IS MIDTERM-merged

CLINICAL IMMUNOLOGY AND SEROLOGY
Topic: Monoclonal Antibody and Transplantation

Lecturer: Melford L. Teodoro, RMT
OVERVIEW
1. Monoclonal antibody
2. Transplantation
I. MONOCLONAL ANTIBODY
Monoclonal antibodies are purified

antibodies cloned from a single cell. These
antibodies exhibit exceptional purity and
specificity and are able to recognize and
bind to a specific antigen.
Discovery of the Technique. In 1975,

Köhler, Milstein, and Jerne discovered
how to fuse lymphocytes to produce a cell
line that was both immortal and a
producer of specific antibodies.
Kohler and Milstein’s technique fuses an

activated B cell with a myeloma cell that
can be grown indefinitely in the
laboratory. This fusion of two different
types of cells is called a hybridoma.
Myeloma cells are cancerous plasma cells.

Normally, plasma cells produce antibody,
so a particular cell line that is not capable
of producing antibody is chosen. In
addition, this cell line has a deficiency of
the enzyme hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) that
makes it incapable of synthesizing
nucleotides from hypoxanthine and
thymidine, which are needed for DNA
synthesis.
CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

The fact that these myeloma cells cannot make their own DNA means that they
will die out unless they are fused to a plasma cell that has the enzymes
necessary to synthesize DNA. This deficiency keeps the myeloma cells from
reproducing on their own.
These hybridoma cells grow continuously, having acquired the immortal

property of the myeloma tumor. From a population of hybridomas, one can
select and expand individual cells that secrete the antibody of desired
specificity; such antibodies, derived from a single B cell clone, are
homogeneous monoclonal antibodies, meaning monoclonal antibodies against
virtually any antigen can be produced.
Most monoclonal antibodies are made by fusing cells from immunized mice with mouse
myelomas. Such mouse monoclonal antibodies cannot be injected repeatedly into human
subjects, because the human immune system sees the mouse Ig as foreign and mounts an
immune response against the injected antibodies
More recently, monoclonal antibodies have been generated by using recombinant DNA
technology to clone the DNA encoding human antibodies of desired specificity. Another
approach is to replace the Ig genes of mice with human antibody genes and then immunize these
mice with an antigen to produce specific human antibodies. Monoclonal antibodies are now in
widespread use as therapeutic agents for many diseases in humans

Inflammatory
(Immunological)
diseases
Target Effect Diseases
CD20 Depletion of B cells Rheumatoid Arthritis, multiple sclerosis,
other autoimmune diseases; B cell lymphoma
IgE Blocking IgE function Allergy-related asthma
IL-6 Receptor Blocking inflammation Rheumatoid arthritis
TNF Blocking inflammation Rheumatoid arthritis, Crohn’s disease,
psoriasis
Cancer
CC52 Depletion of lymphocytes Chronic lymphocytic leukemia
CTLA-4 Activation of T cells Melanoma
EGFR Growth inhibition of epithelial tumors Colorectal, lung and head and neck cancers
HER2/Neu Inhibition of EGF signaling; depletion of Breast cancer
tumor cells
PD-1 Activation of Effector T cells Melanoma, other tumors
PD-L1 Activation of Effector T cells Melanoma, other tumors
VEGF Blocking tumor angiogenesis Breast cancer, colon cancer, age-related
macular degeneration
OTHER DISEASES
Glycoprotein 11b/111a Inhibition of platelet aggregation Cardiovascular disease
II. TRANSPLANTATION
Transplantation is a process whereby an individual (recipient receives cells or tissue from a

second individual (donor). Transplants are classified according to the genetic disparity between
donor and recipient. Grafts in which there is no genetic difference between the donor and
recipient are referred to as isografts, grafts between members of the same species are allografts,
while grafts across a species are referred to as xenografts.
For the most part, transplants are allografts and so there is a genetic disparity between the donor
and recipient, particularly at the MHC loci. To some extent, the MHC genetic disparity
determines whether a graft will be accepted or rejected because the proteins (antigens) that they
encode have been shown to induce the most vigorous rejection episodes.
Graft Rejection
Graft rejection is classified as hyperacute, acute, or chronic. These designations are based on the
time over which the rejection process develops, and on responsiveness to therapy.
Hyperacute rejection occurs minutes to hours following the engraftment. This type of rejection
is an antibody mediated phenomenon and is associated with complement activation, blood clot
formation, and rapid graft failure. A hyperacute rejection response indicates that the recipient has
been previously exposed to donor MHC antigens Individuals at risk are women who have had
several pregnancies or individuals who have had a previous graft that was rejected. The only
treatment option is graft removal.
Acute rejection occurs weeks after tissue transplant, and is caused primarily by helper and
cytotoxic T cell activation Helper T cells secrete cytokines required for the activation of
cytotoxic cells that mediate destruction of the graft Macrophages, activated by the helper T cell
derived cytokines, also play a role in the destructive process.
T cells are activated when they recognize MHC proteins as foreign antigens. For the most
part, recognition of foreign MHC differs from that of the typical antigen because MHC
proteins can be recognized in the absence of antigen presenting cells. Consequently,
numerous T cell clones may be activated, leading to a vigorous immune response. The
severity of graft rejection correlates with the number of T cell clones that are activated
This, in turn, is primarily a function of the genetic disparity at the MHC loci.
Chronic rejection may occur weeks, months, or years post-transplant. It is associated with a
notable increase in the levels of non-T-cell derived non-specific growth factors. There is no
treatment for chronic rejection and the graft must be removed.

Rejection in Bone Marrow Transplantation
Bone marrow transplants are used as treatment for leukemias and lymphomas. These types of
transplants are somewhat unique in that the donor cells attack the recipient's tissues, a
phenomenon referred to as graft versus host disease (GVHD). In GVHD, the T cells present in
the graft are stimulated because the recipient is recognized as foreign. Recipients are severely
immunocompromised and so their immune system cannot attack the graft with the same intensity
Because T cells are the key players in GvHD. deletion of T cells from the bone marrow graft was
attempted to eliminate GvHD Not surprisingly GvHD did not occur, but engraftment of the bone
marrow transplant was severely reduced as well. The present approach for bone marrow
transplants is to delete the T cells, but to supplement the graft with T derived cytokines cell
Immunosuppressive Therapies
Because acute rejection is a T cell mediated event many of the therapies target the T cells.
Treatment for acute rejection consists of increasing the dose of immunosuppressive drugs. This
presents another problem, in that individuals who are immunosuppressed are at risk for infection,
malignancy, and drag associated toxicity. The most commonly used drugs are Cyclosporin A and
FK506, which block the production of a T cell growth factor. Prednisone is used as a nonspecific
anti-inflammatory agent; it targets the macrophage and reduces antigen presentation to T cells
Other drug therapies block the proliferation of activated T cells. Antibodies that target specific
cell surface molecules on T cells are becoming the focus of several clinical trials
Screening to Ensure Compatibility
In clinical practice, recipients and potential donors are pre-screened to ensure the best possible
genetic match at MHC loci to minimize the likelihood of rejection. The pre-transplantation
screening tests are based on serological, cellular, and (newer) molecular typing techniques. Pre-
transplantation testing is more commonly referred to as histocompatibility testing, and includes
tissue matching and tissue typing.
Histocompatibility Testing: Tissue Matching

