipitation tests
• The antigen and antibody are in soluble
form
• Combine to form a visible precipitate
• Presence of electrolytes
• Positive controls and negative controls

11. Precipitation tests
• Precipitation techniques
– Tube precipitation test
– Gel diffusion
• Single radial
• Double
– Immuno electrophoresis
– Counter immuno electrophoresis
Countercurrent electrophoresis (CEP), immuno
electro osmo phoresis (IEOP)

12. Radial Immunodiffusion (Mancini)
• Interpretation
– Diameter of ring is
proportional to the
concentration
• Quantitative
– Ig levels
• Method
– Ab in gel
– Ag in a well
Ag Concentration
Diameter2
AgAgAgAg
Ab in gel

15. Immunoelectrophoresis
• Method
– Ags are separated by electrophoresis
• Interpretation
– Precipitin arc represent individual antigens
Ag
-+
Ag
Ab
Ag
Ab
– Ab is placed in trough cut in the agar

16. 16

17. Countercurrent electrophoresis
• Method
– Ag and Ab migrate toward each other by
electrophoresis
– Used only when Ag and Ab have opposite charges
• Qualitative
–Rapid
Ag Ab
- +

18. Countercurrent electrophoresis:
In this test the antigen and antibody are placed in wells
punched out of an agar gel and the antigen and antibody are
electrophoresed into each other where they form a
precipitation line.
This test only works if conditions can be found where the antigen
and antibody have opposite charges. This test is primarily
qualitative, although from the thickness of the band you can
get some measure of quantity. Its major advantage is its speed.

19. Complement fixation test
• The complement fixation test is an immunological
medical test looking for evidence of infection. It tests for
the presence of either specific antibody or specific antigen
in a patient's serum. It uses sheep red blood cells (sRBC),
anti-sRBC antibody and complement, plus specific antigen
(if looking for antibody in serum) or specific antibody (if
looking for antigen in serum).
• If either the antibody or antigen is present in the patient's
serum, then the complement is completely utilized, so the
sRBCs are not lysed. But if the antibody (or antigen) is not
present, then the complement is not used up, so it binds
anti-sRBC antibody, and the sRBCs are lysed.
• The Wassermann test is one form of complement fixation
test.

21. Coomb's Test (Antiglobulin Test):
a. Direct Coomb's Test
When antibodies bind to erythrocytes, they do not always result in agglutination.
This can result from the antigen/antibody ratio being in antigen excess or
antibody excess or in some cases electrical charges on the red blood cells
preventing the effective cross linking of the cells. These antibodies that bind to
but do not cause agglutination of red blood cells are sometimes referred to as
incomplete antibodies. In no way is this meant to indicate that the antibodies are
different in their structure, although this was once thought to be the case. Rather,
it is a functional definition only. In order to detect the presence of non-
agglutinating antibodies on red blood cells, one simply adds a second antibody
directed against the immunoglobulin (antibody) coating the red cells. This anti-
immunoglobulin can now cross link the red blood cells and result in
agglutination.
b. Indirect Coomb's Test
If it is necessary to know whether a serum sample has antibodies directed
against a particular red blood cell and you want to be sure that you also detect
potential non- agglutinating antibodies in the sample, an Indirect Coomb's test is
performed.
This test is done by incubating the red blood cells with the serum sample, washing out
any unbound antibodies and then adding a second anti-immunoglobulin reagent
to cross link the cells.

23. c. Applications
These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh
factor generally do not agglutinate red blood cells. Thus, red cells from Rh+
children born to Rh- mothers, who have anti-Rh antibodies, may be coated with
these antibodies. To check for this, a direct Coombs test is performed. To see if the
mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed.

