Practical Immunology

& Serology
T. Rashad Saleh M. Alkhwlany
Department of Medical Microbiology
Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

Precipitation Reactions
Precipitations involve combination of soluble antigen with soluble antibody to produce
insoluble complexes that are visible.
Was first noted in 1897 by Kraus who found that culture filtrates of enteric bacteria
would precipitate when they were mixed with specific antibody.

 Combination of antigens with specific antibodies can occur in three phases:

1. Primary phenomenon:

 Binding of an individual Abs with epitope on Ags. Occur in milliseconds.

 Can be measured indirectly by technique such as: immunofluorescence,
radioimmunoassay and enzyme immunoassay.
2. Secondary phenomenon:
 These include precipitation, agglutination and complement fixation test.
3. Tertiary reaction:
 All reaction that occur in vivo.

 Precipitation curve

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 Measurement of precipitation
1. Light scatter
1. Turbidimetry:
Measure of turbidity or cloudiness of solution.

2. Nephelometry:

Measure the light that is scattered at a particular angle (ranging from 10 to about
70Cº) from the incident beam as it pass through a suspension. The amount of
light scattered is an index of the concentration of the solution. It may be recorded
in arbitrary units of relative light scatter. This is a more sensitive method that
turbidity.
 Uses:
Quantification of serum protein such as: IgG, IgA, IgM complement
component C3 and C4, albumin, α–antitrypsin and ceruloplasmin .
This method can be used to detect either Ags or Abs.

2. Passive immunodiffusion technique

In this technique use support media such as gel or agar or agarose. Agarose is
preferred to agar because the agar has a strong negative charge while agarose
almost has none, so the interaction between gel and the reagents are minimized.
Immunodiffusion reaction can be classified according to the number of reactants
diffusing and the direction of diffusion into:

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1. Oudin (single diffusion)

Is a precipitation technique, usually accomplished in a test tube, in which

antibody (or antigen) in a gel is allowed to react with soluble antigen (or
antibody) diffusing through it from a liquid interface.

2. Ouchterlony (double diffusion)

Is a precipitation technique in agar, usually accomplished in a Petri dish, in

which antigen and antibody are allowed to diffuse against each other and permit
the formation of a precipitin line between the sample wells.
If two or more sample (antigen) wells are diffused against a single antiserum
(antibody) well, the following distinctions can be made:
 Lines of identity: a single precipitin line indicating uniformity and identity
of the antigens in the sample wells
 Lines of nonidentity: two distinct and crossing precipitin lines indicating no
cross-reactivity or identity between the antigens in the two sample wells
 Lines of partial identity: a spur formation, indicating that some of the
antigenic determinants (epitopes) are shared between the antigens in the two
sample wells

 Advantages and Limitations

a. The main advantages are simplicity, minimal equipment requirements,
and specificity. If properly carried out, double immunodiffusion assays
are 100% specific as far as detecting an antigen-antibody reaction.
b. The main limitations are lack of sensitivity, and the time required for full
development of visible precipitation (up to 72–96 hours of diffusion in
systems when either reactant is present in small quantities).

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 Applications
In general, double diffusion assays are useful for determining the presence
or absence of a given antigen or antibody in any kind of biological fluid.

3. Radial immunodiffusion
Is a method for estimating antigen concentration.
 Uses the following procedure:
o Antibody to specific antigen is diffused in an agar gel or slab.
o Antigen wells are cut, and samples of various known concentrations are
applied.
o The gel is incubated and precipitin rings are developed.
o The diameter of each precipitin ring is proportional to the initial antigen
concentration; a reference line is determined.
o Unknown concentration is determined by comparison with the reference
line.

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 Advantages and Limitations

Radial immunodiffusion, while not as sensitive as some newer techniques
and relatively slow, is nonetheless reliable for routine quantitation of many
serum proteins.
 Applications
Quantitation of immunoglobulins, complement components, and, in general,
any antigenic protein, that exists in concentrations greater than 5–10 mg/dL
in any biological fluid.

3. Electrophoretic technique
This technique uses the electric current to speed the result. Electrophoresis
separates molecules according to differences in their electrical charge when they
placed in an electric field. This technique can be applied to single and double
diffusion.

1. Immunoelectrophoresis (IEP)

Is an antigen-antibody reaction technique developed to resolve and identify

highly complex mixtures of antigens.
Antigen is placed in a well in agar on a glass slide and subjected to
electrophoresis, using an electric current, to separate the serum proteins at a
given pH (usually pH 8.4) into their constituent parts (albumin, α1, α2, β1, β2,
and γ-globulin fractions) according to their electrical charge.

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The separated proteins react with their specific antibody diffusing from an
antiserum trough, and form a series of arcs of precipitate.
2. Rocket IEP
is an adaptation of radial immunodiffusion in which the antigen is actively
driven through an antibody-containing matrix by an electric potential. As the
equivalence zone of antigen-antibody reaction is reached, the reaction ceases
and elongated precipitin arcs (“rockets”) become visible (Fig. 14.6). The
lengths of those “rockets” are proportional to the antigen concentrations in the
wells. Rocket electrophoresis can be used for the quantitative assay of many
proteins, including immunoglobulins. The reverse modality with antigen
incorporated in the agar can be used for the quantitation of specific antibodies.
This technique is faster and more sensitive than radial immunodiffusion.

3. Counterimmunoelectrophoresis

 Principle
This technique is a variation of double immunodiffusion, in which antigen
and antibody are forced to move toward each other with an electric current
(antibodies move to the cathode while most antigens are strongly negatively
charged and move toward the anode). Precipitin lines can be visualized
between the antigen and antibody wells.
 Advantages and Limitations
The method is faster and more sensitive than double immunodiffusion.
Visible precipitation can be usually observed after 1–2 hours, although for
maximal sensitivity it is necessary to wash, dry, and stain the agar gel in
which the reaction took place, a process that extends the time for final
reading of the results to several days.

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 Applications
Counterimmunoelectrophoresis has been used for detection of fungal or
bacterial antigens in CSF (in patients with suspected meningitis), and for the
detection of antibodies to Candida albicans DNA, and other antigens.

