IMMUNOLOGY-SEROLOGY LAB
antibody takes place
Exercise 7 Serological Test for Syphilis
Primary Level Test
primary stage of reaction
no visible reaction
uses labelled immunoassay for viewing interaction
Secondary Level Test
secondary stage of reaction
visible reaction/agglutination
Serological test for syphilis is a secondary level
test
Tertiary Level Test
consequence of Ag-Ab binding
ex. phagocytosis, complement activation;
opsonization
Terminologies:
Syphilis a venereal disease caused by Treponema
pallidum
Obtained through:
Direct contact
Sexual intercourse
Contact with lesions
Vertical transmission (congenital syphilis)
T. pallidum subsp. pallidum
a spirochete
causative agent of syphilis
corkscrew motility
penetrating organism
Chancre initial lesion of syphilis
Antibodies developed after chancre:
a. Treponemal antibody antibody specific for
Treponema pallidum
b. Non-treponemal or reagin antibody-like
substance formed as a result of the interaction
of the treponema with the body tissues (tissue
phospholipids); non-specific (it is also
produced in other types of infection)
2 Major Groups of Serologic Tests for Syphilis:
I. Non-treponemal or Non-specific Reagin
Tests
Syphilis RPR (Rapid Plasma Reagin) Card Test
Detect presence of reagin
Principle: Flocculation a midway reaction
between agglutination and precipitation
(+) : black clumps against a white
background on a plastic coated card
Flocculation results as coagglutination of the
carbon particle of the RPR card antigen with
the serum sample containing the regain
Nature of the Ag: non cellular particulate AG
Reagents: Serum
Test kit:
o RPR Ag suspension (white cap)
charcoal is one of the contents
o RPR Positive Control (red dropper)
o RPR Negative Control (green dropper)
Glassware and apparatus:
automated/mechanical rotator or agitator
Provided in the kit:
o Disposable test card with 10 cells
(numbered)
o Dispensing needle for RPR AG (16uL)
18 gauge needle
o Dispensing bottle
o Disposable dispensers for serum or
plasma (50 uL) dispense-stir; open on
1 end, closed in other
Cardiolipin reacts with regain; alcoholic
extract; from beefs heart; has reacting site for
reagin
Test kit is semi-quantitative specific regain
titer; perform serial dilution, RPR procedure,
highest Ab titer with positive reaction
Procedure:
1 drop of PC, NC, serum sample
Dispense 1 drop Ag reagent
Stir
Place in mechanical rotator/agitator for 8
minutes at 100 rpm
Interpret result
o Visible flocculation reactive result
o No flocculation non reactive
Reporting:
Reactive large aggregates in the center or the
periphery of the test circle
Weakly reactive presence of small aggregates
around the edge of the test
Non-reactive smooth even appearance with no
aggregates visible
Interpretation:
A negative result is no proof that the individual has
not been infected
No correlation between amount of reagin in
patients serum and severity of the disease
Reagin levels climb to a maximum during
secondary stage of the disease and declines to a
stable during late syphilis
Four-fold or greater increase in reagin titer
suggestive evidence of syphilis
Titer less than 1:8 suggest latent infection,
previous treatment, or late syphilis
II. Treponemal Test
SY-TPHA (Treponema pallidum hemagglutination)
Qualitative test
Presence of anti-treponemal antibody (anti-TPA)
Principle: Passive hemagglutination RBCs;
Ag-carrier + AB
(+) : smooth mat of agglutinated cells at the
periphery
Sensitized cells will agglutinate in the presence
of treponemal Ab
Sensitized cells avian erythrocytes coated
with components of Treponema pllidum
Avian cells carrier particle
Control cell blue container; avian cells with
Reiter strain (non-pathologic strain of
treponema)
Test cell avian erythrocytes with Nichols
strain (pathologic strain of treponema)
W2 must always have a button of non-
agglutinated cell (negative/non-reactive)
W3 must always have a smooth mat of
agglutinated cells at the periphery
Indeterminate result if W2 has
agglutination/reaction
Exercise 9 Serodiagnostic Tests for
Streptococcal Diseases
Genus: Streptococcus
Group A beta-Streptococci
Pathogenicity: extracellular antigens
streptolysin (hemolytic toxin); enzymes:
hyaluronidase and deoxyribonuclease B
(DNase)
ASO Latex Slide Test
Detect ASO in the serum sample
Latex coated with SO Ag
Principle: Passive agglutination
(+) : agglutination ASO content in patients
serum is >200 IU/mL
No agglutination: ASO titer is <200 IU/mL
Exercise 10 C Reactive Protein Test
C Reactive protein
acute phase protein
present in increased amount in serum during an
inflammatory or necrotic process
pentameric (similar to IgM)
primitive Ab
capable of opsonisation, complement activation,
and agglutination
monitor graft rejection
increased CRP indicates rejection
CRP Test
Sensitive indicator for inflammatory processes
Requested to monitor transplanted tissue
Principle: Reverse passive agglutination
Ab-carrier + Ag
CRP reacts as Ag in the test
CRP latex reagent CRP coated with anti-
CRP)
SS with lobar pneumonia + pneumococcus ->
reacts with C polysaccharide
Exercise 11 RF (Rheumatoid Factor) Latex Test
Rheumatoid Factor
Macroglobulinemia
IgM directed against Fc portion of IgG
RF Latex Test
Ab attacking Ab
Similar procedure with ASO
Principle: Passive agglutination
(+) : visible agglutination >20 IU/mL
2 minutes reaction time
Exercise 12 Test for Infectious Mononucleosis
Infectious Mononucleosis
caused by EBV
Pfeiffers glandular disease/fever
kissing disease, intimate or oral contact
IM Quick Test
Principle: Passive agglutination
Detect IM heterophile antibodies
IM-erythrocyte reagent horse RBC; has
reacting sites for IM heterophile Ab
Similar procedure with ASO
Rotate slide for 30 seconds
Leave undisturbed for 90 seconds/1 minute 30
seconds
(+) agglutination within reaction time
(-) wait for another 30 seconds before
declaring a negative result late reaction
Exercise 13 Test for Lupus Erythematosus
Systemic Lupus Erythematosus (SLE)
autoimmune disorder
lesions similar to wolf bite
erythema, lesions, reddening of skin, butterfly rash
on face
SLE Latex Test
Principle: Passive agglutination
Detect presence of anti-DNP
Reacts with DNP-carrier