24. Agglutination
• Visible clumping together of particulate matter by
antigen combining with its specific antibody.
• The clumps will be called agglutinates
• Performed
– Slide
– Tube
– Tile
– Micrtitration plates

26. Agglutination
• Active agglutination test
• The antigen part of of a particulate matter
per se
• examples
• Salmonella, vibrio,
+ 

27. Agglutination
• Passive agglutination test
• Antigen or antibody are not part of
particulate matter but are attached (rided on
inert particles like latex, carbon,)
+ 

30. Immunfluorescence
• Fluorescent dyes illuminated by ultraviolet
light are used to show combination of
antigen antibody .The end point antigen
antibody complexes are seen fluorescing
against a dark background
• Direct
• Indirect

32. Immunofluorescence
• Direct
– Ab to tissue Ag is labeled with fluorochrome
Ag
Fluorochrome
Labeled Ab
Tissue Section

33. Immunofluorescence
• Indirect
– Ab to tissue Ag is
unlabeled
– Fluorochrome-labeled anti-
Ig is used to detect binding
of the first Ab.
Ag
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab
• Qualitative to Semi-
Quantitative

34. Immunofluorescence testing uses fluorescent
Ab either directly or indirectly to visualize
cells or cell aggregates that have reacted
with the FAbs

36. RadioImmunoassay
• The radioactivity of the specific labeled
antibody or antigen is used to quantify the
antigen or antibody in patient’s serum

37. Labels in Immunoassays
• Immunoassays require the use of labeled materials
in order to measure the amount of antigen or
antibody present. A label is a molecule that will
react as part of the assay, and in doing so produce a
signal that can be measured in the solution.
Examples of a label include a radioactive
compound, or an enzyme that causes a change of
color in a solution or its fluorescence (Wild).

38. Noncompetitive Immunoassays
– Noncompetitive (sandwhich) immunoassays generally provide the
highest level of assay sensitivity and specificity. This format is
referred to as a “sandwich” assay because the analyte is bound
(sandwiched) between two highly specific antibody reagents.
– The reaction mixture typically includes an excess of labeled antibody,
so that all drug/metabolite is bound. The amount of antibody-antigen
complex is then measured to determine the amount of drug present in
the sample. The measurement of labeled analyte, usually antibody, is
directly proportional to the amount of antigen present in the sample.

39. Competitive Immunoassays
• The measurement of the analyte using the labels is broadly
categorized into competitive and noncompetitive methods.
– In competitive formats, unlabelled analyte in the test sample is
measured by its ability to compete with labeled antigen for a
limited number of antibody binding sites (Bell). The unlabeled
antigen blocks the ability of the labeled antigen to bind because
that binding site on the antibody is already occupied. Thus, in a
competitive immunoassay, less label measured in the assay
means more of the unlabeled (test sample) antigen is present.
The amount of antigen in the test sample is inversely related to
the amount of label measured in the competitive format (Abbott
Diag.). As one increases, the other decreases.

40. Homogeneous VS Heterogeneous
Methods
• Immunoassay methods that require separation of bound Ab-Ag* complex
are referred to as heterogeneous immunoassays. Those that do not require
separation are referred to as homogeneous immunoassays.
• Homogeneous methods have been generally applied to the measurement of
small analytes such as abused and therapeutic drugs. Since homogeneous
methods do not require the separation of the bound Ab-Ag* from the free
Ag*, they are generally much easier and faster to perform.

41. Types of Immunoassays
• Radioimmunoassays
(RIAs) utilize a
radioactive label
(usually 125I, 3H or
14C), which emits
radiation that can be
measured with a beta or
gamma counter.
• Within the categories of competitive, noncompetitive,
homogenous, and heterogeneous, there are specific
types, which include:

42. Types of Immunoassays Cont’d• In the Enzyme Multiplied
Immunoassay (EMIT), the
drug in the sample and the
drug labeled with G6PD
compete for antibody
binding sites.
• Binding inhibits enzyme
activity, while free enzyme
remains active to interact
with.
• Enzyme activity/absorbance
is directly proportional to
drug concentration.

43. Types of Immunoassays Cont’d
• In the Fluorescent
Polarized Immunoassay,
the drug in the sample
competes with
fluorescein-labeled drug
for antibody binding
sites.
• Reaction mixture is
excited by
planepolarized light.
• As the tracer returns to a
lower energy state, it
emits light; polarization
is measured.
• The polarization value of the sample is
inversely proportional to analyte
concentration.

44. Immunoassay Results
• Qualitative
– Single point calibration at a specific cutoff
– Results are either ‘positive’ or ‘negative’; (i.e. above or below the
cutoff)
– Possible false positives; monoclonal antibodies restrict this
slightly.
• Quantitative
– Provides numeric results that are an estimate of drug/compound
concentration based on the measurement of labeled analyte in the
solution, and taking into consideration the
competitive/noncompetitive nature of the device.
– In terms of use on drugs, this is sometimes complicated by
possible cross-reactivities.