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Agglutination

When bacteria, cells, or large particles in suspension are mixed with antibodies
directed to their surface determinants, one will observe the formation of large clumps;
this is known as an agglutination reaction.
The visualization of agglutination reactions differs according to the technique used for
their study. In slide tests, the non-agglutinated cell or particulate antigen appears as a
homogeneous suspension, while the agglutinated antigen will appear irregularly
clumped. If antibodies and cells are mixed in a test tube, the binding of cells and
antibodies will result in the diffuse deposition of cell clumps in the bottom and walls
of the test tube, while the non-agglutinated red cells will sediment in a very regular
fashion, forming a compact red button on the bottom of the tube.
 Agglutination reactions follow the same basic rules of the precipitation reaction.
a) When cells and antibody are mixed at very high antibody concentrations (low
dilutions of antisera), antibody excess may result, no significant binding with
the cells is seen, and, therefore, the agglutination reaction may appear to be
negative. That dilution at which antibody excess prevents agglutination
constitutes the prozone.
b) When equivalence is approached, larger clumps of cells can be distinguished.
c) At still higher dilutions, when the concentration of antibody is very low, the
zone of antigen excess is reached, and agglutination is no longer seen

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 Biological Consequences of the Antigen-Antibody Reaction

A. Opsonization: After binding to particulate antigens or after forming large
molecular aggregates, antibodies unfold and may interact with Fc receptors on
phagocytic cells. Such interaction is followed by ingestion by the phagocytic cell
(phagocytosis). Substances that promote phagocytosis are known as opsonins.

B. Complement Activation: One of the most important consequences of antigen-

antibody interactions is the activation (or “fixation) of the complement system
C. Neutralization: The binding of antibodies to bacteria, toxins, and viruses has
protective effects because it prevents the interaction of the microbial agents or their
products with the receptors that mediate their ineffectiveness or toxic

 Steps of agglutination:
1. Sensitization or initial binding
Antibody-antigen combination. Sensitization is affected by the nature of the
antibody (IgM with potential valence of the 10 is antibody over 750 times more
efficient in agglutination than is IgG with valence of 2. Also affected by the
natural of antigen-bearing surface, if the epitope are sparse or if they are
obscured by other surface molecules they are less to interact with antibody.

2. Lattice formation
The sum of interactions between antibody is antigen on a particle. Is affected by
physiochemical factors:
1. pH: best between 6.7 to 7.2.
2. Temperature: IgG best at 30 to 37Cº. IgM at 4 – 27Cº.
3. Environment condition.
4. Relative concentration of antigen and antibody.
o Enhancement of lattice formation:
Surface charge must be controlled in order to lattice formation. One method
is the decrease of ionic force of the buffer solution (e.g.: albumin). Other
technique that enhance lattice formation is the:
1. Increase viscosity: adding dextrane, albumin.

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2. Agitation or centrifugation.

1. Direct agglutination (Agglutination of Whole Microorganisms)

o Principle

Bacterial agglutination is observed when a bacterial suspension is mixed with

antibody directed to their surface determinants. Agglutination of whole
microorganisms is commonly used for serotyping isolated organisms and, less
commonly, for the diagnosis of some infectious diseases (e.g., the Weil-Felix test
for the diagnosis of typhus) based on the fact that certain strains of Proteus share
antigens with several Rickettsia species.
o Advantages and Limitations
Agglutination is rapid (takes a matter of minutes) and, by being visible with the
naked eye, does not require any special instrumentation other than a light box.
Its disadvantages are the need for isolated organisms and poor sensitivity,
requiring relatively large concentrations of antibody.

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2. Passive agglutination (Agglutination of Inert Particles Coated with Antigen)

o Principle

Latex particles and other inert particles (polystyrene or bentonite or charcoal) can
be coated with purified antigen and will agglutinate in the presence of specific
antibody. Conversely, specific antibodies can be easily adsorbed by latex
particles and will agglutinate in the presence of the corresponding antigen.

o Applications

1. Immunoglobulin-coated latex particles are used for the detection of anti-Ig

factors (rheumatoid factors) in the rheumatoid arthritis (RA) test
2. Latex particles coated with thyroglobulin are used in a thyroglobulin antibody
test.
3. In some cases, the antigen is bound to latex, and the test detects specific
antibodies (e.g., tests for histoplasmosis, cryptococcosis, and trichinosis).

o Advantages and Limitations

The main advantages of these tests are their simplicity and quick turn-around of
results. The main disadvantages are the need for large amounts of reagents, cost,
and relatively low sensitivity, particularly in the case of the tests for diagnosis of
infectious diseases.

3. Reverse Passive agglutination (Agglutination of Inert Particles Coated with

antibody)

o Principle

Latex particles and other inert particles can be coated with specific antibodies
and will agglutinate in the presence of the corresponding antigen.

o Applications

Detection of microbial antigens: Rapid diagnosis tests for bacterial and fungal
meningitis have been developed by adsorbing the relevant specific antibodies to
latex particles. The antibody-coated particles will agglutinate if mixed with CSF
containing the relevant antigen. This procedure allows a rapid etiological
diagnosis of meningitis which is essential for proper therapy to be initiated.

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o Advantages and Limitations

As passive agglutination test.

4. Agglutination inhibition test:

o Principle

Are based on competition between particular and soluble antigens for

combination with antibody. Lack of agglutination is an indicator of a positive
reaction.

o Applications

Human chorionic gonadotropin (HCG)

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5. Coagglutiation test:
In this test use bacteria as the inert particles to which antibody is attached.
S. aureus is the bacteria used in this method because it has a protein on its outer
surface, called protein A, that naturally attach with Fc portion on antibody leave
the Fab portion free to bind with antigens.

6. Hemagglutination

Hemagglutination tests can be subclassified depending on whether they detect

antibodies against red cell determinants (direct and indirect hemagglutination) or
against compounds artificially coupled to red cells (passive hemagglutination).

1. Direct hemagglutination

o Principle
Red cells are agglutinated when mixed with IgM antibodies recognizing
membrane epitopes.

o Applications
1. Determination of the ABO blood group and titration of isohemagglutinins (anti-
A and anti-B antibodies).
2. Titration of cold hemagglutinins (IgM antibodies which agglutinate RBC's at
temperatures below that of the body
3. The Paul-Bunnell test, useful for the diagnosis of infectious mononucleosis,
detects circulating heterophile antibodies (cross-reactive antibodies that
combine with antigens of an animal of a different species) that induce the
agglutination of sheep or horse erythrocytes.

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o Advantages and limitations

Hemagglutination is simple to execute and requires very simple materials.
However, the tests for cold agglutinins associated with infectious diseases and
the Paul Bunnell test lack specificity.

2. Indirect hemagglutination

o Principle
Indirect hemagglutination detects antibodies that react with antigens present in
the erythrocytes but which by themselves cannot induce agglutination. Usually,
these are IgG antibodies that are not as efficient agglutinators of red cells as
polymeric IgM antibodies. A second antibody directed to human
immunoglobulins is used to induce agglutination by reacting with the red-cell
bound IgG molecules, and consequently, cross-linking the red cells.

o Applications
The best known example of indirect agglutination is the antiglobulin or Coombs'
test, which is used in the diagnosis of autoimmune hemolytic anemia.

o Advantages and limitations

The technique is simple to perform. Poor sensitivity is the main limitation.