• In tissue matching, also referred to as crossmatching, the goal is to ascertain donor-
recipient compatibility as determined by differences at the MHC Serological, cellular and
molecular techniques are available.
• Serological Approach. Serological approaches to tissue matching were considered the

method of choice until the development of molecular techniques. In a serological
approach the donor and recipient tissues are reacted separately with panels of antibodies
for various MHC alleles, and their reactions are compared. The problem with this
approach is that allelic differences can be detected only if an antibody is available for the
allele

• Cellular Approach. The cellular approach to tissue matching uses the mixed leukocyte
reaction (MLR). The MLR is an in vitro test used to mimic the in vivo condition of
transplantation. In the MLR assay and recipient cells are cultured together for several
days to allow T cells to be activated and proliferate in response to disparate MHC
proteins. The amount of proliferation can be measured and used to predict the magnitude
of rejection. Unfortunately, the technique requires an incubation of several days, which is
a limitation for tissues that are not from living donors. Therefore, the MLR is most useful
for bone marrow grafts and in cases of living related donors, where time constraint is not
an issue.
• Molecular Approach. Molecular approaches that have been used for tissue matching
include restriction fragment length polymorphism (RFLP) and the polymerase chain
reaction (PCR). RFLP is a technique in which enzymes are used to cleave genomic DNA
to obtain a pattern of fragmentation with the exception of identical twins, each person has
a unique pattern of fragmentation. Therefore, the degree of genetic disparity between the
donor and recipient can be assessed by comparing patterns of fragmentation. The PCR is
an automated, simple, rapid in vitro technique that allows direct amplification of a
particular DNA sequence, selected by the judicious use of primers (short nucleic acid
sequences) that border the genes of interest. Again, the degree of disparity between the
recipient and donor for selected sequences can be determined. A limitation to the
molecular approach is that the number of disparities may not necessarily predict the
severity of rejection between donor and the recipient because these do not quantitate the
T cell response to the foreign tissue. The molecular tests, however, do not have the time
constraints of the MLR and are superior to serological approaches.
Histocompatibility Testing: Tissue Typing:

• In tissue typing, the individual's HLA genotype is determined. Molecular and serological
tests are both available; however, PCR has become the method of choice in many
laboratories.
• Serological Approach
o Serological testing is a simple and relatively fast method for tissue typing in
which a sample of the individual's blood incubated with a panel of antibodies of
known specificity. In the basic protocol, a blood sample is drawn from the per son
to be typed. Mononuclear cells are isolated and placed in a 96-well microtiter
plate. A panel of antibodies known to react with specific HLA alleles is reacted
with the sample. Complement is then added and the microtiter plates incubated at
37°C.
o If antibodies from the panel bind to the cells in the wells, complement will be
activated and those cells will be lysed. A dye, trypan blue, is added to each well to
determine the number of cells killed. Cells that have been lysed will appear a dark
blue, while those that do not express the relevant antibody will be unaffected. An
aliquot of cells from each well is removed and counted under a light microscope
using a hemocytometer.

Paternity and Histocompatibility

• Testing Histocompatibility testing may also be used to determine paternity. In this
situation, paternity is not confirmed. Rather, the test is one of exclusion. If the child does
not express any of the same HLA 'alleles as the male being tested, then paternity can be
excluded.
HUMAN: HLA

Topic: Serology
OVERVIEW
1. Serology
2. Agglutination method
3. Precipitation method
4. Labeled immunoassays
1. SEROLOGY
• It is the study of antigen-antibody reaction in vitro
Serological test
o Test that involves antigen-antibody reaction
o Detect unknown specimen by using known commercial anti-sera
o Detect the presence of unknown antibodies in the serum of patient by
using known commercial antigen
Serologic testing has long been an important part of diagnostic tests in the clinical
laboratory for viral and bacterial diseases. Immunologic testing is done in many areas
of the clinical •laboratory—microbiology, chemistry, toxicology, immunology,
hematology, surgical pathology, cytopathology, immunohematology (blood
banking)—and a great variety of specimens are tested. Rapid testing is typically used
in the laboratory as well as in home-testing kit.
Preservation of Serum
• Physical: refrigerate for 72 hours at 4-6 degree Celsius
• Chemical: add 0.001g Merthiolate powder per mL of serum or 5% phenol or
tricresol at 0.1 ml/ml of serum
Inactivation of Serum
▪ Some procedures require the use of inactivated serum. Inactivation is the
process that destroys complement activity. Complement is known to interfere
with the reactions of certain syphilis tests and complement components (e.g.,
C1q). It can agglutinate latex particles and cause a false-positive reaction in
latex passive agglutination assays. Complement may also cause lysis of the
indicator cells in hemagglutination assays. Complement in body fluids can be
inactivated by heating to 56° C for 30 minutes. When more than 4 hours has
elapsed since inactivation, a specimen can be reinactivated by heating it to 56°
C for 10 minutes.
• Physical: heat serum at 56 degree Celsius for 30 minutes or heat at 60 -62
degree Celsius for 3-4 minutes only
• Chemical: adding choline chloride
CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

Topic: Serology
Immunologic reactions:
• Primary: combination of antigen-antibody; non visible reaction
• Secondary: demonstrate antigen-antibody reaction; visible reaction
• Tertiary: immunologically in vivo (inside the body); biologic reaction is
detectable
ANTIGEN–ANTIBODY
The primary union of binding sites on an antibody with specific epitopes on an
antigen depends on two characteristics of antibody known as affinity and avidity.
Affinity is the initial force of attraction that exists between a single Fab site on an
antibody molecule and a single epitope or determinant site on the corresponding
antigen. The strength of attraction depends on the specificity of antibody for a
particular antigen.
However, if the epitope and the binding site have a perfect lock-and-key fit, as is
the case with the original antigen, the affinity will be maximal.
Avidity represents the overall strength of antigen–antibody binding and is the

sum of the affinities of all the individual antibody–antigen combining sites.1,3
Avidity refers to the strength with which a multivalent antibody binds a
multivalent antigen and is a measure of the overall stability of an antigen–
antibody complex.2 In other words, once binding has occurred, it is the force that
keeps the molecules together. A high avidity can actually compensate for a low
affinity.
Avidity is the serum of the forces binding multivalent

antigens to multivalent antibodies. In a comparison
between IgG and IgM, IgM has the most potential
binding sites for antigen and thus the higher avidity.
Note that the monomers in IgM can swing up or down
in order to bind more effectively

