SEROLOGY

PRECIPITATION
Involvescombining
soluble antigen with
soluble antibody to
produce insoluble
complexes that are
visible.
PRECIPITATION
Zone of equivalence - maximum precipitation
occurs when the concentration of the antigen
and antibody are about equal
PRECIPITATION
Prozone occurs when excess amount of
antibody is present, and the antigen and
antibody do not combine to form precipitates.
This results in a false negative result
PRECIPITATION
Postzone occurs when excess amount of antigen
is present, and the antigen and antibody do not
combine to form precipitates. This results in a
false negative result.
FLUID-PHASE
PRECIPITATION
Turbidimetry Nephelometry
the measurement a direct measure of
of light light scattered by
transmitted particles suspended
through a in solution.
suspension of Nephelometry is
particles. more sensitive
than
turbidimetry.
PRECIPITATION REACTIONS IN
AN AGAR GEL
Radial immunodiffusion (RID)
RADIAL IMMUNODIFFUSION (RID)
 Mancini or end-point
method: In this technique,
antigen is allowed to diffuse
to completion, and when
equivalence is reached,
there is no further change
in the ring diameter. This
occurs between 24 and 72
hours. The square of the
diameter is then directly
proportional to the
concentration of the
antigen.
RADIAL IMMUNODIFFUSION (RID)
 Fahey and KINETIC ENDPOINT
McKelvey method or
kinetic method: uses
measurements taken
before the point of
equivalence is reached.
In this case, the
diameter is
proportional to the log
of the concentration.
Readings are taken at
about 18 hours.
PRECIPITATION REACTIONS IN
AN AGAR GEL
Double immunodiffusion (Ouchterlony)
PRECIPITATION REACTIONS IN
AN AGAR GEL
Rocket immunolectrophoresis
PRECIPITATION REACTIONS IN
AN AGAR GEL
Countercurrent immunoelectrophoresis (CIE)
PRECIPITATION REACTIONS IN
AN AGAR GEL
Immunoelectrophoresis
the source of the antigens is serum, which is electrophoresed to
separate out the main protein fractions; then a trough is cut in the gel
parallel to the line of separation. Antiserum is placed in the trough, and
the gel is incubated for 18 to 24 hours. Double diffusion occurs at right
angles to the electrophoretic separation, and precipitin lines develop
where specific antigen–antibody combination takes place. These lines
or arcs can be compared in shape, intensity, and location to that of a
normal serum control to detect abnormalities.
PRECIPITATION REACTIONS IN AN AGAR GEL
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
 AGGLUTINATION
Occurs when particles in
suspension clump together due to
antibody-antigen interaction
IgM and IgG antibodies
participate in agglutination
reactions
IgM has more antigen binding
sites, it agglutinates more quickly
CLASSIFICATION OF
AGGLUTINATION REACTIONS
Direct agglutination
CLASSIFICATION OF
AGGLUTINATION REACTIONS
Passive agglutination
CLASSIFICATION OF
AGGLUTINATION REACTIONS
Reverse passive agglutination
CLASSIFICATION OF
AGGLUTINATION REACTIONS
Coagglutination
CLASSIFICATION OF
AGGLUTINATION REACTIONS
Agglutination inhibition
ANTIHUMAN GLOBULIN TEST
 Direct antiglobulin test is used to demonstrate in vivo
attachment of antibody or complement to an individual’s
red blood cells.
ANTIHUMAN GLOBULIN TEST
 Indirect antiglobulin test, or indirect Coombs’ test, is
used to determine the presence of a particular antibody
in a patient, or it can be used to type patient red blood
cells for specific blood group antigens.
COMPLEMENT FIXATION
LABELED REACTIONS
A label producing a
measurable end product is
attached to an antibody or
antigen
Labels include
fluorochromes, enzymes,
chemiluminescent molecules
and radionuclides
LABELED REACTIONS
IMMUNOFLUORESCENCE
 Antibodies labeled with fluorescent dye
are used to detect an antibody or
antigen.
 Commonly used fluorochromes include
fluorescein isothiocyanate (FITC), R-
phycoerythrin, quantum red,
tetramethyl-rhodamine isothiocyanate,
Texas red, phycocyanin, acridine
orange, and propidium iodide.
IMMUNOFLUORESCENCE
DIRECT IMMUNOFLUORESCENCE
 Conjugated (fluorescent labeled) reagent
antibody reacts with an antigen in a clinical
sample to form antigen-antibody complex
IMMUNOFLUORESCENCE
INDIRECT IMMUNOFLUORESCENCE
 Antigens react with unlabeled antibody
forming an antigen-antibody complex that is
then complexed with a labeled antihuman
antibody, creating an antibody-antigen-
antibody “sandwich”
IMMUNOFLUORESCENCE