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3. Passive hemagglutination

o Principle
Passive hemagglutination techniques use red blood cells as latex particles.
Antigen can be coated onto the red cells by a variety of methods, and the coated
cells will agglutinate when exposed to specific antibody.

o Applications
This system has been used as a basis for a variety of diagnostic procedures such
as a test to detect antithyroid antibodies, the Rose Waaler test (RW) for anti-Ig
factors present in the serum of patients with rheumatoid arthritis, and many tests
to detect anti-infectious antibodies.

o Advantages and limitations

This technique shares the relative simplicity of all hemagglutination procedures,
and it is considerably more sensitive than regular hemagglutination. However,
Passive hemagglutination tests are difficult to standardize; specificity is often
problematic, and not too accurate (as all titration tests).

4. Hemagglutination inhibition test:

Hemagglutination Inhibition If the virus and antibody are homologous, the
virus is blocked from attaching to the erythrocytes and no hemagglutination
occurs. Only viruses that agglutinate red blood cells can be identified by this
method (e.g.: rubella, mumps, measles, influenzea, parainfluenzea and
adenoviruses).
Also hemagglutination inhibition tests are difficult to standardize; specificity is
often problematic, and not too accurate (as all titration tests).

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7. Complement Fixation

o Principle
When antigen and antibodies of the IgM or the IgG classes are mixed,
complement is “fixed” to the antigen-antibody aggregate. If this occurs on the
surface of a red blood cell, the complement cascade will be activated and
hemolysis will occur.
In this test, patient's serum is first heated to 56°C to inactivate the native
complement and adsorbed with washed sheep RBC to eliminate broadly cross
reactive anti-red-cell antibodies (also known as Forssman-type antibodies) which
could interfere with the assay. Then the serum is mixed with purified antigen and
with a dilution of fresh guinea pig serum, used as a controlled source of
complement. The mixture is incubated for 30 minutes at 37°C to allow any
antibody in the patient's serum to form complexes with the antigen and fix
complement. “Sensitized” red cells are then added to the mixture.
o If the red cells are lysed, it indicates that there were no antigen-specific
antibodies in the serum of the patient, so complement was not consumed in
the test system and was available to be used by the anti-RBC antibodies,
resulting in hemolysis. This reaction is considered negative.

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o If the red cells are not lysed, it indicates that antibodies specific to the antigen
were present in the test system, “fixed” complement, but none were available
to be activated by the indicator system. This reaction is considered positive.

o Applications
Complement fixation has the advantage of being widely applicable to the
detection of antibodies to almost any antigen. Thus, complement fixation
reactions have been widely used in a large number of tests designed to assist in
the diagnosis of specific infections, such as the Wassermann test for syphilis and
tests for antibodies to Mycoplasma pneumoniae, Bordetella pertussis, many
different viruses, and to fungi such as Cryptococcus, Histoplasma, and
Coccidioides immitis.

o Limitations
Complement fixation tests have technical difficulties and have been progressively
replaced by other methods.

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OVERVIEW OF IMMUNODIAGNOSTIC STUDIES

Immunodiagnostic or serodiagnostic testing studies antigen-antibody reactions for
diagnosis of infectious disease, autoimmune disorders, immune allergies, and
neoplastic disease. These modalities also test for blood groups and types, tissue and
graft transplant matching, and cellular immunology. Blood serum is tested for
antibodies against particular antigens, hence the term blood serology testing.
Antigens are substances that stimulate and subsequently react with the products of an
immune response. They may be enzymes, toxins, microorganisms (eg, bacterial, viral,
parasitic, fungal), tumors, or autoimmune factors. Antibodies are proteins produced by
the body's immune system in response to an antigen or antigens. The antigen-antibody
response is the body's natural defense against invading organisms. Red blood cell
groups contain almost 400 antigens. Immune reactions to these antigens result in a
wide variety of clinical disorders, which can be tested (eg, Coombs' test).
Pathologically, autoimmune disorders are produced by autoantibodies, that is,
antibodies against self. Examples include systemic rheumatic diseases, such as
rheumatoid arthritis and lupus erythematosus.
Immunodeficiency diseases exhibit a lack of one or more basic components of the
immune system, which includes B lymphocytes, T lymphocytes, phagocytic cells, and
the complement system. These diseases are classified as primary (eg, congenital,
DiGeorge syndrome) and secondary (eg, acquired immunodeficiency syndrome
[AIDS]). Hypersensitivity reactions are documented using immediate hypersensitivity
tests and are defined as abnormally increased immune responses to some allergens (eg,
allergic reaction to bee stings or pollens). Delayed hypersensitivity skin tests are
commonly used to evaluate cell-mediated immunity. Histocompatibility antigens
(transplantation antigens) and tests for human leukocyte antigen (HLA) are important
diagnostic tools to detect and prevent immune rejection in transplantation.
o Collection of Serum for Immunologic Tests
Specific antibodies can be detected in serum and other body fluids (eg, synovial
fluid, cerebrospinal fluid [CSF]).

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1. Procure samples. For diagnosis of infectious disease, a blood sample (serum

preferred) using a 4-mL tube should be obtained at illness onset (acute phase),
and the other sample should be drawn 3 to 4 weeks later (convalescent phase).
In general, serologic test usefulness depends on a titer increase in the time
interval between the acute and the convalescent phase. For some serologic tests,
one serum sample may be adequate if the antibody presence indicates an
abnormal condition or the antibody titer is unusually high.
2. Perform the serologic test before doing skin testing. Skin testing often induces
antibody production and could interfere with serologic test results.
3. Label the sample properly and submit requested information. Place specimen in
biohazard bag. Send samples to the laboratory promptly. Hemolyzed samples
cannot yield accurate results. Hemoglobin in the serum sample can interfere
with complement-fixing antibody values.

o Interpreting Results of Immunologic Tests

The following factors affect test results:
1. Previous vaccination (determine time frame)
2. Cross-reactivity: antibodies produced by one species of an organism can react
with an entirely different species (eg, Tularemia antibodies may agglutinate
Brucella and vice versa, Rickettsial infections may produce antibodies reactive
with Proteus OX19)
3. Presence of other serious illness states (eg, lack of immunologic response in
agammaglobulinemia, cancer treatment with immunosuppressant drugs)
4. Seroconversion: the detection of specific antibody in the serum of an individual
when this antibody was previously undetectable

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Streptococcal Antibody Tests

(Anti-Streptolysin O Titer (ASO), Streptozyme, Anti-DNase B (ADB)
Streptodornase)

