Immunology and Serology Serology

•Study of non-cellular portion of the blood.

IMMUNOLOGIC REACTION
(Agglutination, Precipitation, Labelled Immunoassay) • In vitro study of Antigen-Antibody reaction.

•A medical science dealing with blood serum especially in

SEROLOGIC DIAGNOSIS
regards to its reaction and properties.
(Serology of Bacterial and Virus Infection) Agglutination

AGGLUTINATION
SEROLOGICAL REACTION Lattice Formation
ucre11 111U'!PNfW
The'. antibody attaches t o the antigen on •Establishment of cross-links between sensitized particles and
AGGLUTINATION antibodies~ agglutination
the red cell membrane
Clump of Cells (Gruber & Durham, 1896). •Forces involved in Antigen-Antibody Binding:
Sensitized RBC's come close together by

... -
forming bridges created by antibody.
The process by which particulate antigens such as cells aggregate
POTENTIATOR
to form large complexes when specific antibody is present.
1) Electrostatic Forces (Ionic Bonds)
2) Van der Waals Forces (London Dispersion Forces)
3) Hydrogen Binding

PARTICULATE ANTIGEN+ SOLUBLE LARGE COMPLEX >v.J'~ 4) Hydrophobic Binding

~'D RN c,Us agglutinafion

SensltlZed Red Cells

AGGLUTINATION AGGLUTINATION
ANTIGEN AND ANTIBODY REACTIONS
Factors That Influence Aglutlnatlon Reactions Factors That Influence Agglutination Reactions

y y y • -- 00q 0
• Effective way to enhance .
,.
• Optimum serum-to-cell ratio Is 40:1 .
• Usually 2 drops serum to 1 drop of
agglut ination reactions.
=.==-~ =z=.~ 2')(,-5%RBCs.
... ..,..ibedy
Centrifugation • High-speed centrifugation Is one
of the most efficient methods
Antigen-Antibody Ratio • Follow manufacturer's directions.
• Incorrect ratio will likely t o lead to
used in blood banking. Pro-zone & Post-zone
• Reducing ionic strength of Effect of pH • Most abs react at pH 6.S-7.5.

•
_9.,...., - -•-..,n Ionic strength medium facilitates Interaction of
ab with ag Temperature
• Clinically significant abs react best at
37°C. I
 Factors That Influence Agglutination Reactions
Antigen-Antibody Ratio TYPES OF AGGLUTINATION

1. DIRECT AGGLUTINATION
2. INDIRECT AGGLUTINATION
o6of?._
t7 /'11 ~ q, 0
3. AGGLUTINATION INHIBITION

--
4. HEMAGGLUTINATION INHIBITION

_, ·--
( O p t i n a a n ~ al
arid Anliboctr)
..
,.........
...._ .__, + 5. COAGGLUTINATION

TYPES OF AGGLUTINATION ABO Slide Typing TYPES OF AGGLUTINATION

• An antigen-antibody reaction
8.-tB Q B Q g
------
• An antigen-antibody reaction
• A reaction in which partldes • A reaction in which carrier
that occurs when antigens are that results in the clumping of
coated (Latex, RBC. Gelatin. particles coated with antibody
naturally found on a partide. red blood cells. "-IOIII .. 3• 2• h ........
• Base on the Sedimentation ----- °"" ... s....a ......... .... ...... with antigens not clump together because of a
normally found on their surfaces combination with antigen.
Pattern. A - - -
clump together because of
• WidalTesting • ABO Slide Typing
QJ-1
-- -- --
11 11 I combination with antibody.
• Virus Hemagglutlnatlon:
Influenza, Mumps ..,_.,. ... ...._..........,_
@
.,_........_ tk-
0 ••
• ANA, RF, Group AStreptococcus • S. aureus
. . . . . . . . . . . . . . . . . . . . . CNl99 . . . . . . . . . . . . . . . . . . . . . . ..... • CMV, HIV, V7:v • S. pyogenes, S. agalactiae
• DAT/IAT
• • Neisseria spp., Leptospira

PASSIVE AGGLUTINATION REVERSE PASSIVE AGGLUTINATION TYPES OF AGGLUTINATION

'
c...,---
0 • ••• ••
........ -0
c-..---
•
c-..-- 0 • YyY
--...... - ~--
h • An agglutination reaction based
on competition between
antigen-coated particles and
• A test for detecting antibodies to
certain viruses, based on lack of
agglutination as a result of antibo,
neutralizing the virus.

------ ~::.~----
2 soluble patient antigens for a
2
limited number of
antibody-combining sites.
.vvv-~

--
• Detection of lllldt drugs • Detects antibody to Rubella, RSV

......... (Cocaine, Heroin) Influenza, Mumps, Measles,

Adenovlrus
Ac-..---
,1
B - -
 -
AGGLUTINATION INHIBITION TYPES Of AGGLUTINATION COAGGLUTINATION

>Q
-
V V
V V

__ -...._
---~~ .1- • A systems using bacteria as the Inert particles to which antibody is
--v-- --v-- V V attached .
--v-- --v-- • Staphylococcus aureus Is most frequently used ( Protein A).