Topic: Serology
2. AGGLUTINATION METHOD
Agglutination: antigens involved are particulate. Agglutination is the visible aggregation

of particles caused by combination with specific antibody. Antibodies that produce such
reactions are often called agglutinins.
Agglutination, is a two-step process that results in the formation of a stable lattice

network.
The first reaction, called sensitization, involves antigen–antibody combination through

single antigenic determinants on the particle and is rapid and reversible.
The second step, or lattice formation, is the formation of cross-links that form the visible
aggregates. This represents the stabilization of antigen–antibody complexes with the
binding together of multiple antigenic determinants.
Antibody Antigen with multiple Sensitization Lattice formation

determinants (no visible reaction) (visible agglutination)
Phase of agglutination.
Sensitization; antigen and antibody unite through antigenic determinant sites.
Lattice formation; rearrangement of antigen and antibody bonds to form a stable
lattice
IgM with a potential valence of 10 is over 700 times more efficient in agglutination than
is IgG with a valence of 2
Antibodies of the IgG class often cannot bridge the distance between particles because
their small size and restricted flexibility at the hinge region
IgM antibodies, on the other hand, are strong agglutinins because of their larger size
Antibodies belonging to the IgG class agglutinate best at 30°C to 37°C, whereas IgM
Topic: Serology
antibodies react best at temperatures between 4°C and 27°C. Because naturally occurring
antibodies against the ABO blood groups belong to the IgM class, these reactions are best
run at room temperature

Topic: Serology
TYPES OF AGGLUTINATION REACTION:
a. Direct agglutination
• Antigens are found on the surface of the particles
• Example: Blood typing, Kauffman and White scheme (S.typhi), Widal test,
Weil Felix (R. ricketsii), cold agglutination (mycoplasma infection)
b. Passive agglutination
• Antigen is artificially attached to a particulate carrier
• Passive, or indirect, agglutination employs particles that are coated with
antigens not normally found on their surfaces.
• A variety of particles, including erythrocytes, latex, and gelatin, are used
for passive agglutination
• Used to detect antibody, and uses latex bead
c. Reverse passive agglutination
• Antibodies are attached to particulate carriers
• antibody rather than antigen is attached to a carrier particle. The antibody
must still be reactive and is joined in such a manner that the active sites
are facing outward.
• Used to detect antigen, and uses latex bead
d. Coagglutination
• Carrier: bacterium that act as inert particle to attach the antibody
• Uses latex bead
e. Agglutination inhibition
• Reactions are based on competition between particulate and soluble
antigens for limited antibody combining sites. A lack of agglutination is an
indicator of a positive reaction.
• Positive: no agglutination
• Negative: with agglutination
• Example:
HCG + Anti HCG (rgt 1) = Ag-Ab reaction + cells with HCG (rgt2) = NO AGGLUTINATION
NoHCG + Anti HCG (rgt1) = Ab only + cells with HCG (rgt 2) = WITH AGGLUTINATION

Topic: Serology
Antigen Yes NO: positive test
Patient sample Add reagent antibody Immune complexes Add antigen-coated Agglutination
form particles
No antigen No Yes: Negative Test
f. Antiglobulin technique
• Antihuman IgG is added to bridge the gap between the cells to
demonstrate incomplete antibodies
➢ Direct antiglobulin test
▪ Detects in vivo sensitization of cells
▪ Antigen-antibody reaction inside the body
➢ Indirect antiglobulin test

▪ Detects in vitro sensitization
▪ Antigen-antibody reaction outside the body
g. Neutralization
• Antigenic activity is stopped by its specific antibody (no lysis)
• Target: to detect toxins, viral agents or antibodies to the toxin or viral
agents
• Types:
➢ Toxin neutralization:
▪ schick test – diptheria, dick test – scarlet fever, ASO Titer
test
➢ Virus Neutralization

Topic: Serology
Grading Agglutination Reaction

Grade Description
Negative No aggregates
Mixed Field A few isolated aggregates; mostly free-floating cells;
supernatant appears red
Weak (+ -) Tiny aggregates barely visible macroscopically; many free
erythrocytes; turbid and reddish supernatant
1+ A few small aggregates just visible macroscopically; many
free erythrocytes; turbid and reddish supernatant
2+ Medium-sized aggregates; some free erythrocytes; clear
supernatant
3+ Several large aggregates; some free erythrocytes; clear
supernatant
4+ All erythrocytes are combined into one solid aggregate: clear
supernatant
False-Positive Reactions
Contaminated equipment or Store equipment and reagent in clean,
reagent may cause particles to dust-free environment, and handle with
clump care. Use negative quality control
(QC) steps
Autoagglutination Use a control with saline and no
antibody as a negative control.
If positive, patient’s result is invalid
Delay in reading slide reactions Follow procedural directions and read
results in drying out of mixture reactions exactly as specified
Overcentrifugation causes cells Calibrate centrifuge to proper speed
or particles to clamp too tightly and time
False-Negative Reactions
Inadequate washing of red blood Wash cells according to directions.
cells in antihuman globulin (AHG) Use positive and negative QC steps
testing may result in unbound
immunoglobulins neutralizing the
reagent
Failure to add AHG reagent Use positive QC steps
Contaminated or expired reagents Use positive and negative QC steps
Improper incubation Follow procedural protocol exactly.
Use positive and negative QC steps
Delay in reading slide reactions Follow procedural protocol exactly
Use positive and negative control steps
Undercentrifugation Calibrate centrifuge to proper speed and
time
Prozone Phenomenon Dilute serum containing antibody, and
repeat the procedures
Topic: Serology
Alignment of antibody molecules bound to surfaces of a latex

particle and latex agglutination reaction.
Diagram of liposome-enhanced latex

agglutination reactions
INSTRUMENTATION
require no complicated instrumentation to read, several chemistry analyzers have been

developed using automation to increase sensitivity
Nephelometry has been applied to the reading of agglutination reactions and the
term particle-enhanced immunoassay is used to describe such reactions. When particles
are used, the sensitivity can be increased to nanograms/mL. For this type of reaction,
small latex particles with a diameter of smaller than 1 μm are used. One such type of
instrumentation system is called particle-counting immunoassay (PACIA).