Group A ß-hemolytic streptococci are associated with streptococcal

infections or illness.
These tests detect antibodies to enzymes produced by organisms. Group A ß-hemolytic
streptococci produce several enzymes, including streptolysin O, hyaluronidase, and
DNase B. Serologic tests that detect these enzyme antibodies include antistreptolysin
O titer (ASO), which detects streptolysin O; streptozyme, which detects antibodies to
multiple enzymes; and anti-DNase B (ADB), which detects DNase B. Serologic
detection of streptococcal antibodies helps to establish prior infection but is of no
value for diagnosing acute streptococcal infections. Acute infections should be
diagnosed by direct streptococcal cultures or the presence of streptococcal antigens.

o Anti-Streptolysin O (ASO) test

The β-hemolytic group A S pyogenes produce two haemolysins, streptolysin O
(SLO) and streptolysin S (SLS). SLO (MW 60,000) is an oxygen-labile member of
the cholesterol-binding thiol-activated toxin family that elicits antibodies and is
toxic to a variety of cells (hydrolysis of blood cell at different degree α, β and γ).
SLS it is elaborated in the presence of serum, hence the name streptolysin S. It is
not antigenic belongs to the bacteriocin family of microbial toxins.
When ASO titers are low, tests for these latter antibodies can be produced to
substantiate the diagnosis, because they are more sensitive tests.
Elevated levels also are seen in those with rheumatic fever, glomerulonephritis,
bacterial endocarditis, scarlet fever, otitis media, and streptococcal pharyngitis.
Increased in Antibody appears as early as 1 wk after infection; titer rises rapidly by
3-4 wks and then declines quickly; may remain elevated for months to 1 year.
In this test used latex particles coated with Streptolysin O (Ags) that react with
serum antibodies (ASO).

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o M Protein
This substance is a major virulence factor of group A S pyogenes. M protein
appears as hair-like projections of the streptococcal cell wall. When M protein is
present, the streptococci are virulent. S pyogenes that lack M protein are not
virulent. Immunity to infection with group A streptococci is related to the presence
of type-specific antibodies to M protein. Because there are many, perhaps 150,
types of M protein, a person can have repeated infections with group A S pyogenes.
There are two major structural classes of M protein, classes I and II.
It appears that M protein have an important role in the pathogenesis of rheumatic
fever. M protein induce antibodies that react with human cardiac sarcolemma

o Rheumatic Fever
Typical symptoms and signs of rheumatic fever include fever, malaise, a migratory
non-suppurative polyarthritis, and evidence of inflammation of all parts of the heart
(endocardium, myocardium, and pericardium). The carditis characteristically leads
to thickened and deformed valves and to small perivascular granulomas in the
myocardium (Aschoff bodies) that are finally replaced by scar tissue. Erythrocyte
sedimentation rates, serum transaminase levels, electrocardiograms, and other tests
are used to estimate rheumatic activity.

o Indications For Streptococcal Tests

1. Suspected streptococcal infection, to confirm the diagnosis
2. Detection and monitoring of response to therapy for post streptococcal illnesses
such as rheumatic fever and glomerulonephritis
3. Differentiation of rheumatic fever from rheumatoid arthritis, with the former
indicated by elevated levels
o Methods used for determine of ASO test:
1. ASO latex slide agglutination test
This test used to screen for significantly raised ASO titer and to semiquantify
the antibody.
2. ASO microtitration or tube haemolysis test
To titrate the antibody.

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o Procedure
1. Collect a 4-mL blood serum sample in tube. Observe standard precautions.
Place specimen in a biohazard bag for transport to the laboratory.
2. Repeat testing 10 days after the first test is recommended.

o Reference Values
Normal ASO titer: <166 Todd units (or <200 IU)

o Clinical Important
1. In general, a titer >166 Todd units is considered a definite elevation for the
ASO test and may be helpful evidence of a recent infection.
2. The ASO or the ADB test alone is positive in 80% to 85% of group A
streptococcal infections (eg, streptococcal pharyngitis, rheumatic fever,
pyoderma, glomerulonephritis).
3. When ASO and ADB tests are run concurrently, 95% of streptococcal
infections can be detected.
4. A repeatedly low titer is good evidence for the absence of active rheumatic
fever. Conversely, a high titer does not necessarily mean rheumatic fever of
glomerulonephritis is present; however, it does indicate the presence of a
streptococcal infection.
5. ASO production is especially high in rheumatic fever and glomerulonephritis.
These conditions show marked ASO titer increases during the symptomless
period preceding an attack. Also, ADB titers are particularly high in pyoderma.

o Interfering Factors
1. An increased titer can occur in healthy carriers.
2. Antibiotic and adrenal corticosteroids drugs therapy suppresses streptococcal
antibody response result in falsely decreased levels.
3. Increased β-lipoprotein levels inhibit streptolysin O and produce falsely high
ASO titers.

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o Clinical Alert
The ASO test is impractical in patients who have recently received antibiotics or
who are scheduled for antibiotic therapy because the treatment suppresses the
antibody response.

o False positives
Are associated with
1. TB
2. Liver disease (e.g., active viral hepatitis)
3. Bacterial contamination
4. Oxidation of the reagent

o Increases in:
1. Bacterial endocarditis
2. Glomerulonephritis
3. Pharyngitis
4. Reactive arthritis
5. Recent streptococcal infection
6. Rheumatic and connective tissue diseases
7. Rheumatic fever
8. Scarlet fever
9. Upper respiratory tract infections

o Decreased.
Not clinically significant. Levels may decrease with antibiotic therapy.

o Notes
The ESR is usually elevated during the clinical course of ARF and is a useful
indication of current activity of the disease. However, the ESR is very nonspecific
and indicates only that there is an active inflammatory process somewhere in the
body.

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o Other Streptococcal tests:

1. Anti-deoxyribonuclease B Antibody Titer (Anti-DNase B Antibody.

Deoxyribonuclease B is an antigen produced by group A streptococci. The anti-

DNase B test detects antibodies to deoxyribonuclease B, which appear when a
patient has a poststreptococcal infection. The levels increase after patient recovers
from a group A streptococcal infection and thus are a reliable indicator of recent
hemolytic streptococcal infection. This test is more sensitive to streptococcal
pyoderma than the antistreptolysin-O (ASO) test.
Anti–DNase B assay should also be performed because >15% of patients with
acute rheumatic fever do not have an increased ASOT. This assay is superior to
ASOT in detecting antibodies after group A streptococcal skin infections.

o Reference Values
Adult 85 Todd U/mL or <1:85

Child <7 years <60 Todd U/mL or <1:60

Child ≥7 years <170 Todd U/mL or <1:170

o Increases in:
1. Glomerulonephritis (poststreptococcal)
2. Pharyngitis (streptococcal)
3. Poststreptococcal reactive arthritis (PSReA)
4. Pyodermic skin infections
5. Rheumatic fever (acute)

