BASIC SEROLOGICAL TESTS

By: Amare Kiflie (BSc, MSc.)

Oct, 2017
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Serology
Definitions

Serology: is the scientific study of blood Serum.

In practice, the term usually refers to the diagnostic

identification of antibodies in the serum.
Such antibodies are typically formed in response to an
infection (against a given microorganism)
against other foreign proteins, in response to a mismatched
blood transfusion, or to one's own proteins (in instances of
autoimmune disease).
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Serology
Immune system: the structures, cells, and soluble constituents that allow
the host to recognize and respond to foreign stimulus.

Secondary immune response: the cellular and humoral events that occur
when an antigen is encountered for a second or subsequent time.

Antigen (Ag): is a substance (self/non self)which elicits immune response

(Antibody production).
Antibody: A specific protein which is produced in response to an
immunogen and which specifically reacts with an antigen.

Serum: the fluid portion of the blood after the blood clots.

Specificity: the special affinity between an antigen and its corresponding

antibody.
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Serology
Antibody molecules combine reversibly with antigens to form immune
complexes.
Ag + Ab Ag.Ab complex
The detection and measurements of these reactions form the basis of
serology a sub discipline of immunology.

There for Serology is the science of measuring antibody or antigen in body

fluids.

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 Application of serologic tests
Diagnostic purposes
When infection is suspected
autoimmune disorders
Checking an individual's blood type, pregnancy etc.
To diagnose patients with certain immune deficiencies associated with the
lack of antibodies.
In such cases, tests for antibodies will be consistently negative.
To follow treatment out comes.

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A. Syphilis serology
Venereal disease caused by Treponema palladium,
Transmitted by
Direct sexual contact,
Lesion contact
Congenitally and blood transfusion .

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Stages Of Syphilis
1. Primary syphilis:
Development of primary inflammatory lesion
(chancre).
Lesion appears at the point of contact/expossure,
Usually the genitalia i.e. Penis, vagina or rectum
It can also develop on lips or hands
Diagnosed by : - characteristics chancre
- Positive serologic test
- Dark field microscopy
Primary latent stage; all external signs disappear but blood
test is positive
The Lesion arent always noticeable.
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2. Secondary syphilis

Symmetrical generalized rash

Reddish-pink and non-itchy rash on the trunk and

extremities.

Palms of the hands and the soles of the feet

Diagnosed by - skin rash and Positive serologic test

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3. Latent syphilis
Asymptomatic
Diagnosis is only made by serology .

4.Tertiary syphilis
Soft, tumor-like balls of inflammation known as
granulomatous lesion in skin, bone, liver, CNS etc.
Diagnosis: serologic tests.

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Antibodies in syphilis :
1. Non treponemal antibody ( reagin antibody ):
Are auto antibodies against tissue antigens (cardiolipin or
lipoidal) or lipoprotein.
Are non specific and type of IgM and IgA class
Are produced in patients with other infectious disease but can be
positive in syphilis test (measles , hepatitis, leprosy , malaria and
tuberculosis ).
Identified by VDRL and RPR tests.

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2. Treponemal antibody (specific antibody)
Are antibodies directed against T. pallidum antigens
To the spirochete it self or its components
Are IgG type .
Are diagnosed by TPPA (treponema pallidum particle
agglutination), TPHA (Treponema palladum hem agglutination)
FTA-abs and treponemal immuno-chromatographic tests.

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Specimen
Serous fluid from chancres Motile T. pallidum
secondary skin lesion

Blood/Serum/ plasma Serological tests

Cerebrospinal fluid

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Non-treponemal (Non-specific ) Tests
1. Venereal Disease Research Laboratory ( VDRL)
Principle:
Serum + Cardiolipin lecithin cholesterol antigen
After rotating the slide, examine the mixture with
10X objective.
Interpretation:
No clumping/ very slight roughness = non reactive
Small clumping = weakly reactive
Medium/ large clump = reactive.

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For the test to be valid the negative control must be non-reactive and
the positive control must be reactive

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2. Rapid plasma reagin( RPR ) card test
Principle
Serum + reagin reagent
Reagent contains:
Cardiolipin
lecithin
cholesterol antigen
cholin chloride with carbon particles
Clumps visualized as black clumps against white back ground
The result is read macroscopically.
Reporting results
Small to large clumps reactive
No clumping or slight roughness non reactive

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Treponemal (specific) test
1. Fluorescent treponemal antibody absorption test ( FTA Abs)
It is used to confirm syphilis infection after positive screening
test by non treponemal tests
Specimen: heat inactivated serum ( at 56oc for 30 min.)
Interpretation of the serological test for syphilis
Positive result : The patient has syphilis
Negative reaction : Patient doesnt have syphilis: infection is
too recent, effect of treatment, disease is inactive

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B. The pregnancy test
Detect the presence of Human chorionic gonadotrophin
(HCG) hormone, which is produced by the trophoblastic
tissue in the placenta
It has certain importance such as:
To confirm pregnancy
To diagnose ectopic pregnancy
To diagnose trophoblastic tumors
Serum HCG reaches detectable level within 24 hour after
implantation.