.o -==--
V V
• Particles exhibit greater stability than latex particles and are more
--
- h 1•·=
z::::r r::2b""10
z::::r
refractory to changes in ionic strength.
..,__...,
• reactions are often difficult to read.
....,._A
.....,
r::% --v-- --v-- • Coagglutination reagents have been used in identification of

--
r::2--v-- --v--~
--
Streptococci, Nelsserla menlngltldls, Nelsserla gonarrhoeae, Vibrio
cholera 0139, and Haemophilus lnfluenzae

Agglutination Methods Agglutination Methods Agglutination Methods

Naturally occurring Ap/s
Direct on particles. Particles
Agglutination agglutinate In presence
of corresponding ab.
Ag-ab reaction that
Hemagglutlnatlon results in clumping of
RBCs.
Soluble Ap/s bound to
particles, e.g., latex.
Passive (Indirect) Rheumatoid Factor,
Particles agglutinate in
Agglutination Antinuclear Antibody.
presence of
corresponding ab.
Ab attached to carrier Detects abs to certain
Wldal Test for Typhoid
Reverse Passive particles. Particles viruses that agglutinate
Fever. Rapid ID of bacteria.
Agglutination agglutinate in presence Hemagglutination RBCs. In presence of ab, Rubella & other
of corresponding Ag. Inhibition virus is neutralized & viruses.
hemagglutlnatlon doesn't
Competition between
ABO Slide Typing.
particulate ag (reagent) & occur.
Agglutination soluble ag (in specimen) Detection of Illicit Reagent ab attached to
for sites on reagent ab.
Inhibition drugs. carrier bacteria. Visible
Lack of agglutination is Coagglutination Rapid ID of bacteria.
agglutination In presence
positive result.
of corresponding ag.

Antiglobulin-Mediated Agglutination
Antiglobulin-Mediated Agglutination Antiglobulin-Mediated Agglutination
• Direct Antlglobulln Tests:
• Detection of non-agglutinating ab by coupling with Investigation of HON • Indirect Antiglobulin Tests:
2nd ab (antihuman globulin). Investigation of HTR Crossmatching Test
Diagnosis of Autoimmune Hemolytic Anemia Antibody Detection
• Direct & Indirect Antiglobulin Tests. Diagnosis of Drug Induced Hemolytic Anemia Antibody Identification
Penlclllln (Drug Adsorption) Red Cell Antigen Phenotyping: Du Testing
Cephalosporln (Membrane Modification)
Rifampln, Stibophen, Phenacetin (immune Complex)
Methyldopa, Mefenamlc Acid (Autoantlbody Production)
 Antiglobulin-Mediated Agglutination Antiglobulin-Mediated Agglutination Antiglobulin-Mediated Agglutination

• Reagent: • O Check Cell • False Positive Reaction:

Polyspecific AHG: Anti-C3d, Anti-lgG
Prepared by Conventional Technique (rabbits)
Monospecific AHG: Anti C3d or Anti-lgG only
Prepared by Monoclonal antibody (mice)
Added to Negative AHG test to validate the negative Contamination of Reagent
reaction. over centrifugation
After addition of Check Cell, Agglutination Indicates: Strong Agglutinins
(1) AHG was added over incubation with enzyme-treated cell
(2) AHG was not neutralized Improper used of enhancement reagent
Saline stored In glass or metal container
Lack of agglutination Invalidates the results.

Antiglobulin-Mediated Agglutination Agglutination Instrumentation:

• False Negative Reaction: PACIA: Particle Counting Immunoassay
Reagent Failure Involves measurement of the number of residual
Improper washing non-agglutinating particles in the specimen. These are
Failure to add AHG counted by means of a laser beam in an optical particle
Improper centrifugation counter similar to one that is designed to count blood
Serum/Cell ratio is too low cells.
Delayed washing Precipitation

SEROLOGICAL REACTION Antigen-Antibody Binding

Antigen-Antibody Binding
PRECIPITATION AFFINITY
Is the initial force of attraction that exists between a single Fab a Forces involved in Antigen-Antibody Binding:
Soluble Antigen (Kraus, 1897). site on an antibody molecule and a single epitope or
determinant site on the corresponding antigen .
1. Electrostatic Forces (Ionic Bonds)
The combination of soluble antigen with soluble antibody to
produce visible insoluble complexes. CROSS-REACTIVITY 2. Van der Waals Forces (London Dispersion Forces)
Antibodies are capable of reacting wit h antigens that are 3. Hydrogen Binding
SOLUBLE ANTIGEN+ SOLUBLE INSOLUBLE COMPLEX structurally similar to the original antigen that Induced antibody 4. Hydrophobic Binding
production .

l
 Precipitin Curve TYPES OF PRECIPITATION
Antigen-Antibody Binding
AVIDITY
The strength with which a multivalent antibody binds a 1. Precipitation by Light Scatter
multivalent antigen.
2. Passive lmmunodiffusion Method
~$:~ 6~
LAW OF MASS ACTION
A law used to mathematically describe the equilibrium
relationship between soluble reactants and insoluble products.
It can be applied to antigen-antibody relationships.
.,_
---
0 3. Electrophoretic Method

Precipitation by Light Scatter

Electrophoretic Method TURBIDIMETRY
•TURBIDIMETRY • A measure of the turbidity or doudiness of a solution.
•NEPHELOMETRY •ELECTRO-IMMUNODIFFUSION • A detection device is placed in direct line with the incident light,
•IMMUNO-ELECTROPHORESIS collecting light after it has passed through the solution.
Passive lmmunodiffusion Method
•IMMUNO-FIXATION • Measures the reduction In light intensity due to reflection,
•RADIAL IMMUNODIFFUSION absorption, or scatter.
•OUCHTERLONY DOUBLE DIFFUSION
• Scattering of light occurs in proportion to the size, shape, and
concentration of molecules present In solution.

NEPHELOMETRY
• Measures the light that is scattered at a particular angle from
the incident beam as it passes through a suspension.
• Nephelometers measure light scatter at angles ranging from 10
NEPHELOMETRY
--- <•1 Q -(~ )---
~ourc@

--
.•···~-~'.