BIOTIN-AVIDIN
IMMUNOFLUORESCENCE
 Thisis an indirect assay in which the
detection system is modified by using a
biotin-labeled antibody followed by avidin-
labeled fluorochrome. This extra step
increases the specificity and sensitivity of
the assay
LABELED REACTIONS
ENZYME-LINKED IMMUNOSORBENT
ASSAYS (ELISA)
 Enzyme-labeled reagents are used to detect
antigens or antibodies. Enzyme must be stable,
specific and cannot bind to antigen or antibody
independently.
 Typical enzymes that have been used as labels in
colorimetric reactions include horseradish
peroxidase, glucose-6-phosphate dehydrogenase,
alkaline phosphatase, and β-D-galactosidase.
SYSTEMIC LUPUS ERYTHEMATOSUS
Chronic, non-infectious
inflammatory disease
involving many organs
Disease is more likely to
occur in women than
men and in blacks than
whites.
Tissue injury is caused
by autoantibodies and
immune complexes
deposited in the tissues.
ANTI-NUCLEAR ABTIBODIES
(ANA)

Associated with SLE, mixed

connective tissue disease
(MCTD), and RA
Techniques used to detect
ANA: agglutination, indirect
immunofluorescence, and
enzyme immunoassay
INTERPRETATION OF INDIRECT
IMMUNOFLUORESCENT RESULTS
Homogenous (solid, diffuse)
INTERPRETATION OF INDIRECT
IMMUNOFLUORESCENT RESULTS
 Peripheral (ring, membranous, shaggy or
thread)
INTERPRETATION OF INDIRECT
IMMUNOFLUORESCENT RESULTS

Speckled (mottled)
INTERPRETATION OF INDIRECT
IMMUNOFLUORESCENT RESULTS

Nucleolar
INTERPRETATION OF INDIRECT
IMMUNOFLUORESCENT RESULTS
Anti-centromere antibody (ACA)
ANTIBODIES TO DS-DNA
 One particularly sensitive assay
for ds-DNA is an
immunofluorescent test using
Crithidia luciliae, a
hemoflagellate, as the substrate.
This trypanosome has circular ds-
DNA in the kinetoplast. A positive
test is indicated by a brightly
stained kinetoplast with patient Immunofluorescence

serum and an antibody conjugate.

staining pattern of anti-
dsDNA antibodies on C.

This test has a high degree of

luciliaesubstrate. The
kinetoplast located near

specificity, although it is less

the flagellum is stained,
indicating the presence

sensitive than other FANA tests.

of anti-dsDNA
antibodies in a person
with SLE
CRITHIDIA LUCILIAE
RHEUMATOID ARTHRITIS
Chronic, non-infectious,
systemic inflammatory
disease
Women are affected 2 - 3
times more often than men
RHEUMATOID FACTOR
 RF is an anti-antibody, typically
IgM, that binds to the Fc portion
of abnormal IgG
 Usually detected by latex
agglutination. Patients serum is
mixed with IgG-coated latex
particles. Agglutination indicates the
presence of RF.
SYPHILIS SEROLOGY
 Syphilis is caused by Treponema
pallidum subsp. pallidum, a
spirochete
 Sexual transmission is the primary
mode of dissemination, and this
occurs through abraded skin or
mucous membranes coming in
contact with an open lesion.
 Congenital infections can also occur
during pregnancy
DISEASE STAGES
 Primary syphilis
 The initial lesion is
painless, nonbleeding
ulcer called a
chancre.
 Antibodies are
produced 1 - 4 weeks
after the chancre
appears.
 Darkfield analysis of
Example of a primary
syphilis sore.
lesions demonstrates
spirochetes.
DISEASE STAGES
 Secondary syphilis
 Spirochetes are
present throughout
the body during this
stage.
Examples of a
secondary palmar rash