Topic: Serology
Reading Agglutination
Grade Description Description Appearance Appearance
Cells Supernate Macroscopic Microscopic
0 No agglutination Dark, turbid, homogeneous
W+ Many tiny agglutinates Dark, turbid
Many free cells
May not be visible without
microscope
1+ Many small agglutinates Turbid
Many free cells
2+ Many medium-sized Clear
agglutinates
Moderate number of free cells
3+ Several large agglutinates Clear
Few free cells
4+ One large, solid agglutinates Clear
No free cells
For any one grade, readings can be on a scale from weak+ to strong+ (e.g., grade 2 can be scored as 2+w, 2+ or
2+s, depending on the number and size of agglutinates
Microscopic readings are generally performed to differentiate pseudoagglutination (rouleaux) from true agglutination to
detect mixed-field reactions and to confirm a negative reaction

Topic: Serology

Topic: Serology
3. PRECIPITATION METHOD
• involves combining soluble antigen with soluble antibody to produce

insoluble complexes that are visible
• Precipitation reaction: Soluble antigen and soluble antibody react to form an
insoluble product (precipitate), such as double gel diffusion, radial
immunodiffusion, immunoelectrophoresis, immunofixation, nephelometry,
and turbidimetry.
PRECIPITATION CURVE
Zone of Equivalence
• the number of multivalent sites of antigen and antibody are
approximately equal
• precipitation is the result of random, reversible reactions whereby each
antibody binds to more than one antigen and vice versa, forming a
stable network or lattice.
Prozone phenomenon
• occurs when excess amount of antibody is present, and the antigen and
antibody do not combine to form precipitates—the complexes remain
soluble. This results in a false negative result.
• usually only one site on an antibody molecule is used and many free
antibody molecules remain in solution.
• Prozoning should be suspected if a precipitin arc appears to run into a
trough, if an L chain appears fuzzy when an H chain is increased, or if
an arc appears to be incomplete.
Postzone phenomenon
• occurs in which small aggregates are surrounded by excess antigen
• no lattice network is formed
• every available antibody site is bound to a single antigen and no cross-
links are formed.
• occurs when excess amount
of antigen is present, and the antigen and antibody do not
combine to form precipitates—the complexes remain
soluble. This results in a false negative result.
Antigen Concentration
Precipitation curve. The precipitation curve shows how the amount
of precipitation varies with varying antigen concentrations when
the amount of antibody is kept constant. Excess antibody is called
the prozone and excess antigen concentration is called the
postzone
Topic: Serology
TYPES OF PRECIPITATION REACTIONS
1. Measurement of Precipitation by Light Scattering/ Fluid-phase

precipitation
Passive diffusion of soluble antigen and antibody
▪ Turbidimetry
▪ measure of the turbidity or cloudiness of a solution
▪ device measures the reduction in light intensity caused by
reflection, absorption, or scatter
▪ The formation of immune complexes decreases the amount
of light passing through a suspension. The more immune
complexes formed and the larger they are, the greater the
decrease in light able to pass through.
▪ measurements are made using a spectrophotometer or an
automated clinical chemistry analyzer
▪ Nephelometry
❖ measures the light that is scattered at a particular angle
from the incident beam as it passes through a suspension
❖ If a solution has excess antibody, adding increasing
amounts of antigen results in an increase in antigen–antibody
complexes and thus an increase in light scattering.
❖ Nephelometers typically measure light scatter at angles
ranging from 10 degrees to about 90 degrees.
❖ Nephelometry can be used to detect either antigen or
antibody, but typically it is run with antibody as the reagent
and patient antigen as the unknown.
❖ Rate Nephelometry - measurement of serum
proteins; the rate of scattering increase is measured
immediately after the reagent antibody is added.
Principles of nephelometry. The light detection device is at an angle to the incident light, in contrast to turbidity, which
measures light rays passing directly through the solution
2. Passive Immunodiffusion Techniques/ Precipitation reactions in agar

gel
- Agarose, a purified high-molecular-weight complex
polysaccharide derived from seaweed, is used for this purpose
- antigen and antibody diffuse toward one another in a gel matrix,
visible lines of precipitation will form
- Antigen and antibody are added to wells in the gel and antigen–
antibody combination occurs by means of diffusion.
- When no electrical current is used to speed up this process, it is
known as passive immunodiffusion.

Topic: Serology
Radial immunodiffusion. The amount of precipitate formed is in proportion to the antigen present in the sample. In
The Macini end-point method, concentration is proportion to the diameter squared.
▪ Radial Immunodiffusion (single diffusion technique)

❖ antibody is uniformly distributed in the support gel and
antigen is applied to a well cut into the gel. As the antigen
diffuses out from the well, antigen– antibody combination
occurs in changing proportions until the zone of equivalence
is reached and a stable lattice network is formed in the gel.
The area of the ring obtained is a measure of antigen
concentration that can be compared with a standard curve
obtained by using antigens of known concentration.
❖ precision of the assay is directly related to accurate
measurement of samples and standards
❖ Sources of error include overfilling or underfilling the wells,
nicking the side of the wells when filling, spilling sample
outside the wells, improper incubation time and temperature,
and incorrect measurement
❖ used to measure IgG and IgA subclasses as well as
complement components.
❖ Immunodiffusion is simple to perform and requires no
instrumentation, but it has largely been replaced by more
sensitive methods such as nephelometry and enzyme-linked
immunoassay
❖ End-point method radial immunodiffusion

❖ antigen is allowed to diffuse to completion; when
equivalence is reached, there is no further change in
the ring diameter
❖ Equivalence occurs between 24 and 72 hours. The
square of the diameter is then directly proportional
to the concentration of the antigen
❖ major drawback to this method is the time it takes to
obtain results
❖ A graph is obtained by plotting concentrations of
standards on the x axis versus the diameter squared
on the y axis, creating a smooth curve to fit the
points
❖ Kinetic or Fahey method
❖ uses ring diameter readings taken at about 19 hours
before equivalence is reached
❖ diameter is then proportional to the log of the
concentration and a graph is plotted using semi-log
paper.
❖ The diameter is plotted on the x axis and the
concentration is on the y axis, which automatically
gives a log value
Topic: Serology
▪ Double immunodiffusion (Ouchterlony technique)