--... ........ ·
I- ~
closod s.ample or b l•nk.
-
-.;:
M
l
~,::'"

degrees to about 90 degrees.

iuvn•I
• Light scatter may be recorded in arbitrary units of "relative light ..._·····- ·,. o=tpe::,hunw, ,...m
E p hoo,
iblan
)k p ,oc....,,
( * 4.' ) - - -- --- ,

A-
scatter," or it may be directly extrapolated by a computer to give { bl C ) _ _ _
V \ .I u on,11
actual concentrations in milligrams per deciliter (mg/dL) or
international units per milliliter (IU/ml). sou.co mo~;;;;;,..,or "'°-' ~)' - - ; ~ : ,~

det-.c.10, · .
 GEL PRECIPITATION: IMMUNODIFFUSION
SINGLE IMMUNODIFFUSION TECHNIQUE
One reactant (Ag or Ab) remains fixed in gel.
• Other reactant is allowed to moved.
Interaction with the reagent that is immobilized.
• Reagents become fixed in the gel if they are added to the gel
medium while it is in liquid form .
DOUBLE IMMUNODIFFUSION TECHNIQUE
Both reactants (Ag and Ab) diffuse within a gel.
Both reagents are added after gel has set.
GEL PRECIPITATION: IMMUNODIFFUSION RADIAL IMMUNODIFFUSION
Classlfled Into 4 types: A simple, specific method for identification and quantification of
a number of proteins found in serum and other body fluids.
• Internal reactants like specific antibodies added to buffered
Single Diffusion Single Dimension agarose medium, and serum containing standard volume of
Single Diffusion Double Dimension CHON or Ag is placed in well, centered in agarose.
Double Diffusion Single Dimension Result: Preclpltln Ring
Interpretation: Diameter of the ring Is proportional to the
Double Diffusion Double Dimension
concentration of the Antigen.
Quantitative: lg Levels, Serum CHON's, Serum Complement

RADIAL IMMUNODIFFUSION RADIAL IMMUNODIFFUSION llltY.l~lll''1llUn >IJN

( _; \,:) 0 O
,,,,~-'-
I .·:• .. . \/ .-.. :_ O
.
O O 0
·~···
,,. '. / Anl,rtl

..
,, ,,·.
I, I I
...... t
\'
,•' i..J_ ~ ,.......__

Altlneot
1 !: - ,L

Antigen is allowed to
diffuse to completion and
Measurement taken before Longer Reaction Time (Sensitive) Shorter Reaction Time •
.la<olnl! - - c1 ht1t
1
v.-1•• ..
I ••• •• •' • ••••• '.
•t\
•
@ ... .
the point of equivalence is Occur 24 hours to 72 hours Read within 18 hours (24 hours)
1\1 .~,e I•" -
i•"'l"<><~ ····• , :~\~ .... .---- - .....,.
when equivalence Is
reached, there is no further
reached, Antigen is not Ring Diameter =Antigen Cone. Ring Diameter =Log Cone. (Ag) •'!'IC
.•••• 'l •• -
·~ • : • ~( i
l
allowed to diffuse I.Jses Graphing_Paper Uses Semi-log paper
chan_ge in the ring completely. ,, .,.l.•
...... ··~·, "-i
,,
. ,..,.,._
diameter.
~,/ rom. ,q
Ac Concentration

OUCHTERLONY DOUBLE DIFFUSION

OUCHTERLONV DOUBLE DIFFUSION
\1· - .
\
Ag/Ab Complexes form and precipitate when the 2 reactants

--·--·
meet at the equivalence zone. '~ .
• Use to determine the relationship between Ag and Ab Identity : SINGLE SMOOTH ARC .
.-- '
• Both Ag and Ab diffuse
Ab is precipitating identical Ag specificities lliu • Aal. Ab . ... 1
Incubation period: 12 and 48 hours in a moist chamber
Clinical application: Detection of antibody associated with Non-Identity : CROSS EACH OTHER DOua5-

autoimmune disease (RA, SLE Sjogren's). fungal antigens No common antigen determinants .,"'"'--" -----\

..•:•:~:::·~
1-,~
.~~~~""\', '.! \
-
such as Asperglllus, 8/astomyces, Coccldloldes, and Candida ~- :~"'. ,.\ ~'V'lr't.;q
Disadvantage: Semiquantitative only Partial Identity : SPUR FORMATION.
............ <

--
Antigens are Not Identical but Common Antigen determinant

......
l\o • Aol. AQ2
?t<f~ ]_A}: )<, 1
All • , . 11. 1,,t, .. -,.1
A,b .. . . . , . . .2
Agta "'a 091 ot AQt
M is hMta.,r ll,Q
• :•. : ;~•.

I

ELECTRO-IMMUNODIFFUSION
• lmmunodiffusion reaction In a support medium with the use of
electric current to enhance mobility of reactants and to increase
movement toward one another.
PRINCIPLE:
Antibody: Positively Charged: Migrates toward the cathode(-)
Antigen: Negatively Charged: Migrates toward the anode (+)
ELECTRO-IMMUNODIFFUSION
COUNTERCURRENT IMMUNOELETROPHORESIS
ONE DIMENSIONAL DOUBLE ELECTROPHORESIS
•Antigen and antibody migrate toward each other by
electrophoresis.
only when Ag and Ab have opposite charges.
•Detection of the semi-quantitative antigen of the body fluid
•Faster and more sensitive than Ouchterlony Method.
ELECTRO-IMMUNODIFFUSION
COUNTERCURRENT IMMUNOELETROPHORESIS
1. Wells cut in a row in the agar mixed with antibody.
2. Antigen is placed In the wells. an electrical current applied, and precipitation begins.
3. As the concentration of antigen changes, there ls dissolution and reformation of the
precipitate at ever Increasing Intervals.
4 . The end result Is a predpltln line with a conical shape.
5. The helaht Is measured and Is directly proportional to the concentration of
antl&en.
6. If standards are run a standard curve is constructed and concentration determined .
7. Advantage over RID Is results are obtained In a few hours.
8. Primarily used to quantltate lmmunoglobulins and to assay proteins whose
concentrations are to low for nephelometry, but too high for RID.

ELECTRO-IMMUNODIFFUSION Ill ,._

ELECTRO-IMMUNODIFFUSION ROCKET ELECTROPHORESIS/ LAURELL TECHNIQUE

'. t}
ONE DIMENSIONAL SINGLE ELECTROPHORESIS
\oo,it 5CM JS1', ,~
ROCKET ELECTROPHORESIS/ LAURELL TECHNIQUE +
,.: -~-

, _;. Lt l
J~ttlltE
.

