2. Serology
Serology is the study of immune reaction in human blood.
These immune system is body defense mechanisms against
disease-causing organisms.
The principle involved with serology is the antibody-antigen
reaction.
Antigen is the substance which "provokes" the body to produce
antibodies.
Antibody is the substance which fights the invading organism.
Tassew Tefera
2
3. Serology…
The samples are analyzed in a laboratory to detect
either antigen or antibody.
There are several serology techniques that can be
used depending on the suspected antibodies.
Serology techniques include agglutination,
precipitation, complement-fixation, fluorescent
antibodies, and others.
Tassew Tefera
3
4. What Abnormal Results Mean?
As the disease gets worse, more antibodies will be
present.
If antibodies are found, you may:
Have a current infection
Have been infected in the past
Have immunity to a certain organism and are unlikely to
become sick
Tassew Tefera
4
5. Serologic diagnostic methods
Infectious diseases can be definitively diagnosed in only
three ways:
1. By visualizing the agent directly in clinical material
obtained from the patient.
2. By detecting a specific product of the infectious agent
in clinical material obtained from the patient.
3. By detecting an immunological response specific to the
infecting agent in the patient´s serum(antibody).
Tassew Tefera
5
6. Antigen - antibody reactions
Antigen - antibody reactions are the methods by which
antigens and antibodies are measured.
Ag - Ab reaction can be visualized in different ways according
to the type of the antigen, conditions of the reaction and the
medium the reaction takes place in.
When an antibody combines with a corpuscular antigen
(forming part of a cell - e.g. bacteria, virus, blood cell or inert
part with bound antigen) the cells agglutinate (form
clumps).
When an antibody combines with a non corpuscular
antigen (toxin, enzyme, microbial extract) a precipitate is
formed.
Tassew Tefera
6
7. When an antibody combines with an antigen
which forms part of the surface of certain cells
(e.g. red blood cells) the cells are lyzed.
Other types of serological reactions used for
antigen or antibody detection are used in
microbiology - e.g. immunofluorescent test,
EIA, ELISA (enzyme immunosorbent assays),
RIA (radioimmunoassay), which use specific
detection systems.
Antigen - antibody reactions…
Tassew Tefera
7
8. Serological tests can be used in two ways:
1. A known antibody can be used to detect and measure
an unknown antigen
2. A known antigen can be used to detect and measure an
unknown antibody.
Serologic tests are performed as:
a) Qualitative tests - find out presence of antibodies or
antigens in the serum.
b) Quantitative tests – conducted by serial dilutions.
Quantitative results are normally expressed in terms
of the titer of the serum.
Antigen - antibody reactions…
Tassew Tefera
8
9. Types of serologic reactions.
A. Agglutination
A corpuscular antigen (agglutinogen) is
agglutinated with specific antibody (agglutinin).
Agglutination can be read either visually or by
microscope.
Presence or absence of clumping is noted.
Tassew Tefera
9
10. I) Direct agglutination
Tests the presence of unknown microbial antigen structure in the
sample.
Slide agglutination is most often used method.
Specific antisera are prepared by immunization of animals with
bacterial strain is used as reagent.
II) Indirect agglutination
Tests the presence of antibodies in the serum.
Known antigen is used as a reagent.
E.g., Widal reaction for diagnosing typhoid and paratyphi.
Types of serologic reactions...
Tassew Tefera
10
11. B. Precipitation
When a specific antibody - precipitin combines with a
colloidal antigen - precipitinogen in solution or in gel it
forms precipitation.
Antigen and antibody are fully combined at the
equivalence zone.
State of optimum relation between antigen and antibody to
carry out the reaction called flocculation.
Tassew Tefera
11
12. Some of the serologic tests include:
Widal weilflex
Human immunodeficiency virus (HIV)
Measles
Rubella
Syphilis
Viral hepatitis (A,B,C etc )
Pregnancy tests (HCG)
Malaria RDT
Blood grouping
Anti-stereptolysin O(ASO)
Tassew Tefera
12
13. Syphilis
Syphilis is venereal disease caused by infectious organism called
Treponema pallidum.