❖ both antigen and antibody diffuse independently through a
semisolid medium in two dimensions, horizontally and
vertically.
❖ Wells are cut in a gel and reactants are added to the wells.
Most Ouchterlony plates are set up with a central well
surrounded by four to six equidistant outer wells.
❖ Antibody that is multispecific is placed in the central well
and different antigens are placed in the surrounding wells to
determine if the antigens share identical epitopes.
❖ Diffusion takes place radially from the wells. After an
incubation period of between 12 and 48 hours in a moist
chamber, precipitin lines form where the moving front of
antigen meets that of antibody and the point of equivalence is
reached.
❖ The density of the lines reflects the amount of immune
complex formed
❖ The position of the precipitin bands between wells allows
for the antigens to be compared with one another.
❖ Several patterns are possible:
(1) Fusion of the lines at their junction to form an arc
represents serological identity or the presence of a common
epitope
(2) a pattern of crossed lines demonstrates two separate
reactions and indicates that the compared antigens share no
common epitopes
(3) fusion of two lines with a spur indicates partial identity.
▪ Ouchterlony double diffusion is still used to identify fungal
antigens such as Aspergillus, Blastomyces, Coccidioides,
and Candida.
▪ Common errors include overfilling of wells, irregular well
punching, unlevel incubation area, gel drying, increased
room temperature, and antigen or antibody contamination
by bacteria or fungi.
Ouchterlony diffusion patterns. An antibody mixture is placed in the central well. Unknown antigens are placed in
the outside wells. The antibodies and antigen diffuse radially out of the wells. (A) Serologic Identity. If the antigens
are identical, they will react with the same antibody and the precipitate line forms a continuous arc. (B) Nonidentity. If
the antigens share no identical determinants, they will react with different antibodies and two crossed lines are formed.
(C) If antigen 3 has a determinant in common with antigen 1, one of the antibodies reacts with both antigens. Another
antibody that reacts with different determinants on antigen 1 (absent on antigen 3) passes through one precipitation
line and forms the spur on the other line. The spur formed always points to the simpler antigen with fewer antigenic
determinants

Topic: Serology
▪
Electrophoretic Techniques
❖ Countercurrent immunoelectrophoresis (CIE)
❖ Diffusion can be combined with electrophoresis to
speed up or sharpen the results
❖ Electrophoresis separates molecules according to
differences in their electric charge when they are
placed in an electric field.
❖ direct current is forced through the gel, causing
antigen, antibody, or both to migrate.
❖ As diffusion takes place, distinct precipitin bands
are formed
❖ Immunoelectrophoresis is a double-diffusion
technique that incorporates electrophoresis to
enhance results.
❖ On an agar gel plate or slide, antigen is added to one
well and antibody is added to another well. An
electric current accelerates the of the antigen and
antibody toward each other, resulting in
precipitation sooner than if an electric current is not
applied.
❖ CIE can be used to detect antibodies to infectious
agents and microbial antigens. CIE has generally
been replaced by easier to perform assays, such as
agglutination tests.
❖ Immunofixation electrophoresis
❖ similar to Immunoelectrophoresis except that after
electrophoresis takes place, antiserum is applied
directly to the gel’s surface rather than placed in a
trough
❖ Serum, urine, or CSF is electrophoresed. Antisera
contained in a cellulose acetate strip is then placed
on top of the electrophoresis gel. The antibodies
diffuse into the electrophoresis gel and combine
with the antigens, forming a precipitate.
❖ Detects the presence of an immunoglobulin in
serum or urine
Comparison of Immunoelectrophoresis and Immunofixation Electrophoresis
Feature IEP IFE
Ease of use Easy More complex
Sensitivity Less sensitive More sensitive
Monoclonal Better for typing large monoclonal gammopathies Used for difficult to characterize anomalous
gammopathies proteins
Interpretation Challenging Easier

Topic: Serology
Comparison of immunofixation electrophoresis (IFE) and immunoelectrophoresis (IEP) for two patients with monoclonal gammopathies. A, Patient specimen with an IgG
(k) monoclonal protein, as identified by IF. NOTE the position of the monoclonal protein (arrow). After electrophoresis each track except serum protein electrophoresis
(SPE) is reacted with its respective antiserum, then all tracks are stained to visualize the respective protein bands. Immunoglobulins G, A and M (IgG, IgA, IgM) KAPPA
(k) and lambda indicate antiserum used on each track. B same specimen as in A. with proteins identified by IEP. Note the
▪ Rocket immunoelectrophoresis
❖ Used to quantify antigens
❖ Antigens are electrophoresed in agar-containing antibody. A
pH is selected so that the antibodies are immobile. The
antibody and antigen combine to form precipitates in the
shape of a "rocket."
❖ The height of the rocket is proportional to the concentration
of antigen in the specimen.
Rocket Immunoelectrophoresis of human serum albumin. Patient samples were applied in duplicate.
Calibrators were placed at opposite ends of the plate

Topic: Serology
4. LABELED IMMUNOASSAYS
▪ labeled immunoassays are designed for antigens and antibodies that may
be small in size or present in very low concentrations. The presence of
such antigens or antibodies is determined indirectly by using a labeled
reactant to detect whether or not specific binding has taken place
▪ substance to be measured, often called the analyte, typically is a protein;
bacterial antigens, hormones, drugs, tumor markers, specific
immunoglobulins, and many other substances.
FORMATS FOR LABELED ASSAYS
Competitive Immunoassays
• reactants are mixed together simultaneously; labeled antigen competes with
unlabeled patient antigen for a limited number of antibody-binding sites.
• involves using a solid phase surface to which specific antigen is attached.
• The patient’s potentially containing antibody and an enzyme-labeled antibody
specific to the test antibody (conjugate) are mixed.
• Chromogenic substrate is then added, which changes color in the presence of
the enzyme.
• The amount of color that develops is inversely proportional to the amount of
antibody in the patient’s serum.
Principles of a competitive immunoassay

1. Unknown concentration of analyte in patient sample (red dots) competes with labeled analyte (yellow stars) for binding sites on
immobilized antibody. Sample A is a negative control
2. Wash to remove unbound material
3. After substrate is added, a colored product (signal) is generated with an intensity proportional to the amount of enzyme-labeled analyte
bound to the antibody
The signal strength is usually inversely to the analyte concentration

Topic: Serology
Format for competitive immunoassays

Noncompetitive Immunoassays
• a specific antigen is attached to a solid-phase surfaces (plastic bead or
microtiter well)
• The patient’s serum that could contain antibody is added to the solid-phase
surface, followed by an enzyme-labeled antibody specific to the test
antibody.
• The added chromogenic substrate changes color if the enzyme is present.
• The amount of color that develops is proportional to the amount of
• Capture Enzyme Immunoassay has the same principle with
noncompetitive immunoassay
Noncompetitive Immunoassay
1. For sample B, immobilized reagent

antibodies capture analyte in patient sample
(red dots) while in the Ab capture
technique; immobilized reagent antigen
captures patient antibodies. Sample A is a
negative control
2. Wash to remove unbound materials
3. Add enzyme-labeled (Detection) antibody
that binds to a different epitope on analyte
for sample B (or binds Fc region on human
Ig for antibody capture)
4. Wash to remove unbound materials
Add substrate and measure color change. Color
intensity is directly related to analyte (Sample B) or
human antibody (Antibody capture)