2. Antihyaluronidase (AH) test

Hyaluronidase is an extracellular enzyme antigen produced by group A beta-
hemolytic streptococci. This test measures levels of antibodies to hyaluronidase
that appear in patient who are recovering from group A beta-hemolytic
streptococcal infections. Levels increase after patient recovers from a group A
beta-hemolytic streptococcal infection (about the second week of infection) and

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decrease 3–5 weeks after infection. Levels are thus a reliable indicator of recent
group A beta-hemolytic streptococcal infection. A better test than the
antistreptolysin-O (ASO) test for detecting antibodies in acute glomerulonephritis,
which follows a streptococcal pyoderma.

o Reference Values
<128 U/mL

o Increases in:
1. Recent group A streptococcal disease
2. Glomerulonephritis (acute)
3. Rheumatic fever (acute)

3. Streptozyme test
A nonspecific screening test for the detection of antibodies to multiple exoenzymes
of various species of streptococci using a commercial reagent containing
erythrocytes coated with streptococcal antigens (DNase, streptokinase, streptolysin
O, and hyaluronidase). This test can determine current or recent streptococcal
infection earlier than the ASO titer, but it cannot determine the location or type of
streptococcal infection. In a positive test, antibodies begin increasing by week 3
after infection and decrease by week 10.
This test is not as sensitive in children as it is in adults.

o Reference Values
Titer <166 Todd units or <100 streptozyme units.
o Increases
As ASO

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

C-REACTIVE PROTEIN (CRP)

C-reactive protein (CRP) is a glycoprotein produced during acute inflammation or

tissue destruction. The protein gets its name from its ability to react (or cross-react)
with Pneumococcus somatic C-polysaccharide and precipitates it. CRP is one of
the most sensitive acute-phase reactants (e.g., fibrinogen, ceruloplasmin Alpha-1
antitrypsin, haptoglobin, Apolipoproteins). It begins to rise about 4-6 hours after
onset of inflammation and reach peak in 48–72 hrs (usually 25–35 mg/dL) and has
a half-life of 5-7 hours, less than one-fourth that of most other proteins that react to
acute inflammation.
There are two types of CRP assays.
 One measures a wide range of CRP levels to include those found in patients
with acute infections. The reportable range is typically 0.3 to 20 mg/dL.
 The second is a high-sensitivity CRP (hs-CRP) assay. The latter can detect a
lower level of CRP to include those that may be of value in measuring the risk
for a cardiac event. The sensitivity is to 0.01 mg/dL. The hs-CRP is useful,
therefore, for assessment of risk for developing myocardial infarction in
patients presenting with acute coronary syndromes.

o Procedure
1. Collect a 4-mL blood serum sample in test tube.
2. Place the specimen in a biohazard bag for transport to the laboratory.

o Reference Values
Normal <0.8 mg/dL (<8 mg/L)

o Clinical Important
1. A positive test indicates active inflammation but not its cause. CRP is an
excellent tool for monitoring disease activity. hs-CRP is a tool for assessing
cardiovascular risk. ESR determination is preferred in chronic inflammation.
2. The CRP has several important advantages over the erythrocyte sedimentation
rate (ESR):

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

 ESR may be influenced by altered physiologic states e.g.: high area.

 ESR is affected by anemia, polycythemia, spherocytosis, macrocytosis,
congestive heart failure, hypergammaglobulinemia or serum protein
changes in which ESR is increased.
 CRP has fewer technical problems, and greater sensitivity to acute
inflammation because of shorter half-life of the protein CRP.
 CRP tends to increase (begins in 4–6 hrs) before rises in antibody titers
and ESR levels.
 CRP levels also tend return to normal before ESR.
 CRP and ESR disappear when steroids or salicylates suppress
inflammatory process.
3. The CRP is elevated in rheumatic fever, RA, myocardial infarction,
malignancy, bacterial and viral infections, and postoperatively (declines after
fourth postoperative day).
 There is some evidence that CRP levels are useful in evaluation of
postoperative recovery. Normally, CRP begins to increase in 4–6 hrs and
reaches a peak value 48-72 hours after surgery and then begins to fall,
entering the reference range 5-7 days after operation. Failure to decrease
significantly after 3 days postoperatively or a decrease followed by an
increase suggests postoperative infection or tissue necrosis. For maximal
information and easier interpretation of the data, a preoperative CRP
level should be obtained with serial postoperative CRP determinations.
 Infection:
Indicate presence of infection (30–35 mg/dL in 80–85% of acute
bacterial infections and Lower <20 mg/dL in viral infections, but not
useful to differentiate bacterial from viral infections).
4. Increased In:
 Inflammatory disorders: RA, rheumatic fever, vasculitis syndromes (e.g.,
hypersensitivity vasculitis).
 Inflammatory bowel disease: Significantly higher in ulcerative colitis
 Chronic inflammatory diseases: Usually <8.0 mg/dL.

29
Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

 Tissue injury or necrosis:

o AMI: CRP appears within 24–48 hrs, peaks at 72 hrs, and becomes
negative after 7 days; correlates with peak CK-MB levels, but CRP
peak occurs 1–3 days later. Failure of CRP to return to normal
indicates tissue damage in heart or elsewhere.
o Ischemia or infarction of other tissues.
 Rejection of kidney or marrow transplant but not of heart transplant.
 Malignant (but not benign) tumors, especially breast, lung, GI tract; >10
mg/dL in one-third of cases. May be a useful tumor marker because a
high CRP is often present when CEA (Carcinoemberyonic antigen) and
other tumor markers are not increased.
 Burns, trauma.
 Infections
Infections in various sites (e.g., newborns; GU, GI, and biliary tracts,
pelvic inflammatory disease [PID], CNS) or due to other organisms (e.g.,
parasites, fungi). In children younger than 6 yrs with meningitis, CRP
>20 mg/dL after 12 hrs (50 mg/dL in older patients) suggests bacterial
rather than viral cause.
5. Not Increased In
 Autoimmune diseases (e.g., SLE, mixed connective tissue disease):
Little or no increase unless infection is present.
 Pregnancy
 Strenuous exercise
 Angina
 Cerebrovascular accident
 Seizures
 Asthma
 Common cold
 Rejection of heart transplant