45. Immunoassay Results Cont’d
Typical 4-parameter logistic
graph for a competitive-format
immunoassay.
Dose-response curve for a non-
competitive CL immunoassay.
A pregnancy test is an
example of a
commercially produced
immunoassay that
produces a positive or
negative qualitative
response.

46. Immunoassays and Forensic
Science
• Forensic toxicology encompasses the determination of the presence and concentration of
drugs, other xenobiotics and their metabolites in physiological fluids and organs and the
interpretation of these findings as they may impact on legal issues. These include
medical examiner investigations, driving under the influence and other transportation
accident investigations, workplace pre-employment, random and for-cause drug testing
and judicial monitoring of arrestees and parolees.
• For the most part, forensic toxicologists use commercial immunoassays directed
primarily towards abused drugs. Commercial immunoassays developed for therapeutic
monitoring of other drugs, veterinary drugs and pesticides, as well as immunoassays
developed in research laboratories for specialized studies, may find a role in the forensic
toxicology laboratory for specialized cases.

48. Solid Phase RIA for antigen/direct
• Ag detection
– Immobilize Ab
– Incubate with sample
– Add labeled antibody
– Amount of labeled Ab
bound is proportional to
the amount of Ag in the
sample
• Quantitative
Solid
Phase
Ag
Immobilized
Ag in
Patient’s
sample
Labeled
Ab

49. Solid Phase RIA for antibody/indirect
• Ab detection
– Immobilize Ag
– Incubate with sample
– Add labeled anti-Ig
– Amount of labeled Ab
bound is proportional
to amount of Ab in the
sample
• Quantitative
Solid
Phase
AgImmobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig

50. ELISA
• Uses an enzyme system to show the specific
combination of antigen antibody
• An enzyme labeled or linked to a specific antigen
• A substrate
• A color reader
• Double antibody technique to detect and assay
antigen
• Indirect technique to Assay and antibody

51. Double Antibody ELISA
• Ag detection
– Immobilize Ab
– Incubate with sample
– Add labeled antibody
– Amount of labeled Ab
bound is proportional to
the amount of Ag in the
sample
• Quantitative
Solid
Phase
Ag
Immobilized
Ag in
Patient’s
sample
Labeled
Ab

52. Indirect ELISA
• Ab detection
– Immobilize Ag
– Incubate with sample
– Add labeled anti-Ig
– Amount of labeled Ab
bound is proportional
to amount of Ab in the
sample
• Quantitative
Solid
Phase
AgImmobilized
Ab in
Patient’s
sample
Labeled
Anti-Ig

53. Radioimmuoassays (RIA)
Enzyme-Linked Immunosorbent
Assays (ELISA)
Lattice formation not required

54. Competitive ELISA for antigen
• Method
– Determine amount
of Ab needed to bind
to a known amount
of labeled Ag
+ 
Prior to Test
Labeled
Ag
+ 
Test
+
Patient’s
sample
Labeled
Ag
+
– Use predetermined
amounts of labeled
Ag and Ab and add a
sample containing
unlabeled Ag as a
competitor

55. Competitive ELISA for Ag
• Method cont.
– Determine amount
of labeled Ag bound
to Ab
• NH4SO4
• anti-Ig
• Immobilize the Ab
• Quantitative
– Most sensitive test
+ 
Test
+
Patient’s
sample
Labeled
Ag
+
– Concentration determined from a standard curve
using known amounts of unlabeled Ag
Solid
Phase
Solid
Phase

56. Enzyme-Linked ImmunoSorbantEnzyme-Linked ImmunoSorbant
Assay (ELISA)Assay (ELISA)
Indirect ELISA Sandwich ELISA

57. Enzyme-Linked ImmunospotEnzyme-Linked Immunospot
(ELISPOT)(ELISPOT)
• Detect cells secreting a specific antigen
• Most commonly used to detect cytokine
secretion by T cells upon stimulation (IFNγ,
IL2, IL4, etc.)
• Can detect cytotoxic activity (perforin)
IFNγ
http://www.protcol-online.org/