 Ulcers develop on
mucous membranes.
 Serologic tests are
positive.
DISEASE STAGES
Latency

Stage of syphilis with

no signs or symptoms
Nontreponemal and
treponemal serologic
tests are positive
DISEASE STAGES
 Tertiary syphilis
 Symptoms occur 2 - 40 years after
initial infection.
 Gummas (syphilis lesions due to
hypersensitivity reaction to treponemal
antigens) are found throughout the
body.
 Three major manifestations:
gummatous syphilis, cardiovascular
disease, and neurosyphilis.
CONGENITAL SYPHILIS
 Treponema pallidum can cross the
placenta during any stage of the
disease.
 Infection of the fetus causes late
abortion, stillbirth, neonatal death,
neonatal disease or latent infection.
 The outcome depends on the stage of
the mother’s disease - primary or
secondary syphilis causing the worst
outcome - and the age of the fetus at
infection.
DIRECT DETECTION
 T. palllidum is detected
using darkfield
microscopy or silver
stain
 Fluorescent Antibody
Testing of a Specimen
 The use of a fluorescent-
labeled antibody is a
sensitive and highly
specific alternative to
dark-field microscopy. Darkfield micrograph of
Treponema pallidum.
SEROLOGICAL TESTS
Treponema pallidum
infection cause the host to
produce nonspecific
antibody, reagin, and
specific treponemal
antibodies
SEROLOGICAL TESTS
 Nontreponemal antigen tests
 Detect reagin and are only used for screening because
of this antibody will cross-react with similar antigens
present in SLE, autoimmune disease, pregnancy and
chronic infections such as hepatitis.
 The percentage of false positives in these tests is
high, so all reactive results must be confirmed
using a test that detects antibodies specifically
directed at T. pallidum, so-called treponemal
antigen tests
 Treponemal antigen tests

 Use T. pallidum cells as the antigen source. These

assays are highly specific.
NONTREPONEMAL ANTIGEN TESTS
VDRL test
•This test measure the antibody (reagin) a
patient has formed against cardiolipin
•Tests are read microscopically for
flocculation
•Mainly limited to use on CSF now, this is
the only serologic test approved for testing
CSF
RPR test
•The assay uses CDRL antigen with
charcoal particles. The charcoal is trapped
in the reaction, which allows the reaction
to be seen macroscopically.
•The test can be qualitative or
semiquantitative. Dilutions are made to
semiquantify the amount of antibody
present.
TREPONEMAL ANTIGEN TESTS
Fluorescent treponemal
antibody absorption
(FTA-ABS) test
Agglutination tests

Particle agglutination tests

such as the Serodia TP-PA test
have largely replaced
microhemagglutination tests.
HUMAN IMMUNODEFICIENCY
VIRUS SEROLOGY

HIV is a member of the

family Retroviridae
Two serogroups: HIV-1 is the
predominant strain, and is
found worldwide. HIV-2 is
limited to West Africa.
STRUCTURAL GENES
 The genome of HIV includes three main structural genes— gag, env,
and pol—and a number of regulatory genes.
 The env gene
 Codes for the glycoproteins gp160, gp120, and gp41, which are
found in the viral envelope
 The gag gene
 Codes for p55, a precursor protein from which four core structural
proteins are formed: p6, p9, p17, and p24.
 The pol gene
 Codes for enzymes necessary for HIV replication, namely p66 and
p51 - these are subunits of reverse transcriptase
 p31, or integrase, which mediates integration of the viral DNA
into the genome of infected host cells
 p10, a protease that cleaves protein precursors into smaller active
units.
 These proteins are located in the core, in association with the HIV
RNA.
STRUCTURAL GENES
CD4 T-CELL ENUMERATION
 CDC has used CD4 T-cell counts to classify
patients into various stages of HIV infection,
with those whose counts are below 200/μL
being classified as having AIDS.
Absolute # CD4 T cells
= WBC count X % Lymphocytes
X % CD4 T cells
 The absolute CD4 T-cell count is then
compared to the reference range, which is
typically from 500 to 1300 cells/μL peripheral
blood.
DETECTION OF HIV
ANTIBODIES