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

Rheumatoid Arthritis (RA)

o Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a chronic systemic disease whose most prominent
symptom is inflammation of joints. The small joints of the hands and feet,
especially the proximal joints, are most frequently affected; involvement of larger
joints of the extremities is somewhat less frequent, and occasionally non-extremity
joints may be affected. Polyarticular involvement is much more common than
monoarticular disease. Articular disease activity may or may not be preceded or
accompanied by systemic symptoms such as low-grade fever, myalgias, malaise,
and fatigue. Rheumatoid arthritis tends to be a slow, intermittently active,
migratory process that is frequently symmetric. Onset is gradual in 75%-80% of
affected adults and more severe and abrupt in 20%-25%. Inflammatory
involvement of non-articular organs or tissues such as the heart or lungs may
sometimes occur. Patients with RA have increased of the antigen HLA-DR4.
Rheumatoid factor and related diseases are associated with production of a group
of immunoglobulins called rheumatoid factors (RFs) that include IgG, IgM, and
IgA varieties. These immunoglobulins (antibodies) have specificity for IgG. It is
still not certain whether the altered IgG is the cause of the inflammatory
abnormalities in RA or is a body response against the inflammatory process. From
the laboratory standpoint, the most important is the one that is an IgM
macroglobulin. RF combines with its altered IgG antigen in vivo, accompanied by
complement fixation. IgM RF, like other antibodies, is produced by lymphocytes
and plasma cells of B-cell origin. In some persons, especially in infants, IgM
antibody production against some infectious organism not associated with
rheumatoid disease may result in concurrent production of varying amounts of IgM
RF. Outside the body, IgM RF can combine with normal IgG without complement
fixation (in fact, some patient serum contains excess C1q component of
complement, which may cause a nonspecific RF test reaction that can be avoided
by heat inactivation of complement before the test).

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

 Four of the following clinical criteria must be present to diagnose rheumatoid

arthritis:
1. Morning stiffness for at least 6 weeks
2. Pain on motion or tenderness in at least one joint for at least 6 weeks
3. Swelling in at least one joint for at least 6 weeks
4. Swelling in at least one other joint for at least 6 weeks
5. Symmetrical joint swelling with simultaneous involvement of the same
joint on both sides of the body
6. Subcutaneous nodules
7. X-ray changes, including bony decalcification

 Laboratory findings
 Anemia is present in about 40% of men and 60% of women. The anemia
usually appears within 2 months after onset of clinical disease, usually does
not become more severe, and is usually of mild or moderate degree, with a
hemoglobin value less than 10 gm/100 ml (100 g/L) in fewer than 10% of
cases. There are some correlation between the degree of anemia and the
initial severity of illness. The anemia of RA is form of the anemia of chronic
disease, which typically is normocytic and normochromic. However, anemia
in RA is more likely to be hypochromic (reported in 50%-100% of cases),
although microcytosis is found in less than 10% of cases.
 White blood cell (WBC) counts are most often normal or only minimally
elevated. About 25% of RA patients are said to have leukocytosis, usually
not exceeding 15,000/mm3 (15 × 109/L). Leukopenia is found in about 3%
of cases, usually as part of Felty's syndrome (RA plus splenomegaly and
leukopenia).
Anemia and leukocytosis are more common in juvenile-onset RA than
adult-onset RA.
 In active RA, nonspecific indicators of acute inflammation, such as the
erythrocyte sedimentation rate (ESR) and C-reactive protein level, are
elevated in most (but not all) patients. The serum uric acid level is normal

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in most patients. The serum iron and iron-binding capacity level are
generally normal or decreased.
 Serologic tests. Serologic tests are the usual method of laboratory diagnosis
in adult-onset RA. Various types of serologic tests may be set up utilizing
reaction of IgM RF with IgG gamma globulin, differing mainly in the type
of indicator system used to visually demonstrate results.

Rheumatoid Factor (RF) Test

The blood of many persons with RA contains a macroglobulin-type antibody called

rheumatoid factor (RF). Evidence indicates that rheumatoid factors are anti–gamma-
globulin antibodies (IgM).
This test is useful in the diagnosis of RA. It measures RFs (antibodies directed against
the Fc fragment of IgG). These are usually IgM antibodies, but they may also be IgG
or IgA.
In this test, the reagent is the latex particles coated with human IgG (anti-IgM) that
used for the detection of serum antibody called RF (IgM). The slide tests in general
have a slightly greater sensitivity than tube tests but also produce more false positive
results. Therefore, slide tests should be used mainly for screening purposes.

o Procedure
1. Collect a 4-mL blood serum sample in a test tube. Observe standard
precautions.
2. Place specimen in biohazard bag for transport to laboratory.

o Reference Values
Normal 0–20 U/mL or 0–20 kU/L, based on rate nephelometry

o Clinical Implications
1. A positive RF test result often supports a tentative diagnosis of early-onset RA
(e.g., versus rheumatic fever).
2. Absence of RF does not exclude the diagnosis or existence of RA.

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3. RFs frequently occur in a variety of other diseases, such as SLE, endocarditis,

tuberculosis, syphilis, sarcoidosis, cancer, viral infections, Sjögren's syndrome,
and diseases affecting the liver, lung, or kidney as well as in patients who have
received skin and renal allografts.
4. Some patient serum contains excess C1q component of complement, which
may cause a nonspecific RF test reaction that can be avoided by heat
inactivation of complement before the test

o False positive results

Certain diseases, especially those associated with increased IgG
(“hyperglobulinemia”), produce a significantly high number of positive reactions.
These include:
 Collagen diseases
 Sarcoidosis
 Syphilis
 Viral hepatitis and cirrhosis
 Bacterial infections (especially subacute bacterial endocarditis[SBE])
 Old age (as many as 10%-25% positive over age 70).
 Those who have received multiple vaccinations and transfusions.
The incidence of reactive RA tests is higher with the slide than the tube tests. The
percentage of positive reactions in the diseases listed ranges from 5%-40%.
Sjögren's syndrome (75%-96%) and SLE (50%) are most likely to produce false
positive results.

Rose-Waaler test

The original method for detection of RF. Also known as the “sheep cell agglutination
test.” Anti-sheep red blood cell (IgG) antibodies were reacted with tannic acid-treated
sheep RBCs, and then the RF in the patient's serum was allowed to combine with the
antibody IgG coating the sheep cells. Clumping of RBCs indicated a positive test
result.

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

Plotz-Singer latex test

The latex fixation tube test for RA, known also as the “Plotz-Singer latex test,”
currently is considered the standard diagnostic method. The average sensitivity in
well-established clinical cases of adult RA is about 76% (range, 50%-95%). Clinically
normal controls have about 1%-2% positive results (range, 0.2%-4%). Latex slide tests
offer an average sensitivity of approximately 85% (range, 78%-98%), with positive
results seen in approximately 5%-8% of normal control persons (range, 0.2%-15%). It
may take several weeks or months after onset of clinical symptoms, even as long as 6
months, before RA serologic test results become abnormal.

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Placental Hormones
During pregnancy, the placenta secretes estrogens, progesterone, human chorionic
gonadotropin (hCG), and human placental lactogen (hPL).
Estrogens and progesterone, which are not specific to pregnancy. In contrast, hCG
and hPL are fairly specific to pregnancy, but levels may also be altered in
individuals with trophoblastic tumors (e.g., hydatidiform, choriocarcinoma) and
tumors that ectopically secrete placental hormones.