58. 1. Indirect ELISA
• The steps of the general, "indirect," ELISA for determining serum
antibody concentrations are:
1. Apply a sample of known antigen of known concentration to a surface,
often the well of a microtiter plate. The antigen is fixed to the surface
to render it immobile. Simple adsorption of the protein to the plastic
surface is usually sufficient. These samples of known antigen
concentrations will constitute a standard curve used to calculate antigen
concentrations of unknown samples. Note that the antigen itself may be
an antibody.
2. The plate wells or other surface are then coated with serum samples of
unknown antigen concentration, diluted into the same buffer used for
the antigen standards. Since antigen immobilization in this step is due
to non-specific adsorption, it is important for the total protein
concentration to be similar to that of the antigen standards.
3. A concentrated solution of non-interacting protein, such as
Bovine Serum Albumin (BSA) or casein, is added to all plate wells.
This step is known as blocking, because the serum proteins block non-
specific adsorption of other proteins to the plate.

59. 4. The plate is washed, and a detection antibody specific to the antigen
of interest is applied to all plate wells. This antibody will only bind
to immobilized antigen on the well surface, not to other serum
proteins or the blocking proteins.
5. The plate is washed to remove any unbound detection antibody.
After this wash, only the antibody-antigen complexes remain
attached to the well.
6. Secondary antibodies, which will bind to any remaining detection
antibodies, are added to the wells. These secondary antibodies are
conjugated to the substrate-specific enzyme. This step may be
skipped if the detection antibody is conjugated to an enzyme.
7. Wash the plate, so that excess unbound enzyme-antibody conjugates
are removed.
8. Apply a substrate which is converted by the enzyme to elicit a
chromogenic or fluorogenic or electrochemical signal.
9. View/quantify the result using a spectrophotometer,
spectrofluorometer, or other optical/electrochemical device.

60. To detect antibody (indirect ELISA):

61. 2. Sandwich ELISA
• A sandwich ELISA:
 Plate is coated with a capture antibody
 sample is added, and any antigen present binds
to capture antibody
 detecting antibody is added, and binds to
antigen
 enzyme-linked secondary antibody is added,
and binds to detecting antibody
 substrate is added, and is converted by enzyme
to detectable form.

62. A less-common variant of this technique, called "sandwich" ELISA, is
used to detect sample antigen. The steps are as follows:
1. Prepare a surface to which a known quantity of capture antibody is
bound.
2. Block any non specific binding sites on the surface.
3. Apply the antigen-containing sample to the plate.
4. Wash the plate, so that unbound antigen is removed.
5. Apply primary antibodies that bind specfically to the antigen.
6. Apply enzyme-linked secondary antibodies which are specific to the
primary antibodies.
7. Wash the plate, so that the unbound antibody-enzyme conjugates
are removed.
8. Apply a chemical which is converted by the enzyme into a color or
fluorescent or electrochemical signal.
9. Measure the absorbance or fluorescence or electrochemical signal
(e.g., current) of the plate wells to determine the presence and
quantity of antigen.

63. To detect antigen (sandwich ELISA):

64. 3. Competitive ELISA
• A third use of ELISA is through competitive binding.
The steps for this ELISA are somewhat different than
the first two examples:
1. Unlabeled antibody is incubated in the presence of its antigen.
2. These bound antibody/antigen complexes are then added to an
antigen coated well.
3. The plate is washed, so that unbound antibody is removed. (The
more antigen in the sample, the less antibody will be able to
bind to the antigen in the well, hence "competition.")
4. The secondary antibody, specific to the primary antibody is
added. This second antibody is coupled to the enzyme.
5. A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal.
• For competitive ELISA, the higher the original antigen
concentration, the weaker the eventual signal.

65. Competitive binding

66. Applications
• Because the ELISA can be performed to evaluate either the presence of
antigen or the presence of antibody in a sample, it is a useful tool both for
determining serum antibody concentrations (such as with the HIV test [1] or
West Nile Virus) and also for detecting the presence of antigen. It has also
found applications in the food industry in detecting potential food allergens
such as milk,peanuts,walnuts,almonds, and eggs [2]The ELISA test, or the
enzyme immunoassay (EIA), was the first screening test commonly employed
for HIV. It has a high sensitivity.In an ELISA test, a person's serum is diluted
400-fold and applied to a plate to which HIV antigens have been attached. If
antibodies to HIV are present in the serum, they may bind to these HIV
antigens. The plate is then washed to remove all other components of the
serum. A specially prepared "secondary antibody" — an antibody that binds to
human antibodies — is then applied to the plate, followed by another wash.
This secondary antibody is chemically linked in advance to an enzyme. Thus
the plate will contain enzyme in proportion to the amount of secondary
antibody bound to the plate. A substrate for the enzyme is applied, and
catalysis by the enzyme leads to a change in color or fluorescence. ELISA
results are reported as a number; the most controversial aspect of this test is
determining the "cut-off" point between a positive and negative result.