The standard screening

method for HIV antibody
has been the ELISA, and
the standard
confirmatory test is the
Western blot.
INTERPRETATION OF WESTERN
BLOT RESULTS
Most laboratories follow the criteria of the
Association of State and Territorial Public Health
Laboratory Directors and CDC. According to
these criteria, a result should be reported as
positive if at least two of the following three

p24, gp41, and

bands are present:

gp120/gp160
INTERPRETATION OF WESTERN
BLOT RESULTS

Western blot,
showing results
from a negative
sample, an
indeterminate
sample, and a
positive sample.
 HEPATITIS A
LABORATORY TESTS
 Aspartate aminotransferase (AST) and especially
alanine aminotransferase (ALT) levels are
increased and peak before jaundice occurs
 Anti-HAV antibodies are present at onset of
symptoms and for years afterward.

SEROLOGICAL/MOLECULAR MARKERS
IgM anti-HAV Acute hepatitis A
Total anti-HAV Immunity to hepatitis A
HAV RNA Detection of HAV in clinical, food, or water
samples
HEPATITIS B
LABORATORY TESTS
 The first marker that appears at the end of the incubation
period is HBsAg..
 Soon after HBsAg is detected in the blood, HBeAg appears.
 The next marker to appear is anti-HBc, which begins to rise a
couple weeks into the acute infection.
 The anti-HBc IgM antibody peaks a few weeks after the acute
infection stage, then disappears in about 6 months during
recovery. Anti-HBc IgG will persist for several decades.
 At the end of the acute stage, anti-HBe begins to rise and
peaks about 2 - 16 weeks later.
 The last marker to appear is anti-HBs. It appears at the end
of the acute stage and the beginning of the recovery stage. Its
concentration peaks, then plateaus during recovery and never
disappears. Presence of this antibody indicates immunity.
 In chronic infections, patients do not produce anti-HBs, and
HBsAg persists. These patients become chronic carriers of the
virus and are at risk for cirrhosis and hepatocellular carcinoma.
 HEPATITIS B

SEROLOGICAL/MOLECULAR MARKERS
HBsAg Active hepatitis B infection
HBeAg Active hepatitis B infection with high degree of infectivity
IgM anti-HBc Current or recent acute hepatitis B
Total anti-HBc Current or past hepatitis B
Anti-HBe Recovery from hepatitis B
Anti-HBs Immunity to hepatitis B
HBV DNA Acute, atypical, or occult hepatitis B, viral load may be used to monitor
effectiveness of therapy
 HEPATITIS C
LABORATORY TESTS
 Anti-HCV is diagnostic of HCV infection.

 Anti-HCV IgM does not distinguish between

acute and chronic disease because both IgM and
IgG antibodies are detectable for years.
 ELISA tests have false positive results, so the
best test to use for diagnosis is an immunoblot
assay.

SEROLOGICAL/MOLECULAR MARKERS
Anti-HCV Current or past hepatitis C infection
HCV RNA Current hepatitis C infection; viral load may be used
to monitor effectiveness of therapy; also used to
determine HCV genotype
 DELTA HEPATITIS
LABORATORY TESTS
 Only HBsAg positive patients are tested for HDV.
 HDV-Ag is the first marker to appear, detectable
about 1 - 4 days before symptoms start.
 IgM anti-HDV appears next followed by low levels of
IgG anti-HDV.
 The switch to high levels of IgG anti-HDV indicates
past HDV infection.

SEROLOGICAL/MOLECULAR MARKERS
IgM anti-HDV Acute or chronic hepatitis D

IgG anti-HDV Recovery from hepatitis D or chronic hepatitis D

HDV RNA Active HDV infection; viral load may be used to monitor
effectiveness of therapy
 HEPATITIS E
LABORATORY TESTS
 Acute infection is indicated by the presence of
IgM anti-HEV, which is detectable at clinical
onset but declines rapidly in the early recovery
period.
 Immunoassays for IgG anti-HEV, which persists
longer, may be performed to determine previous
exposure and seroprevalence of the infection.