HUMAN CHORIONIC GONADOTROPIN

Human chorionic gonadotropin (hCG) is a glycoprotein that is produced by the
developing placenta. Its presence in blood and urine has been used for decades to
detect pregnancy. hCG can be detected in the urine of pregnant women 26 to 36
days after the first day of the last menstrual period (ie, 5 to 7 days after
conception). Pregnancy tests should return to negative 3 to 4 days after delivery.
The glycoprotein hormones hCG is composed of two different subunits. The α-
subunit is similar in all of the glycoprotein hormones, and the ß subunit is unique to
each hormone. Highly specific assays measure the ß subunit of hCG.
Human chorionic gonadotropin prevents the normal involution of the corpus
luteum at the end of the menstrual cycle and stimulates it to double in size and
produce large quantities of estrogen and progesterone. It is also thought to
stimulate the testes of the male fetus to produce testosterone and to induce descent
of the testicles into the scrotum.

o INDICATIONS FOR HUMAN CHORIONIC GONADOTROPIN TEST

 Early detection of pregnancy (i.e., within 8 to 10 days of conception), especially in

women with a history of infertility or habitual abortion

 Prediction of outcome in threatened abortion (levels below 10,000 mIU/mL are highly

predictive that abortion will occur)

 Suspected intrauterine fetal demise or incomplete abortion as indicated by decreased

levels

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

 Suspected hydatidiform or choriocarcinoma as indicated by elevated levels

 Suspected testicular tumor as indicated by elevated levels

 Support for diagnosing nonendocrine tumors that produce hCG ectopically (e.g.,

carcinoma of the stomach, liver, pancreas, and breast; multiple myeloma; and

malignant melanoma), as indicated by elevated levels

 Monitoring for the effectiveness of treatment for malignancies associated with ectopic

hCG production, as indicated by decreasing levels

o Reference Values
 Normal

o Positive: pregnancy exists

o Negative: nonpregnant state

o Clinical Implications
1. A positive result usually indicates pregnancy.

2. Positive results also occur in

i. Choriocarcinoma

ii. Hydatidiform mole

iii. Testicular and trophoblastic tumors in males

iv. Chorioepithelioma

v. Chorioadenoma destruens

vi. About 65% of ectopic pregnancies

3. Negative or decreased results occur in

i. Fetal demise

ii. Abortion, threatened abortion (test remains positive for 1 week after

procedure)

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o Interfering Factors
1. False-negative test results from

i. Dilute urine (low SG)

ii. Using a specimen obtained too early in pregnancy.

2. False-positive tests are associated with

iii. Proteinuria

iv. Hematuria

v. The presence of excess pituitary gonadotropin

vi. Certain drugs (eg, chlorpromazine, phenothiazines, methadone)

FERTILITY TESTS
Tests include evaluation of amenorrhea, anovulation, sperm count (angiosperm,

oligospermia), hormone testing, hysterosalpingogram, laparoscopy, hysteroscopy,

fertiloscopy, semen analysis, postcoital test, endometrial biopsy, and chromosome karyotype

to exclude Kallmann's syndrome. Hormone testing rules pregnancy in or out (eg, chorionic

gonadotropin, prolactin, luteinizing hormone [LH], follicle-stimulating hormone [FSH],

thyroid-stimulating hormone [TSH], postcoital test, and antisperm antibodies).

o NOTE
A postcoital examination is done to assess cervical mucus and competent sperm motility.

A specimen is obtained from the endocervical canal within 2 to 12 hours of coitus and is

examined for viscosity (stretching to 6 cm is normal) and for ferning effect of estrogen.

The presence of >50% sperm confirms male competence.

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Treponemataceae
Treponema pallidium

‫٘بب ا‬ٚ Syphilis‫ بالسااس ل‬ٚ‫ اٌّعببف‬Venereal disease‫تسبب ٘بب ٖ البكخشَااالالضااشزلالض ااشٌل‬
ٓ‫ عب‬ٚ‫ٌٕتمً عبٓ رفٌبك االحصاا لالنسساٍ ِب يبٍٓلمصاا لولا ُ أ‬ٚ ، ‫لوألدي‬ٚ‫ْلمكخسب أ‬ٛ‫الضشز لد ٌى‬
. ‫ع ِٓ الس‬ٛٔ ‫ْ أي‬ٛ‫٘ ٖلالبكخشَا ال تى‬ٚ ، ) ‫رفٌك األملالضصابت إٌى النسُن (لوألدي‬

: ‫اٌغٍف ِع ٌج ٌّف يثالثة أطىاس‬ ‫السس‬

.‫لل‬Primary Syphilis‫السس لاالبخذائٍل‬ 
.‫ل‬Secondary Syphilis‫السس لالثانىٌلل‬ 
.‫ل‬Tertiary Syphilis‫السس لالثالثٍلل‬ 

 Lab . diagnosis :
 Specimens :
Fluid from chancres , all skin lesion in all stages and genital ulcers .

ً‫ ف فب‬ٛ‫وب‬ِٛ ْٛ‫ي الضاشَ لل ضاااداثلالوُىَات فبئْ ٘ب ٖ البكخشَاا ال تىب‬ٚ ٕ‫يعد س ع ت لٍٍٍة ِٓ ت‬ 

ً ‫بُ أٌاب‬ٌّٙ‫ِٓ ا‬ٚ ، ً ِّٙ ْٛ‫ ٘ فً لائللالعقذلال ُضساوَت ٌى‬ٛ‫و‬ٚ ‫ ا فئْ تحدٌد‬ٌٙٚ ، ‫عُساثلالقشه‬
. ‫ي أي مااداثلحُىَت‬ٚ ٕ‫ عٓ ت‬: ً‫ال‬ٚ‫سؤاي الضشَ أ‬
.Blood‫ فًلالذملل‬T. pallidium ٛ‫و‬ٚ ‫ال ٔستطٍع تحدٌد‬ 

Blood sample ( 3 – 5 ml ) is required for serological testing . The serum or

plasma must not be haemolyzed , lipaemic or contaminated .