67. Flow CytometryFlow Cytometry

68. Flow CytometryFlow Cytometry
• FACS – Fluorescence Activated Cell
Sorter is the generic term used for flow
cytometry (even without sorting)
• Simultaneous analysis of different
physical parameters in a single cell
• Can analyze up to several thousands of
cells per second
• Versatile, sensitive

69. What is in a Flow Cytometer?What is in a Flow Cytometer?
• Fluidics
– To introduce and focus the cells for interrogation by a laser
• Optics
– To generate and collect the light signals (scatter and
fluorescence)
• Electronics
– To convert the optical signals to proportional electronic
signals and digitize them for computer analysis (PMTs)

70. Principle of Flow Cytometry
 Cell sample labeled with appropriate
fluorescent Abs
 Cells in suspension are passed through
machine in single file in a stream of fluid
 Stream is focused through one or more laser
beams, measuring light scatter and
fluorescence characteristics
 Fluorescence detected by photomultiplier
tubes (PMTs)
 Signals sent to computer for analysis

71. Cell Parameters Analyzed in a Flow CytometerCell Parameters Analyzed in a Flow Cytometer
 Forward Scatter (FSC): cell size
 Side Scatter (SSC): cell internal complexity (relative
granularity)
 Fluorescent labeling of cell surface or intracellular
structures using fluorescent antibodies: allows
investigation of cell molecules and function (FLI, FL2,
FL3, FL4, etc..)

72. Properties of FSC and SSCProperties of FSC and SSC
• Forward Scatter
– Diffracted light
– Size of the cell
– Related to cell surface area
• Side Scatter
– Reflected light
– Light reflecting from cellular
components
– Related to cell granularity and
complexity
– Detected at 90° to the laser
beam
Incident Light
Source FSC Detector
SSC Detector

73. Whole Blood-RBCs lysedWhole Blood-RBCs lysed
Largest and most
complex population
1000
SideScatter
Forward Light Scatter
0 200 400 600 800
02004006008001000
Smallest and least
complex population
Neutrophils
Eosinophils
Monocytes
Lymphocytes

74. Fluorochromes: Excitation andFluorochromes: Excitation and
Emission SpectraEmission Spectra
• Antibodies can be conjugated to fluorochromes
• The amount of fluorescent signal detected is proportional to
the number of fluorochrome molecules on the particle.

75. Flow Cytometry AnalysisFlow Cytometry Analysis
• Single parameter analysis:
– Histogram plot
– Horizontal axis: level of
fluorescence - brighter cells
further right
– Vertical axis: number of events
per channel number
– Analyze level of expression of
marker
• Two parameter analysis:
– Dot-plot
– One axis shows first color
– Second axis shows second color
– Analysis of individual
populations of cells
100 101 102 103 104
CD86 PE
Isotype
MUTZ-3
iDC
mDC.031

76. Intracellular Cytokine StainingIntracellular Cytokine Staining
• To detect cytokine
production by a specific cell
upon stimulation
• Used to define T cell
activation by epitope
recognition and the T cell
polarization

77. MHC Tetramer Staining
• Identify T cells specific for a certain
MHC-peptide complex
PE
CD8+
T
lymphocyte
(CTL)
Mature
Dendritic
Cell
HLA
Peptide
TCR

78. Common Apllications of FlowCommon Apllications of Flow
Cytometry in ImmunologyCytometry in Immunology
• Phenotype of cell, surface molecules
• Intracellular cytokine staining
• Antigen specificity
• Cell proliferation (e.g. CFSE, BrdU incorporation)
• Cell sorting
• Apoptosis analysis
• Cytotoxicity assays
• Phagocytosis assays
• Cell cycle analysis (DNA content analysis)
• Cell signalling molecules, Calcium flux assays
• Organelle-specific studies (e.g. lysosome)
• Cellular transport assays
• Transfection efficiencies