Ji. 1i::_ <

ONE DIMENSIONAL SINGLE ELECTROPHORESIS
•When RID is applied with current, it becomes
electroimmunoassay/ rocket electrophoresis .
'. .... ., . 1
. ff
..
. -,:,, . '
•Ag and Ab different charges at selected pH
coverage finally meet at the middle, when all
Antigen
migration II ... ..
{) :t ) \)F(H ft (
i :_.·_,•_·.
~--.:·.<'·;I:_ ,~:i_.:·J
ii
-
'
antigen is precipitated , the precipitin patter resembles
that of a shooting rocket.
' '
~· Q): •@: :®: © ·'®'- :®: ·i1
.,.
•
,:.
I , .·
,'; ' . l
t,. ,
i- ,
- ,.-., . :: tt- .· :,, .. ,..,. .,- -~-

(+I
--
1-1
IMMUNO-ELECTROPHORESIS IMMUNO-ELECTROPHORESIS
~ ·-p Jp1¢J.:. _.,. ::,
.,.Z:-_s ,.-·,
A semiquantitative gel precipitation technique in which
proteins are first separated by electrophoresis and then •ADVANTAGE: (1) Reliable and accurate method fro
detecting structural abnormalities and .
subjected to double diffusion with antibodies directed
concentration changes in proteins
against the individual proteins.
(2)Useful in screening circulating immune
•METHOD complexes/ Myeloma proteins
Ag is separated by electrophoresis,
Ab is placed in trough cut in the agar, •APPLICATIONS
Serum: detection of Monoclonal Gammopathy
Trough is filled with an anti-serum
Urine: detection of Bence Jones Protein
Incubation: 18 to 24 hours
 IMMUNO-ELECTROPHORESIS IMMUNO-ELECTROPHORESIS
IMMUNO-FIXATION ELECTROPHORESIS

.
1. A two step double diffusion technique. ( - ) 1- - - .... ( +- ) • A semiquantitative gel precipitation technique similar to that
2. Proteins are first electrophoresed to separate them. of lmmuno-electrophoresis. except that antibody js added
,., , .,
3. A trough is cut in the gel parallel to the line of separation, antiserum ;>
. .,, directly to the surface of the gel after electrophoresis has

-
C

taken place,

-
is added to the trough and the gel Is incubated overnight.
4. Double diffusion occurs as the antibody and separated proteins • A cellulose acetate strip impregnated with antiserum is placed
diffuse towards one another In the gel and form preclpltln lines In the .,.. over the separate proteins after serum , urine or CSF are
gel.
5. The lines can be compared in shape, intensity and location to that of C:
--==--=-= •- .... electrophoresed.

normal serum control to detect abnormalities and is

...,,.. , A • Diffusion of antiserum into the gel occurs rapidly, resulting in
precipitation.
semiquantitative.

A _, B
.. IMMUNO-FIXATION ELECTROPHORESIS

= -·-- . - 14 WESTERN BLOT

I
-
SP G A M K A.
L ,M ft ij i, PRINCIPLE:

-
i
.
I ,,
are separated by Poly Acrylomlde Gel

D-n. .,
SPE G A M >. SPE G A M >.
Electrophoresis (PAGE) and trans-blotted onto
c ., "' nitrocellulose/nylon membranes.

ti -
•Antibodies in serum react with specific antigens.
1

SP£ G A M . >.
'

SPE 0 E
-
>. F>.
It=
•Signals are detected according to the principles of test
systems.
•Antibodies against microbes with numerous
cross-reacting antibodies identified more specifically.

--- -~"-r-,-.,_
·•iflf .\ \i·~.
.. . .
~1•.-·t-:
... ;I' --- ----- Precipitation Methods
..,,. a.. • • • • • l . i-----
t .. r -__. •~-•·"". -........ l,·...
t • . \.. • .,.-- Precipitation Methods
Light scattering by Ag-Ab lg's, Complement, ROCKET IMMUNO Electrical charge applied to
Nephelometry lg's, Complement,
complexes. RID to facilitate migration of
C•reactive protein ELECTROPHORESIS ag into agar. AFP
Ag diffuses out of well in No longer commonly Proteins separated by
Radial
lmmunodiffusion
gel containing ab. performed except for IMMUNO electrophoresis then double Largely replaced by
Precipitin ring forms. low-volume testing of ELECTROPHORESIS (IEP) diffusion with reagent abs In IFE

•
(RID) trough In agar.
lgD&lgG.
• Ouchterlony
Af!:s & Ab's diffuse from
wells in gel & form EYDBi11 a!J1111:!li, IMMUNOFIXATION
Proteins separated by
electrophoresis. Antiserum
placed directly on gel. Ag-ab
ID of Ip In
monoclonal
gammopathles,
Double Diffusion precipitin lines where ll!IS1l!l;l1!zlll !lY!:llli!r ELECTROPHORESIS (IFE) complexes precipitate. Bence Jones
they meet. inllgllns,
I proteins. I
 LABELLED IMMUNOASSAY LABELLED IMMUNOASSAY
1. Rapid, specific, and sensitive assays to determine the Ligand: Substance being measured in immunoassay.
presence of Important biologically active molecules Can be Antigen or Antibody.
2. This are designed for antigens and antibodies that may
be small In size or present In very low concentrations. Isotopic: Immunoassay that uses radioisotope as label.
3. Antigens or antibodies Is determined Indirectly by using a
labeled reactant to detect whether of not specific binding
Non-Isotopic: Enzyme, Fluorochrome, Chemiluminescent
has taken place.
Labelled Immunoassay

Characteristics of Labelled Immunoassay Characteristics of Labelled Immunoassay Characteristics of labelled Immunoassay