SEROLOGICAL/MOLECULAR MARKERS
IgM anti-HEV Current hepatitis E infection

IgG anti-HEV Current or past hepatitis E

HEV RNA Current hepatitis E infection

EPSTEIN-BARR VIRUS SEROLOGY
EPSTEIN-BARR VIRUS (EBV)
 DNA virus
 Member of the herpes virus group
 Transmission is through saliva
DISEASES
Infectious mononucleosis (IM)
 A disease of the reticuloendothelial system
 Common findings are lymphocytosis, with reactive
(atypical) lymphocytes, and enlarged cervical lymph
nodes
 Infected individuals have abnormal white blood cell
differentials and sometimes abnormal liver function
tests
Burkitt’s lymphoma
 Malignant neoplasm of B lymphocytes
Nasopharyngeal carcinoma
 Nasopharyngeal squamous cell carcinoma
LABORATORY TESTS
 Heterophile antibodies
 Paul-Bunnell presumptive test
Screening test to detect heterophile

antibodies that is not specific to IM.

Serial dilutions of serum are

incubated with a 2% suspension of

sheep RBCs. Agglutination is a
positive reaction.
A titer of 28 or less is normal

Titer of >56 is suggestive of IM

LABORATORY TESTS
Davisohn differential test
 Differentiates among three different heterophile antibodies based
on adsorption onto beef RBCs and guinea pig kidney cells.

TYPE OF HETEROPHILE ADSORBED BY ADSORBED BY

ANTIBODY GUINEA PIG KIDNEY BEEF ERYTHROCYTES
CELLS
Forssman Yes No
Serum sickness Yes Yes
Infectious No Yes
mononucleosis

 The absorbed agglutinins can be removed by centrifugation and

aspiration of resultant fluid, which is then tested with sheep
erythrocytes
LABORATORY TESTS
Monospot
 Uses the principle of agglutination of horse erythrocytes by
heterophil antibody present in infectious mononucleosis
LABORATORY TESTS
EBV-specific tests
 Can detect anti-viral capsid antigen (VCA), anti-early
antigen/diffuse (EA/d), anti-early antigen/restricted
(EA/r), and anti-Epstein-Barr nuclear (EBNA)
antibodies
 EBV-specific antibodies can be detected by ELISA,
immunofluorescnet assay and immunoblot techniques
SIMPLE DILUTIONS
 A dilution involves two entities: the solute,
which is the material being diluted, and the
diluent, which is the medium making up the
rest of the solution.
 The relationship between these two is expressed
as a fraction
 For example, a 1:20 dilution implies 1 part of
solute and 19 parts of diluent. The number on
the bottom of the fraction is the total volume,
reached by adding the volumes of the solute and
diluents together.
1/Dilution = Amount of Solute/Total Volume
COMPOUND DILUTIONS
 To calculate a compound dilution problem, the
first step is to plan the number and sizes of
simple dilutions necessary to reach the desired
end point.
 To use the preceding example, a 1:500 dilution
can be achieved by making a 1:5 dilution of the
original serum, a 1:10 dilution from the first
dilution, and another 1:10 dilution. This can be
shown as follows:
Serum:
1:5 dilution 1:10 dilution 1:10 dilution
0.1 mL serum 0.1 mL of 1:5 dilution 0.1 mL of 1:10 dilution
0.4 mL diluent 0.9 mL diluent 0.9 mL diluent
SERIAL DILUTIONS
 Serial dilutions are a set of dilutions in
which the dilution factor is exactly the
same at each step.
 This is commonly done to determine the
strength or titer of a particular antibody
in patient serum as a part of the diagnosis
of a disease state.
 The most common serial dilution is a
doubling dilution, in which the amount of
serum is cut in half with each dilution.
SERIAL DILUTIONS
For example, six test tubes can be set up with 0.2
mL of diluents in each.
If 0.2 mL of serum is added to the first tube, this
becomes a 1:2 dilution.
0.2 mL serum/0.2 mL serum + 0.2 mL diluents
= 0.2 mL/0.4 mL = 1/2
Then when 0.2 mL of the 1:2 dilution is added to 0.2
mL of diluent, a 1:4 dilution is obtained. The final
dilution is obtained by counting the number of tubes
and setting up a multiplication series in which the
original dilution factor is raised to a power equal to
the number of tubes. In this example, if the first tube
contains a 1:2 dilution, the dilution in tube number
six is:
½ x ½ x ½ x ½ x ½ x ½ = 1/64