When syphilis is present, the body produces Reagin antibody.
There are other disorders which can also produce Reagin.
Therefore, when the person has a positive test for Reagin, further
testing is needed to determine if the person has syphilis or some
other disorder such as leprosy, tuberculosis, malaria, etc.
False positive result is common.
Tassew Tefera
13
14. Non specific Tests for syphilis:
a) VDRL (Venereal Disease Research Lab)
These test is rapid and screening tests.
If positive (reactive): needs further analysis
If negative (non-reactive): syphilis absent.
Utilizes an antigen which consists of cardiolipin, cholesterol
and lecithin.
Serum must be heated to 56 C for 30 minutes to remove anti-
complementary activity which may cause false positive, if serum
is not tested within 4 hours must be reheated for 10 minutes.
VDRL used primarily to screen cerebral spinal fluid.
Results reported as reactive/non-reactive.
Tassew Tefera
14
15. b) RPR(rapid plasma reagin)
General screening test.
Cannot be performed on CSF.
The VDRL cardiolipin antigen is modified with choline
chloride to make it more stable and is attached to charcoal
particles to allow visual reading, the antigen comes prepared
and is very stable.
Serum or plasma may be used for testing, serum is not heated.
Non specific Tests for syphilis…
Tassew Tefera
15
16. Specific Treponemal Tests
1. Treponema pallidum Hemagglutination (TPHA)
Adapted to micro techniques.
Tanned sheep RBCs are coated with T. pallidum antigen.
Agglutination of the RBCs is a positive result.
2. Fluorescent Treponemal antibody absorption test (FTA-ABS)
One of the most used confirmatory test.
Highly sensitive and specific, but time consuming to perform.
3. ELISA
Tubes coated with T. pallidum antigen.
Antibody in serum attaches to antigen.
Detectable color change occurs.
Tassew Tefera
16
17. Typhoid and Paratyphoid Fever
Typhoid and Paratyphoid Fever is caused by Salmonella
species.
Some times it is termed as enteric fever since they
colonize the intestine.
Medically important Salmonella species are S.typhi (typhoid
fever), S.paratyphi A and B (paratyphoid fever).
Tassew Tefera
17
18. Salmonella have three different antigenic structures.
a) O- antigen (somatic antigen)
It is lipopolysaccharide of the outer membrane.
b) H-antigen (flagellar antigen)
H-antigen is protein, which makes the peritrichous flagella.
c) Vi- Antigen:
This is the antigen that determines the virulence, the ability to
cause disease of the organism.
Typhoid and Paratyphoid Fever
Tassew Tefera
18
19. Widal test
Widal test is an agglutination test for the detection of
agglutinins (antibodies) for H and O antigen for salmonella in
patients with typhoid fever and paratyphoid fever.
Is an agglutination reaction that can performed on slide or in
test tubes.
Principle: The patient serum is tested for those anti-O and
anti-H antibodies against commercially prepared O and H
antigen suspensions. Visible agglutination is seen if the anti
salmonella antibody present.
Tassew Tefera
19
21. Rickettsial Disease
Rickettsia is obligate intercellular parasite.
Based on their antigenic structure, the genus Rickettsia has
been divided into three main groups:
Typhus group (R. prowazeki, R. typhi),
Scrub typhus group (R. tsutsugamushi),
Spotted fever group (R. conori, R. siberica, R. rickettsi).
Rickettsia causes typhus
Tassew Tefera
21
22. Weilfelix test
A Weil Felix test is a type of agglutination test most
commonly used to diagnose typhus.
The reaction is based on similarity of particular antigenic
determinant, which occur in most species of pathogenic
rickettsia and in the OX-19, OX -2 strains of Proteus
vulgaris and OX-K strains of Proteus mirabilis.