• Immunoassay in which
patient Ag & labeled reagent
Competitive
ag compete for binding sites
on reagent Ab.
• Immunoassay that doesn't
Non-competitive involve competition for
binding sites.
• Immunoassay with
Heterogeneous separation step to remove
free from bound analyte.
Immunoassay that doesn't
Homogeneous require separation step.
Easier to automate.
• Standards (calibrators), are unlabeled analytes that are made up
in known concentrations of the substance to be measured.
• Most immunoassays use a solid-phase vehicle for separation.
(Polystyrene Test Tubes, Microtiter Plates, Glass or Polystyrene
Beads, Magnetic Beads, Cellulose Membranes).
Separation: Physical Means Washing

Characteristics of Labelled Immunoassay Labelled Immunoassay Labelled Immunoassay

Detection of the Label: Quality Control:
Run a blank tube, usually phosphate-buffered saline, with every RADIOIMMUNOASSAY
Radio lmmunoassays, this involves a system for counting test.
radioactivity. Any readings indicative of label in the blank are known as ENZYME IMMUNOASSAY
background FLUORESCENT IMMUNOASSAY
Enzymes, fluorescence, or Chemiluminescence, typically a A negative control and a high and a low positive control should
change In absorbance in a substrate Is measured by be run in addition. CHEMILUMINESCENT IMMUNOASSAYS
spectrophotometry. • All controls and the patient sample are usually run in duplicate.
 RADIOIMMUNOASSAY RADIOIMMUNOASSAY RADIOIMMUNOASSAY
• The first type of immunoassay developed was
• Competitive Binding Assay
Analyte being detected competes w ith a radiolabeled analyte for a \U. H-~~

u
radioimmunoassay (RIA), pioneered by Yalow and Berson In limited number of binding sites.
~-..._._,. ~..... S.,.C:tficbindiftg
the late 1950s. The amount of label In the bound phase Is INVERSELY
proportional to the amount of patient antigen present. A - -

A technique used to measure small concentrations of an ..........

--
analyte, using a radioactive label on one of the immunologic Non-competitive Binding Assay • •.o. ---
A6A c-.-
reactants .
The amount of label In the bound phase Is DIRECTLY
proportional to the amount of patient antigen present.
•
A A

.....
........,._........., ---~
~_.....

Application of RIA Application of RIA

RADIOIMMUNOASSAY
3
2. RADIOIMMUNOSORBENT TEST (RIST}
131 1; 1251; and Tritiated hydrogen or H 1. RADIOALLERGO SORBENT TEST (RAST)
Labels •Measures the total lgE concentration in serum (allergic and
Detection Radioisotopes emit radioactivity. •Used to detect allergenic antibodies (allergic) parasitism)
Scint illation Gamma Counter •Employs a paper disc+ Patient serum 121 a paper + Patient serum 121 lgE in the
Type(s) of Assays Competitive/ Heterogeneous Washed with saline Iii serum reacts specifically with disc Ill Disc is washed Ill
Sensitivity. Specificity. Radioactively labelled anti serum Addition of radiolabeled antibody (sheep or
Advantages
specific for human lgE is applied to the disc Ill rabbit) to human lgE Iii Measure the
6Disadvantages Radiation hazard.
Measure the SPECIFIC lgE total lgE
Short shelf life of reagents.
Disposal problem.
Application TSH, Total lgE Level

ENZYME IMMUNOASSAY ENZYME IMMUNOASSAY

ENZYME IMMUNOASSAY
Advantages Sensitivity. Specificity. • Any immunoassay that uses an enzyme as label.
Labels ALP, Horseradish Peroxidise,
No health hazard .
D-galactosidase, G6PD No disposal problems.
• A substrate is added to measure enzyme activity.

Detection Enzymes react with substrate to Reagents with long shelf life.
• Direct EIA: First type of EIA developed.
produce color change. Can be automated. Competitive. Enzyme-labeled reagent
Type(s) of Assays Mostly noncompetitive now. Disadvantages Natural inhibitors in some specimens. is part of initial Ag-Ab C' All reactants added at same
Heterogeneous & homogeneous. Nonspecific protein binding. time.
1 incubation & 1 wash.
 DIRECT ENZYME IMMUNOASSAY ENZYME IMMUNOASSAY INDIRECT ENZYME IMMUNOASSAY
Principle Enzyme-labeled ligand & unlabeled patient Prlndple Ag attached to solid phase. Ab In specimen
• Any Immunoassay that uses an enzyme as label.
ligand compete for binding sites on ab attaches. Unbound ab removed by washing.
attached to solid phase. Free labeled ligand Enzyme-labeled antiglobulin added. Attaches
• A substrate is added to measure enzyme activity.
removed by washing. Substrate added. Color to ab on solid phase. Substrate added. Color
Inversely proportional to concentration of
• Indirect EIA: Noncompetitive EIA.
dfrectlyproportlonal to ab concentration.
ligand In specimen. Enzyme-labeled reagent Isn't involved in Initial Ag-Ab C'. 2
Application Used to detect abs to viruses,
incubations & 2 washes. E.G, HIV, HAV, HCV, EBV.
Application Used to measure small relatively pure ags,
More sensitive than direct assays.
E.G., insulin, estrogen.

INDIRECT ENZYME IMMUNOASSAY ENZYME IMMUNOASSAY CAPTURE ENZYME IMMUNOASSAY

0.
Solld..pt,o!&O • nogon Pn1inn1 u nllbody
l§
Sptteillc binding
• Any immunoassay that uses an enzyme as label.

• A substrate is added to measure enzyme activity.