Topic: Serology
HETEROGENEOUS VERSUS HOMOGENEOUS ASSAYS
Heterogeneous enzyme immunoassays

• require a step to physically separate free from bound analyte
• involve a solid phase (microwell, bead) and require washing steps
to remove unbound antigens or antibodies.
• can have a competitive or noncompetitive format
Homogeneous enzyme immunoassays

• do not need a separation step
• involve an enzyme label, chosen so that the enzyme is inactivated
when bound to an antibody
• This type of assay is simpler to perform because there is no
washing step
• consist only of a liquid phase and do not require washing steps
• faster and easier to automate
• have competitive formats
Homogenous Immunoassay. Reagent antibody is in solution. Patient antigen and enzyme-labeled antigen are added to
the test tube. Patient antigen and enzyme-labeled antigen compete for a limited number of binding sites on the antibodies.
When patient antigen is present, the enzyme label on the reagent antigen is not blocked, so color development is
observed. Sample A has a low concentration of patient antigen, whereas Sample B contains more patient antigen and has
stronger color development
IMMUNOASSAYS
1. RADIOIMMUNOASSAY
• pioneered by Yalow and Berson in the late 1950s to determine the level of
insulin–anti-insulin complexes in diabetic patients.
• valuable in measuring a number of substances such as hormones
• serum proteins, and vitamins that either occur at very low levels in blood plasma
or are so small that they could not be detected otherwise.
• uses a radioactive substance as a label
• Iodine 125 has been the most popular radioactive labels
o easily incorporated into protein molecules and emits gamma radiation,
which is detected by a gamma counter.
o Very low quantities of radioactivity can be easily measured.
• extremely sensitive and precise technique for determining trace amounts of
analytes that are small in size
• the chief disadvantage is the health hazard involved in working with radioactive
substances
Topic: Serology
2. ENZYME IMMUNOASSAYS
• using enzymes as labels, were developed as alternatives to RIAs
• react with suitable substrates to produce breakdown products that may be
chromogenic, fluorogenic, or luminescent.
• some type of spectroscopy can then be used to measure the changes involved.
• labels for immunoassay, enzymes are cheap and readily available, have a long
shelf life, are easily adapted to automation
• sensitivity can be achieved without disposal problems or the health hazards of
radiation
• Enzyme labels can either be used qualitatively to determine the presence of an
antigen or antibody or quantitatively to determine the actual concentration of an
analyte in an unknown specimen.
• enzymes that have been used as labels in colorimetric reactions include
horseradish peroxidase, alkaline phosphatase, and β-D-galactosidase.
o Alkaline phosphatase and horseradish peroxidase have the highest
turnover (conversion of substrate) rates, high sensitivity, and are easy to
detect, so they are most often used in such assays.
Enzymes used in enzyme

immunoassays
Enzyme Source
Acetylcholinesterase Electrophorous
electicus
Alkaline phosphatase Escherichia coli
B-Galactosidase Escherichia coli
Glucose oxidase Aspergillus
niger
Glucose-6-phosphate- Leuconostoc
dehydrogenase (G6PD) mesenteroides
Lysozyme Egg white
Malate dehydrogenase Pig heart
Peroxidase Horseradish
a. Heterogeneous
Enzyme Immunoassays
i. Competitive Enzyme Immunoassays
• first enzyme immunoassays were competitive assays based on the
principles of RIA
• involves using a solid phase surface to which specific antigen is
attached.
• The patient’s potentially containing antibody and an enzyme-
labeled antibody specific to the test antibody (conjugate) are mixed.
• Chromogenic substrate is then added, which changes color in the
presence of the enzyme.
• The amount of color that develops is inversely proportional to the
amount of antibody in the patient’s serum.
• This method is typically used for measuring small antigens that are
relatively pure, such as drugs and hormones.
Topic: Serology
ii. Noncompetitive Enzyme Immunoassays

• specific antigen is attached to a solid-phase surface (plastic bead or
microtiter well.
• The patient’s serum that could contain antibody is added to the
solid-phase surface, followed by an enzyme-labeled antibody
specific to the test antibody.
• The added chromogenic substrate changes color if the enzyme is
present.
• The amount of color that develops is proportional to the amount of
• Most noncompetitive assays are indirect immunoassays, or so-
called indirect enzyme-linked immunosorbent assays (ELISA)
o because the enzyme-labeled reagent does not participate in
the initial antigen–antibody binding reaction.
o designed to detect antigens or antibodies by producing an
enzyme-triggered color change.
o Antigen is typically bound to solid phase (microtiter plates,
nitrocellulose membranes, and magnetic latex or plastic
beads.
• most frequently used immunoassays in the clinical laboratory
because of its sensitivity, specificity, simplicity, and low cost
• This type of assay is used to measure antibody production to
infectious agents that are difficult to isolate in the laboratory and for
autoantibody testing.
• Viral infections especially are more easily diagnosed by this method
than by other types of testing.
iii. Capture Assays

• antibody, rather than antigen, is bound to the solid phase
• often called sandwich immunoassays or capture assays
• Antigens captured in these assays must have multiple epitopes
• the sample antigen binds to an antibody fixed onto a solid phase; a
second antibody, labeled with a chemiluminescent label, binds to
the antigen-antibody complex on the solid phase
• the emitted light is directly proportional to the analyte concentration.
• The detection device for analyses is a simple photomultiplier tube
used to detect the emitted light.
• Capture assays are best suited to antigens that have multiple
determinants, such as antibodies, cytokines, proteins, tumor markers,
and microorganisms, especially viruses
• Chemiluminescent labels can be divided into five major groups: (1)
luminol; (2) acridinium esters; (3) peroxyoxalates; (4) dioxetanes;
and (5) tris(2,2′−bipyridyl)-ruthenium (II).