In other word, Proteus antigen is used to detect
rickettsial antibody.
This could be an example of hetrophile antigen
antibody reaction.
Weil Felix test has similar principle and procedure with
Widal test.
Tassew Tefera
22
23. Serology of Hepatitis virus
Hepatitis virus causes inflammation of the liver.
There are 5 distinct hepatitis viruses, A, B, C, D and E
which cause chronic and acute hepatitis.
Chronic carrier state may develop and may result in liver
failure due to cirrhosis, hepatocellular carcinoma, or
fulminant hepatitis.
Tassew Tefera
23
24. Hepatitis A virus (HAV)
Transmitted by fecal-oral route.
First and most clinically useful is IgM antibody to HAV.
IgM indicates acute infection and disappears in 3-6
months, replaced by IgG anti-HAV.
IgG peaks during convalescence and may remain
detectable for life.
Immuno-chromatographic test are available for serologic
diagnosis of HAV.
Tassew Tefera
24
25. Hepatitis B (HBV)
Route of infection is usual parenteral, direct inoculation,
blood transfusions, maternal to fetus or newborn.
Laboratory diagnosis involve the detection of three marker
systems.
1. Hepatitis B surface antigen (HBsAg) is the first to appear,
appears 2 to 4 weeks during late incubation, marker of choice
for recent infection.
2. Anti-hepatatis B surface antigen (anti-HBs) is the last
antibody to appear, may persist for life.
3. IgM antibody to hepatitis B core antigen (anti-HBc) may be
the only detectable marker during the core window, differentiates recent
infection from chronic carrier state.
Tassew Tefera
25
26. Hepatitis C Virus
Clinically and epidemiologically similar to HBV.
Sixty to 70% of HCV patients will develop chronic
hepatitis, 10-20 % cirrhosis and 15% hepatocellular
carcinoma.
HCV and HBV may be present as co-infections.
Present of HCV antibodies only indicates present or
past infection.
Tassew Tefera
26
27. Human Immunodeficiency Virus (HIV)
Causes Acquired Immunodeficiency Syndrome (AIDS).
Characteristics of the virus
Icosahedral, enveloped virus of the lentivirus
subfamily of retroviruses.
Retroviruses transcribe RNA to DNA.
Tassew Tefera
27
28. HIV test algorism
1) KHB
If the test for KHB is non reactive then the test is reported
negative because the test is very sensitive.
If the test for KHB is reactive we proceed to the next step or test
to which is very specific.
2) Stat pack
The test is more specific than the former one.
If the test result is reactive and agree with KHB test result; then
the test is reported positive but if the test result is negative and not
agree with KHB test result we proceed to third test.
3) Unigold (tie-breaker)
The test is more specific and sensitive.
Any results of the test is reportable. i.e. if reactive the test is
reported positive and if non reactive reported as negative.
Tassew Tefera
28
30. Pregnancy tests
Human chorionic gonadotropin (HCG) is a hormone
secreted by placenta during pregnancy.
Its production stimulates secretion of progesterone by the
ovary.
Human chorionic gonadotrophin appears in urine, blood and
amniotic fluid.
The serum and urine level rise rapidly during gestation,
reaching a peak at six to eight weeks, after which there is a
steady decline.
Tassew Tefera
30
31. Immunologic test of pregnancy
Immunologic test could be qualitative and quantitative.
Qualitative estimation of HCG in urine is used for early
detection and confirmation of pregnancy.
The test is less expensive and quicker test.
Sample could be urine and blood.
Tassew Tefera
31
32. Factors Affecting Pregnancy Tests
False Negative may occur in conditions like:
test is performed too early.
Urine too diluted -falsely low levels of HCG.
Ectopic pregnancy.
False positive may occur in conditions like:
Proteinuria and hematuria
Test after abortion
Bleeding
Turbidity of urine
Bacterial contamination
Tassew Tefera
32