Principle Ab attached to solid phase. Ag in specimen
attaches. Enzyme-labeled ab added, attaches
to different determinant. Enzymatic activity
directly proportional to amount of ag in

f3 ·
• Solid phase EIA: Reagent Ag or Ab bound to support medium. sample.
E.G., polystyrene test tubes, microtiter plates, Used to measure lgs, hormones, proteins &
Application
cellulose membranes, glass beads.
detect tumor markers, viruses, parasites,
fungi.
T u be I• w;u;h ed E n %)'mo-t.belod B ln ch n o to
t o r arnova unbo und a nti-fm m u noglobUlin patk m t antibody
a n tibody

MElHOO DISCl1PllON PIINCIPII OTHII

CAPTURE ENZYME IMMUNOASSAY BA HtiOfO'l'fl'OUS.

romi,1111... dh<t
[nzynNbtl<d ligand& "1•bei!d pilloal
llgandromp,t1 lor llndlngsim"'obal-
O!lgiJ>alEIA.Ulldto,......,.,.U
lt\atlftlyplftilQl,~g. lmull~
ENZYME IMMUNOASSAY
ladlld 10ddplw.fl!f i,beltd llgaod1t- t<IIOCJII.
""td b!"""~-addtd.Calor ll1IIOO DOOIIIOII PRIIKIPU 0111!1

U· --
ln"""1propoll1onal l0"""'1tAt1Dnof
liglnd ~!jlldmt,I.
P.ipdB..ISA M!ltmbHd ~l,11Xib00illdto11l!fllmn!in lil- Mly ha'I! btilt [Of\lrol.llwy
Hot-,. Ag a!U<htd 10 IOlld im11. Ab spodlllffl U!fdlOddl<lahl tnmoe,,.g. gle U!f OO!lt!.limpe ldded.~!!!II(! of quilitiliv!.

/A -~-~
Enzyme-lilkrd
lmOKJl10IOl'oon1 noncllffl!)!lft lY,, -...11nboundablfll'<lftdb)'m1ng. HIY,HAV,HCV.E8'1.
asuyllUSA) 1nc11,a !n,yme-bbtl<d "1tlgialluHn ,cid!d. Al- ag-ab mm~ root!d col111!d mt

oo -~ ·
bdles 1oab on solld pl!B.SUl>slmudd!d.
c,~rdlr!ctlyproportioNlro,b<0""'111>- ll!ed fix d!l!llililion olklw mo-
lngeftew!

-·
11on. Moll 111111t1v, lhan comp,tllll, !IA.
One of most common immoousS3YS- '4in - biml·
p!l!MI WSIXI~ IJWhen
lflgllllib. (00\- l!cuW!IJII NJl!! lllll rffdly

-
innuloi!liy

Siocijillase fi,ear.:bn<lng E,.,,,,.w,,Jed Br,cqto

SaodollchB.ISA
O!aplllf""I 11011(0fflp,tllw,
Ab1~10IOlldilla11,Ag • ,,..i....
,_..,.,.._,b,cld!d,at-
Ag!IIMl""'ffllllilrlt-
li"'110"'"""'igl."'""""'-pio-
-(!Mil) !l1Zllf'libll!dooid!
iffit1Hn~ri~=,~oi--
Ktirily mmr!rl lllll!r nilillltl,tg,
mllXl!I, th!rlp!W(INJ!,~
.,~ omixxlt polieman6Jon lJdlo!IOdlllnlt dellllllNIILEn!Jmatit
a<11111ydn<tlyprDpOIUWlto"'10llllof ;rg
tmldell<ll\l!IOIIIIWf\ffllll!,
~ l.aql.ll!jlClll(-of tioNl10CLUlllllionof 1glnlp!dm!n. ol abra. !lml1!d.

""--"'"'-
;rganau,elloall!ff!CI.Too nu:h;rg
'°""""'· forllirmJ llle .. - .....
 ENZYME IMMUNOASSAY FLUORESCENT IMMUNOASSAY FLUORESCENT IMMUNOASSAY

B~
--
Labels Fluoresceln (Green @ 490-495 nm)

§~§~
Advantages Sensitivity. Specificity.

ff Detection
Rhodamine (Orange @ 550 nm)
Fluorochromes absorb energy from light
No health hazard .
No disposal problems.
source, convert to longer wavelength Reagents with long.shelf life.
(lower energy). Can be automated.
Type(s) of Assays Competitive. Disadvantages Autofluorescence from organic substances in serum.

Q!._O: Heterogeneous & Homogeneous. Nonspecific binding to substances in serum.

Expensive, dedicated Instrumentation .

FLUORESCENT IMMUNOASSAY FLUORESCENT IMMUNOASSAY FLUORESCENCE POLARIZATION

IIE1HOD PIIIKIPU

_,.. ,_,
:=...,~..,3~=. I_,...,...,_
onu WMPWOFAIW.fflS

** @MMUNOASSAV *
.AA _,AJ.. A
*
Dna11ucnK!nt ~angimllldo....i.Jd• - -,,og,.Fu,15(... loclei.1,w~ ..11geo
••nllbody(llfA)
Amlng .....__.___._ A .il!t.!,:i_._
® .®'7®® ®
+ ®® -
lid...........
lnOO!<Opl.
~11111gimlldt-'"dwlllll)Ml1 Solid-phase L.a:>eled antibody Antigen-antibody
® ®*®
-s..dwidll«h"""."
.......
FIIIM'fM:fnt,ntiludt• Antibody Labeted and

--
lncr.,.sed
11111bodJ(lfA) ant)Qen oombin.ation
bdmtDlt:
S8Wll.1fcan~abpnsemilsamn.a1-
ftuaiecfiHDW,r,_ilai,..n
De!emJbsklstnrn. ontllodJ{FANA),ft"°""""
tt!p)llmontlbody(ITA) A fluorescence A
patient antigen po\arlza lion
glal,IA, ,Ollldle5 1Dlb.-
® ® __,..l.,_, __,..l.,_,
obs<r,,dwitll-tnia-
.AA_,AAA . A A _ ~
"'=--=r-~i.e:i
·=·~ L,b<l,d"'""'r.':wltll"ln~-r.r .... ~
=- :=:::r"~:.::ii--
.. NYbeiod '!I - J,pily,
-""-" llonpotalcGugs.-
...._._.__
Soli<k>tme
A A A .MA.
Unlahelftd 11nlibody AnllQM-antlbody labeled Fklore!Cefloe
* * + ®®®-®''®®'®+®®
*
® ®® ®® '-SI -.!:..,
®® 'F'*f'c'\
antigen combination anti-inununoolobuin Antibody Labaa.d and Decruased
B patient entigen ~arizatlon