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Methods of Detection
A. Direct agglutination slide test( direct latex slide
test )
Principle:-
Latex reagent coated with anti-HCG antibodies +
urine
Presence of visible agglutination positive for HCG.
No visible agglutination indicates Negative for HCG
B. Indirect agglutination slide test
Principle:
Urine + anti-HCG antibody + latex HCG
Presence of visible agglutination negative for HCG.
No visible agglutination indicates positive for HCG
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 C. Strip test
Principle:-
The test reaction is between the HCG in the urine
and gold coated antibody on the strip .
A positive control is also impregnated on the strip
so that interpretation will be very easy.
key:
The preferential sample for HCG test is first
morning urine

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C. Serological test for typhoid fever
The causative agent is mainly salmonella typhi.
Transmission:-
ingestion of contaminated food or water
Salmonella species have three distinct antigenic
structures :
O antigen ( somatic) oligosaccharide
H- antigen (flagellar) - protein
Vi- antigen ( K- antigen) /capsular antigen =
polysaccharide .

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Two types of test are available to diagnose typhoid fever

Rapid slide test
Tube agglutination test.
Rapid side test (Widal test)
Detect agglutinating antibodies to O and H antigens of
salmonella typhi from serum
On clean slide , a drop of serum is placed (non-
hemolyzed sample)
Then a drop of antigen suspension is added
Finally mix , look for agglutination .

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 Tube agglutination test:-
Serial dilution of patient serum is made in physiologic
saline .
An antigen suspension is added to the mixture and
incubated for certain period of time (18hr) .
Finally the highest dilution (titer) giving agglutination
reaction is determined.
Widal test is reported by giving the titer for both O and
H antibodies.

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Tube dilution

0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

0.5
0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

initial
Tube 1 2 3 4 5 6 7 8 9 10

Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120

Dilution
factor 10 20 40 80 160 320 640 1280 2560 5120

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Limitation of widal test

False positive reactions

Due to past infection
Cross reaction (malaria, relapsing fever, non-typhoidal
salmonella)

False negative reaction

Early typhoid cases
Poor antigen preparation
Poor transport of reagents &storage.
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 Rickettssial disease
Rickettsiae:-
Resembles viruses in that
They are obligate intracellular parasites (not free
living)
They are about the size of larger virus
They resemble bacteria because
They contain both RNA and DNA
They multiply by binary fusion
They have cell wall
Transmission is through:
Inhaling dried infected vector feces and through
Bitten by an infected vector on the skin
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Weil-Felix reaction
It is an antibody test.
Antibodies of acute Rickettssial infection cross react
with somatic antigen of OX19 and OX2 strains of
protues vulgaris, and OXK strains of P.mirabilis.
Limitation of Weil-Felix test
The test is not specific and less sensitive in early phase
of the disease
False negative reaction is common in scrub typhus.
False positive may occurs in Protues infection,
Brucellosis, Borreliosis
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 HIV TESTS

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Rapid tests
An antigen is coated on the strip with positive control .
up on addition of serum /plasma or whole blood depending
on the test procedure , there will be reaction between
antigen and antibody if present in the sample.
Reactive results : two colored bars (one for the control &
the other for the patient )
Non- reactive : single colored bar ( positive control only )
Invalid result : without having any line.

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 Add 2 drops of
serum and
wash solution

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 Positive Negative

Read results in 10 -12 minutes

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 Current HIV test algorithm
BEIJING WANTAI

1 drop sample + 1drop buffer

for 30

Reactive
Non reactive
Do confirmatory Uni-Gold
Report Negative
test

2 drop sample + 2 drop buffer for 10

Non reactive Reactive

Do tiebreaker test Report Positive

3 drops sample + 1 drop buffer for 10 VIKIA

Non reactive Reactive

Report Negative Report Positive
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 Previously used HIV test algorithm (Ethiopia)
Blood
Blood Sample
Sample

Test 1 (Determine) KHB Beijing Wantii

Non-reactive
Non-reactive Reactive
Reactive

Report
Report Negative
Negative Test 2 (Capillus) STATpak Uni-Gold
Non Reactive
Reactive
Non Reactive
Reactive
Report
Report Positive
Positive

Test
Test 33 (Unigold)
(Unigold) Uni-Gold VIKIA
Reactive
Reactive Result
Result Non-reactive
Non-reactive Result
Result
Report
Report Positive
Positive Report
Report Negative
Negative
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