‫ريللب لالقساصلالىاقٍل‬ٚ‫ ا فئٔٗ ِٓ اٌاف‬ٌٙٚ ‫تستطٍع اخخشاقلالن ذ‬ٚ ، ‫٘ ٖلالبكخشَا ع ٌٍةلالعذوي‬ 

. ‫الوزس عٕد عٍٍّة وّع العُساث‬ٚ ‫و ٌه أخ الوُطت‬ٚ ‫ل‬Protective gloves

 Morphology :
T. pallidium is thin delicate spiral measuring about 0.2 µm in width and 5
– 15 µm in length with 6 – 12 coils ( the distance between two coils are 1
µm ) . They are actively motile .
، ‫ال فبً مساوتلمصابى تلبصاُجتلماشام‬ٚ ، ‫ فبً الخوااُشاثلالشطبات‬ٙ‫ ٘ ٖ البكخشَا ال ٌّىٓ رؤٌت‬
: ‫اسطة‬ٛ‫إّٔ تش ٘د ي‬ٚ
. Dark – field microscopy‫لالضنهشلرولالوقللالضظ ل‬-
. Fontana stain : ً‫ ِث‬Fluorescent stain‫ استخداَلصبجتلللل‬-
. Burris Indian ink : ً‫ ِث‬Negative stain‫ الصبغلالسالبل‬-

. 42ºC ‫ق‬ٛ‫و ٌهلدسمتلالوشاسة ف‬ٚ ،‫٘ ٖ البكخشَا تمتً بالنسافل‬ 

 Culture :
T. pallidium can not be cultured on artificial media .

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 Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

T. pallidium is a microaerophilic organism , it survives best in 1 – 4 %

oxygen
. ) ‫ٌىٓلحسضًلمخخبشَا ًً فً مضاسعلنسُنُت ( خصٍلاألسانب‬ٚ ‫ودلولطلصسعٍ ِٕ س لخسضُخها‬ٌٛ ‫ال‬

o Serological diagnosis of syphilis :

A person infected with T. pallidium produce two types of antibody :
1. Non – specific antibody that reacts with cardiolipin antigen in
non – specific syphilis test .
2. Specific treponemal antibody that reacts with treponemal
antigen in specific syphilis test .

 Non – specific tests ( cardiolipin antigen test ) include :

1. VDRL ( Venereal Diseases Research Laboratory ) which
is read microscopically .
2. RPR ( Rapid Plasma Reagin ) test which is read
macroscopically .
3. TRUST ( Toluidine Red Unheated Serum Test ) which is
read macroscopically .

: ًّ‫ٕ٘ نلفوىصاثلتستخدَ يدروة ألً تش‬ٚ 

1. Wassermann test.
2. USR ( Unheated Serum Test ) .
3. RST ( Reagin Screen Test ) .

‫ ٌعتّببد عٍببى الخوااشٌ عببٓ األمساااملالضاااادة‬Cardiolipin antigen test‫٘ب ٖ السوىصاااثل‬ 

‫لمع ا ِحابف‬ٚ‫ فً األشخاص اٌّصب يٍٓلبضاشزلالساس ي سبتخداَ مو اى أ‬Reagin ‫اٌّسّ ف‬
: ِٓ
‫اٌ ب ي ٌعت ببف كااتنخنُن ي ٌطفٌمببة السذفُااتلللللللللللللللللللل‬ٚ Cardiolipin – Cholesterol - Lecithin
.‫اسطة العُنلالضنشدة‬ٛ‫ ي‬ٚ‫تمفأ إٌتٍجةلمنهشَا ًً أ‬ٚFlocculation test

‫ ل‬:‫ مالحظتل‬o
‫ ِستخٍصبة ِببٓ أنساانتل‬Phospholipid ٓ‫ ( ِب ف ع ب رف عبب‬Cardiolipin ‫ٌبتُ اسببتخداَ ِب ف‬ 
‫ ب تتحببفر ِببٓ األنساانتلالضصااابت‬ٌٙ ‫ة‬ٙ‫ق اابلالخااشوف ) كااتنخنُن ألٔببٗ ٌعتمببد أْ ٕ٘ ب ن مااادة ِش ب ي‬
‫٘ ٖ الضادةلتحفز عٍى إنخاجلأمساملمااادة‬ٚ ، ٙ‫أٌا ً ِٓ البكخشَا ٔفس‬ٚ ‫ل‬T.pallidium ‫اسطة‬ٛ‫ي‬
. Luetic reagin ‫تسّى‬
. ) ‫ ٌتحسٍٓ قشاءة إٌتٍجة ( الخساعل‬Lecithin ٚCholesterol ‫ٌتُ إض فة‬ 
. Complement ٍُ‫ ٌتحط‬Serum‫ فً ٘ ا االخخباس ٌتُ تسخٍٓ عُستل‬VDRL 
ٓ‫ٌّىب‬ٚ Choline chloride‫لٌبه يئضب فة ماادةل‬ٚ ٍٓ‫ فً ٘ الاالخخباس ٔستغًٕ عٓ اٌتسبخ‬RPR 
. Black carbon particles َ‫ٌتُ استخدا‬ٚ Plasma ٚ‫ أ‬Serum‫استخداَ عُستل‬
Black carbon ٓ‫ يبدالً ِب‬Red particles َ‫ ي ستثٕ ء اسبتخدا‬RPR ‫ ٔفس فحص‬TRUST 
. ‫ضٍح رؤٌة الخساعل‬ٛ‫ ٌت‬particles
. Complement fixation test ‫ ع رف عٓ فوصلحثبُجلالضخضضتل‬Wassermann 

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Practical Immunology & Serology T/ Rashad Saleh Alkhwlany

 Specific treponemal antigen tests :

Non – specific cardiolipin ‫ْ ٔتٍجبةل‬ٛ‫ عٕبدِ تىب‬، ‫٘ ا الخساعل ٌستخدَ إلث ت اإلصاابتلبالساس‬
Non – specific cardiolipin ‫ فبً اٌّفالبً اٌّتبةخفف ِبٓ اإلصاابت لٍب أْ ٔتٍجبةل‬ٚ‫ مىمبات أ‬test
. ‫ْللالبت‬ٛ‫ تى‬test

Specific treponemal antigen tests include :

 TPHA (T. pallidium Haemagglutination Assay ) .
 TPPA (T. pallidium Particle Agglutination Assay ) .
 IC ( ImmunoChromatographic ) rapid strip test syphilis .
 Latex agglutination syphilis fast test .
 FTA – ABS ( Fluorescent Treponemal Antibody Absorption )
test .
 Various EIA ( Enzyme Immunoassays ) .

‫ ي سببتخداَ أنخُنُساااث‬IgG ٓ‫عبب‬ٚ IgM‫٘ ب ٖ السوىصاااث تعتّببد عٍببى اٌتحببفي عببٓ األمساااملالضاااادةل‬ٚ
‫ حبُبااااثلمااانلالنُالحاااُنل‬ٚ‫ أ‬Latex‫ حبُبااااثل‬ٚ‫تغطبببً كشَااااثلالاااذملالوضاااشاء أ‬ٚ ‫ِستخٍصبببة ِبببٓ البكخشَاااا‬
. Gelatin

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