FLUORESCENT IMMUNOASSAY CHEMILUMINESCENT IMMUNOASSAYS CHEMILUMINESCENT IMMUNOASSAYS

Labels luminol, acridium esters, ruthenium
• Chemiluminescence is another technique employed to follow derivatives, nitrophenyl oxalates.
antigen-antibody combination. Detection Chemiluminescent molecules produce
light from chemical reaction.
• It Is the emission of light caused by a chemical reaction, Type(s) of Assays Competitive & noncompetitive.
typically an oxidation reaction, producing an excited molecule
Heterogeneous & homogeneous.
that decays back to its original ground state.
Advantages Same with IEA & IFA
Disadvantages Quenching of light emission by some
biological materials
 COMPLEMENT FIXATION COMPLEMENT FIXATION
•PRINCIPLE:
•Complement fixation occurs after the binding of antigen
and antibody. Ag+ Ab = Ag-Ab complex+ COMPLEMENT = FIXED
•METHOD:

•Uptake of complement can be used as an indicator of the TEST SYSTEM: Rgt (Ag) + Unknown (Ab) + Guinea Pig Serum
presence of either specific antigen or antibody. INDICATOR: Sensitized Sheep RBC + Hemolysisn

Guinea Pig Serum - Best Source of Complement

•This technique has been used in the detection of viral,
Other Assay & Skin Tests fungal, and rickettsia antibodies . Rabbit Anti-sera - Best Source of Hemolysin
Sensitized Sheep's RBC - Indicator Cells

,1,-- FLOCCULATION NEUTRALIZATION TEST

+C
• Neutralizing antibodies can destroy
,1,- •A specific type of precipitation that occurs over a narrow

.
range of Ag concentration. the infectivity of viruses, which
provides the basis for assays that
can determine the amount of viral
_£.~ •Aggregation of colloidal particles (clumping) in a serological
reaction : VDRL
NEUTRALIZATION
antibody present.
+ + TESTS
• These techniq ues are often used to

g-Lysa
RPR
detect antibodies against herpes

Noey..-~---·
simplex virus Types 1 and 2 (HSV-1
and HSV-2) and echovirus.
L..,_ - nega- - •

CELLULAR TEST CELLULAR TEST CELLULAR TEST

• Positive In CGD
NITROBLUE • Assess the intracellular killing activity of • Use bacteria-infected tumor cells to • Are used to assess the human
ADCCASSAYS
TETRAZOLIUM neutrophil assess the killing ability of NK cells. MIXED-LYMPHOCY leukocyte antigen D (HLA-D)
• Positive: YELLOW
• Uses chemotactic substances to TE CULTURES compatibility of donor and recipient
• Fluorescent immunoassay BOYDEN
assess the chemotactic response of lymphocytes.
• FITC - dye conjugated to specific antibody on CHAMBER
FLOW CELL cell
neutrophils. PHAGOCYTOSIS • Mix bacteria with neutrophils to
CYTOMETRY • Cell + fluorescent Ab = cell fluoresce LYMPHOLYSIS • Assess the ability of tc to lyse labeled ASSAYS assess the cells' phagocytic ability.
• Scattered light TESTS target cells.

..
• T & B cell, NK cell, granulocyte, RBC count
 SKIN TESTS
SKIN TESTS SKIN TESTS
• PPD (purified protein derivat ive) ID Area of
definite palpable induration of edema after
. For Lymphogranuloma v·enerum (LGV)
• Brucella Ag
BRUCELLERGIN TEST • (+) Edematous plaque (1-6mm)
• Chlamydlal Ag- ID • (+) Brucellosis
48 hours FREI TEST • (+) 7mm diameter papule (read after 48
TUBERCULIN TEST ~
• (+) More than or equal 10mm TB but not hours) Histoplasmin,
•
• (+) Wheal (48 hours)
definite Coccldiodln
~
• (·) Less than 1-mm rule out TB
• Dlphteria anti toxin

.
• Palnl!:ss ski!! teit fo[ tl,!b!:[Culosls
PPD tape patch on arm
SCHICK TEST ..
• Diphteria toxin ID
(+) Redness (24-36 hours)
(+) indicates lack of immunity to diphteria
Toxoplasmin • Ag

• Allergen
lnduratlon (48 hours)
(20mins)
VOLLMER'S PATCH • (+) Red area with tiny vesicle (48 hours) • Delayed HPS skin test, measure T cell function
• Erythrogenlc Trlchlnella Skin Test • Most simple procedure to evaluate T cell
TEST • (+) TB Infection but not definite DICKS TEST • (+) Redness (24-36 hours)
.
• (-) Cannot rule out TB
For children
function

Allergen solution Positiv e test: Skin POLYMERASE CHAIN REACTION (PCR)

is placed on skin is red and itchy
FOUR PROCESS:

~@
POLYMERASE CHAIN REACTION (PCR)
A means of amplifying tiny quantities of nucleic acid using
a heat-stable polymerase enzyme and a primer that is
specific for the DNA sequence desired.
1. Initiation
2. Denaturation: dsDNA is heated to 9S"C to separate the
DNA into single (20 to 40 seconds).
3. Annealing: DNA is cooled to SIC to allow primers to
bind/anneal to complimentary sequence on the
separate DNA strands.
4. Elongation: at 7:tC, the heat stable DNA polymerase
(Taq polymerase) binds to the 3' end of each
primer and synthesize new strand of DNA

SEROLOGY OF BACTERIAL INFECTION Syphilis

Human syphilis is caused by the spirochete Treponema pollidum.
Serology of Syphilis
• Antibodies against treponemal antigens and nontreponemal
Streptococcal Serology cardiolipin antigens (Wasser-mann antigens) develop and elicit a
Febrile Disease Serology cell-mediated and humeral Immune response .
Helicobacter pylori Infection • Transmission of Disease: Sexual Contact
Mycoplasma pneumoniae Infection Serology of Syphilis Blood or blood-product transfusion
Woman to her Fetus.
 Syphilis FOUR STAGES OF SYPHILIS FOUR STAGES OF SYPHILIS
Inflammatory lesions (chancres)
FOUR STAGES OF SYPHILIS: Primary Syphilis appear 2 to 8 weeks after infection Contagious and is generally
latent Stage considered to begin after the
and last for 1 to 5 weeks.
second year of infection.
1. Primary (Early) Syphilis Usually occurs 6 to 8 weeks after
chancres first appear. This stage is Is characterized by granulomatous
2. Secondary Syphilis
lesions known as gummata . These
3. Latent Stage of Syphilis Secondary Syphilis
characterized by a generalized rash ,
Tertiary Syphilis
and secondary lesions may develop lesions may develop in skin, mucous
4. Tertiary Syphilis membranes, joints, muscles, and
in the eyes, joints, or central
bones.
nervous system (CNS).