Topic: Serology
• Direct labels include luminol, acridinium ester, and

electrogenerated luminescent chelate from ruthenium and
tripropylamine (TPA) complex [Ru(bpy)3+].
o These labels are attached directly to antigens, antibodies, or
deoxyribonucleic acid (DNA) probes, depending on the
assay format.
o Oxidation of isoluminol by hydrogen peroxide (H2O2) in
the presence of a catalyst (e.g., microperoxidase) produces a
relatively long-lived emission at 425 nm.
o Oxidation of acridinium ester by alkaline H2O2 in the
presence of detergent produces a rapid flash of light lasting
from 1 to 5 seconds at 429 nm. Peak intensity can be used
for the measurement.
• Indirect labels are attached to antibodies, antigens, and DNA
probes, depending on the assay format.
o Enzyme labels often used in indirect procedures include the
following:
▪ Alkaline phosphatase (ALP)
▪ Horseradish peroxidase (HRP)
▪ Beta-galactosidase (β-galactosidase)
▪ native or recombinant apoaequorin (from the
bioluminescent jellyfish, Aequorea).
• It is activated by reaction with coelenterazine.
Light emission at 469 nm is triggered by
reaction with calcium chloride.
Format for sandwich Immunoassay

Topic: Serology
b. Homogeneous Enzyme Immunoassays

• less sensitive than heterogeneous assays, but they are rapid, simple to perform, and
adapt easily to automation
• chief use has been in the determination of low-molecular-weight analytes such as
hormones, therapeutic drugs, and drugs of abuse in both serum and urine
• example of a homogeneous immunoassay is the enzyme-multiplied immunoassay
technique (EMIT) developed by the Syva Corporation
• based on the principle of change in enzyme activity as specific antigen–antibody
combination occurs.
• Reagent antigen is labeled with an enzyme tag
• sensitivity of homogeneous assays is determined by the following: (1) detectability
of enzymatic activity; (2) change in that activity when antibody binds to antigen; (3)
strength of the antibody’s binding; and (4) susceptibility of the assay to interference
from endogenous enzyme activity, cross-reacting antigens, or enzyme inhibitors
• Disadvantages
o the fact that some specimens may contain natural inhibitors.
o Additionally, the size of the enzyme label may be a limiting factor in the
design of some assays.
o Nonspecific protein binding is another difficulty encountered with the use of
enzyme labels.
i. Rapid Immunoassays
• membrane based, easy to perform, and give reproducible results
• designed primarily for point-of-care or home testing
• designed as single-use, disposable assays in a plastic cartridge
• Immunochromatography
- analyte is applied at one end of the strip and migrates toward the
distal end where there is an absorbent pad to maintain a constant
capillary flow rate
- labeling and detection zones are set between the two ends
- ss the sample is loaded; it reconstitutes the labeled antigen or
antibody and the two form a complex that migrates toward the
detection zone.
- An antigen or antibody immobilized in the detection zone captures
the immune complex and forms a colored line for a positive test,
which may be in the form of a plus sign
- Excess labeled immunoreactant migrates to the absorbent pad.
- results are most often qualitative rather than quantitative

Topic: Serology
3. FLUORESCENT IMMUNOASSAYS
• 1941, Albert Coons demonstrated that antibodies could be labeled with molecules that
fluoresce
• used as substitutes for radio isotope or enzyme labels
• first used for localization of antigen in cells or tissues
• fluorescent compounds, called fluorophores or fluorochromes, can absorb energy from an
incident light source and convert that energy into light of a longer wavelength and lower
energy as the excited electrons return to the ground state
• fluorescent antibody technique consists of labeling antibody with fluorescein isothiocyanate
(FITC), a fluorescent compound with an affinity for proteins, to form a complex (conjugate)
• Fluorescent techniques are extremely specific and sensitive.
• Fluorescein absorbs maximally at 490 to 495 nm and emits a green color at 520 nm.
o Tetramethylrhodamine absorbs at 550 nm and emits red light at 585 nm.
o phycobiliprotein, europium (β-naphthyl trifluoroacetone), and lucifer yellow VS
• Immunofluorescent assay
o Antibodies used to identify such antigens are highly specific; when bound to antigen
in the tissue, the fluorescent probe attached to the antibody is detected under
ultraviolet light using a fluorescent microscope
i. Direct Immunofluorescent Assays

• a conjugated antibody is used to detect antigen-antibody reactions at a
microscopic level
• Fluorescein-conjugated antibodies bound to the fluorochrome FITC are
used to visualize many bacteria in direct specimens
• HRP conjugated to antibody, the immunoperoxidase stain, can be used
to detect CMV, other viruses, or nucleic acids in cells
• Biotin bound to avidin or antibody can be complexed to fluorescent
dyes or to color-producing enzymes to form specific detector systems.
o This system can be applied to the detection of nucleic acids in
organisms such as CMV, hepatitis B virus (HBV), Epstein-
Barr virus (EBV), and Chlamydia.
ii. Indirect Immunofluorescent Assays

• antibodies (immunoglobulins) not only react with homologous antigens,
but also can act as antigens and react with antiimmunoglobulins
• serologic method most widely used for the detection of diverse
antibodies.
• first step: patient serum is incubated with a known antigen attached to a
solid phase
• 2nd step: the slide is then washed and an anti-human immunoglobulin
containing a fluorescent tag is added
• 3rd step: immunoglobulin combines with the first antibody to form a
sandwich, which localizes the fluorescence

Topic: Serology
Immunologic Assays performed by indirect fluorescent antibody technique

Antiadrenal antibodies
Antibody (histone-reactive [HR]-ANA)
Anticentriole antibodies
Anticentromere antibodies
Anti-glomerular basement membrane antibodies
Anti-islet cell antibodies
Anti-liver-kidney microsomal (LKM) antibodies
Antimitochondrial
Antimyelin
Antimyocardial
Antinuclear antibody
Anti-parietal cell
Antiplatelet
Antireticulin
Antiribosome
Antiskin (dermal-epidermal)
Antiskin (interepithelial)
Anti-smooth muscle
Antistriational
Cytomegalovirus (IgM antibody)
Histone-reactive antinuclear antibody (HR-ANA)
Human immunodeficiency virus (total and IgM antibody)
Immunoglobulin M (IgM) Antibodies (Antigen specific)
Lymphocyte typing
Rubella virus antibody
Toxoplasma gondii antibody
•
iii. Inhibition Immunofluorescent Assay

• The inhibition immunofluorescent assay is a blocking test in which an antigen is first exposed
to unlabeled antibody and then to labeled antibody, and is finally washed and examined.
• If the unlabeled and labeled antibodies are both homologous to the antigen, there should be no
Topic: Serology
fluorescence.
• Antibody in an unknown serum can also be detected and identified by the inhibition test.
iv. Fluorescence Polarization Immunoassays

• based on the change in polarization of fluorescent light emitted from a labeled molecule when
it is bound by antibody
Incident light directed at the specimen is polarized with a lens or prism so that the waves are
aligned in one plane.
o If a molecule is small and rotates quickly enough, the emitted light is unpolarized
after it is excited by polarized light.
o If, however, the labeled molecule is bound to antibody, the molecule is unable to
tumble as rapidly and it emits an increased amount of polarized light

Topic: Serology
4. CHEMILUMINESCENT IMMUNOASSAYS
• emission of light caused by a chemical reaction, typically an oxidation reaction, producing an
excited molecule that decays back to its original ground state
• molecules capable for chemiluminescence: luminol, acridinium esters, ruthenium derivatives,
and nitrophenyl oxalates
• have an excellent sensitivity; the reagents are stable and relatively nontoxic
• very little reagent is used, they are also quite inexpensive to perform
• relatively high speed of detection also means a faster turnaround time.
• Ruthenium, one of the common chemical substances used as an indicator, can be conjugated
with antibody and applied to sandwich type assays.
5. EMERGING LABELING TECHNOLOGIES

a. Quantum Dots (Q dots)
- semiconductor nanocrystals used as fluorescent labeling reagents for biological
imaging
b. SQUID Technology
- novel method of target labeling is to tag antibodies with superparamagnetic
particles, allow the tagged antibodies to bind with the target antigen, and use a
superconducting quantum interference device (SQUID) to detect the tagged
antigen-antibody complex
c. Luminescent Oxygen-Channeling Immunoassay
- when these particles are in proximity during excitation, singlet oxygen moves
from the donor bead to the receptor bead, where it triggers the generation of a
luminescent signal
d. Signal Amplification Technology
- Tyramide signal amplification (TSA)
o provides a messenger RNA (mRNA) in situ hybridization protocol that
is effective in detecting B cell clonality in plastic-embedded tissue
specimens
e. Magnetic Labeling Technology
- an application of the high-resolution magnetic recording technology developed
for the computer disk drive industry.
- inherently safe, instrumentation is less expensive, signals are almost permanent,
and spatial resolution is increased
f. Time-Resolved Fluoroimmunoassay
- fluorescence is measured after a certain period to exclude background
interference fluoresce
g. Fluorescence in Situ Hybridization
- uses fluorescent molecules to brightly “paint” genes or chromosomes. The rapid
expansion in the availability of polyclonal and monoclonal antibodies has
fostered a dramatic increase in light microscopic immunohistochemistry (IHC)
and in situ hybridization

Topic: Serology
Classification of various Immunoassays and Their Characteristics
Labels (Reporter B/F Separation Signal Detection Sensitivity
Groups)
Precipitation Not Required Not Required Naked eye, turbidity, nephelometry 10
immunoassays
Particle immunoassays Not Required Naked eye, pattern analyzer, 5
spectrophotometry, particle counting
Radioimmunoassays Required Photon counting 5
Enzyme immunoassays Required Spectrophotometry, fluorometry photon, 0.1
Counting (CL-EIA)
Fluorescence Required Photon counting 5
immunoassays
Chemiluminescence Required Photon Counting 5
immunoassays
Particles Used as Labels for Particle Agglutination Immunoassays

Assay Method Supply
Human erythrocyte Direct hemagglutination (Landsteiner) ABO Blood Type
Erythrocyte antibody hemagglutination: titer plate/slide Human Immunodeficiency virus antibody
Avian erythrocyte Direct hemagglutination Human influenza virus antibody
Fixed animal erythrocyte Passive hemagglutination: titer plate Treponema pallidum antibody
Reverse Passive hemagglutination: titer plate Hepatitis B surface antigen (HBsAg)
Latex Reverse Passive agglutination: slide Chorionic gonadotropin
Reverse Passive agglutination: turbidimetry Immunoglobulin (Ig)E
Reverse Passive agglutination Ferritin
Latex (color) Immunochromatography Human Chorionic gonadotropin hCG
Microcapsule Passive agglutination: titer plate T. pallidum antibody
Gelatin particle Passive agglutination: titer plate HIV, T. pallidum antibody
Reverse Passive agglutination: charge-coupled device (CCD) Human hemoglobin (hHb)
camera
Polypeptide particle Passive and reverse agglutination T. pallidum antibody, HBs antibody
Silicate Particle Passive agglutination: titer plate/CCD camera T. pallidum antibody
Gold particle Reverse agglutination enhancement photometry Total estrogen
Metal sols Reverse agglutination hCG, hHb

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CLINICAL IMMUNOLOGY AND SEROLOGY

Topic: Monoclonal Antibody and Transplantation

Monoclonal antibodies are purified

Discovery of the Technique. In 1975,

Kohler and Milstein’s technique fuses an

Myeloma cells are cancerous plasma cells.

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

These hybridoma cells grow continuously, having acquired the immortal

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

Transplantation is a process whereby an individual (recipient receives cells or tissue from a

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

Rejection in Bone Marrow Transplantation

Screening to Ensure Compatibility

Histocompatibility Testing: Tissue Matching

• Serological Approach. Serological approaches to tissue matching were considered the

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

Histocompatibility Testing: Tissue Typing:

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

Paternity and Histocompatibility

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Monoclonal Antibody and Transplantation

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

Avidity represents the overall strength of antigen–antibody binding and is the

Avidity is the serum of the forces binding multivalent

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

Agglutination: antigens involved are particulate. Agglutination is the visible aggregation

Agglutination, is a two-step process that results in the formation of a stable lattice

The first reaction, called sensitization, involves antigen–antibody combination through

Antibody Antigen with multiple Sensitization Lattice formation

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

TYPES OF AGGLUTINATION REACTION:

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

➢ Indirect antiglobulin test

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

Grading Agglutination Reaction

Alignment of antibody molecules bound to surfaces of a latex

Diagram of liposome-enhanced latex

require no complicated instrumentation to read, several chemistry analyzers have been

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

• involves combining soluble antigen with soluble antibody to produce

TYPES OF PRECIPITATION REACTIONS

1. Measurement of Precipitation by Light Scattering/ Fluid-phase

2. Passive Immunodiffusion Techniques/ Precipitation reactions in agar

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

▪ Radial Immunodiffusion (single diffusion technique)

❖ End-point method radial immunodiffusion

▪ Double immunodiffusion (Ouchterlony technique)

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

Feature IEP IFE

Ease of use Easy More complex

Sensitivity Less sensitive More sensitive

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

FORMATS FOR LABELED ASSAYS

Principles of a competitive immunoassay

CLINICAL IMMUNOLOGY AND SEROLOGY | Topic: Serology

Format for competitive immunoassays

1. For sample B, immobilized reagent