Syphilis Syphilis Syphilis

CONGENITAL SYPHILIS
Syphilis can be transmitted to a fetus after the 18th week of
gestation.
Treatment of the infected mother before the 18th week will
prevent infection; treatment after the 18th week will cure it.
Detection: PCR
Note:
Treatment. Penicillin is the drug of choice, although tetracycline
or erythromycin can also be used.
Primary Stage: A seropositive patient in the primary stage of
disease usually becomes nonreactive approximately 6 months
after treatment.
Note:
Secondary Stage: If treatment occurs during the secondary
stage, the patient usually becomes non-reactive within 12 to 18
months after treatment.
Patients treated 10 years or more after infection may always
remain seropositive.

Serological Test of Syphilis DIRECT DETECTION OF SPIROCHETES DIRECT DETECTION OF SPIROCHETES

Tests for Syphilis are based on the detection:
1. DIRECT DETECTION OF SPIROCHETES Dark-Field Microscopy:
TEST ANTIGEN ANTIBODY COMMENTS
a. Dark-Field Microscopy Diagnose: Primary & Secondary Syphilis
b. Fluorescent Antibody Testing Oi1'dMkroscopi(
• Dark-field Condenser: is used to keep all incident light out of
2. NON-TREPONEMAL ANTIBODY DETECTION: Oanfxij t piliinl!IMpilxnl
the field except for that captured by the organisms. Non! actM lt!ion. t.'111111ft ,xii \
a. Venereal Disease Research Laboratory (VDRL) ~tml!ll,!ljltrM~

:«,~~~ \
b. Rapid Plasma Reagin (RPR) Test
• Specimen: obtained by cleaning the lesion with sterile saline
3. TREPONEMALANTIBODY DETECTION: and rubbing it with clean gauze.
RIIOll!C!!llantmdy 1pal/M frolll pibtnl Mti~ ant~
a. Fluorescent Treponemal Antibody Absorption Test (FTA-ABS) • Pathogenic treponemes: Corkscrew Shape & Flexing Motility
wi1hHUOlmllllag lhindltfld4;1ptm111 doll oot
b. Treponema pallldum Immobilization (TPI) Test • FALSE NEGATIVE: (1) delay In evaluating the slides, (2) an
I\Mtob!lht
c. Antibody Capture Enzyme-linked lmmunosorbent Assay (ELISA) Insufficient specimen, (3) pretreatment with antibiotics.
d. Hemagglutination Tests
 NON-TREPONEMAL ANTIBODY DETECTION NON-TREPONEMAL ANTIBODY DETECTION NON-TREPONEMAL ANTIBODY DETECTION
Venereal Disease Research Laboratory (VDRL) Slide Test: Venereal Disease Research Laboratory (VDRL) Slide Test: Venereal Disease Research Laboratory (VDRL) Slide Test:
The VDRL is a qualitative and quantitative agglutination test • Antigen Delivery Needles: • Antigen Delivery Needles:
using heat-inactivated patient serum.
• Inactivation of Serum: 56•c for 30 minutes. Qualitative Serum VDRL: 18 gauge needle without bevel Quantit at ive Serum VDRL: 19 gauge needle without bevel
• Reactivation of Serum: 56•c for 10 minutes. that will deliver 75 drops of
that will deliver 60 drops of
• CSF can also be used. antigen suspension per ml antigen suspension per ml, 23 gauge needle
• Uses a slides with ceramic ring. that with or without bevel that will deliver
Antigen: 0.03% Cardlollpln, 0.9% Cholesterol, 0.21% Lecithin 100 drops of saline per ml.
Ring Diameter = 14 mm
Ring Diameter = 14 mm

NON-TREPONEMAL ANTIBODY DETECTION NON-TREPONEMAL ANTIBODY DETECTION NON-TREPONEMAL ANTIBODY DETECTION

Venereal Disease Research Laboratory (VDRL) Slide Test: Rapid Plasma Reagin (RPR):
• Antigen Delivery Needles:
• The RPR is an agglutination test. Modified VDRL Test. Method Flocculation Flocculation
CSF VDRL: 21 or 22 gauge needle that will deliver Antigen is similar to the VDRL antigen with the addition of the
Detects Reagin Reagin
100 drops per ml. following: Charcoal, EDTA, Thimerosal, Choline Chloride
Unheated serum is the specimen of choice, although plasma Antigen Cardiolipin Cardiolipin with charcoa l
Ring Diameter = 16 mm maybe used. Positive Microscopic Macroscopic
1.75 mm depth • Uses a plastic cards. Reaction Clumps Agglutination
Antigen delivery: 20 gauge Specimen Inactivated Serum Serum
Serum: 100 rpm for 8 minutes CSF

NON-TREPONEMAL ANTIBODY DETECTION NON-TREPONEMAL ANTIBODY DETECTION NON-TREPONEMAL ANTIBODY DETECTION

Reactivity during
lwqmm
May be neg in primary stage. Same as False Positives Biologic false pos with Same as
disease Titers usually peak during VORL. infectious mononucleosis (IM),
\ffi Crilf~ Pllgin Wtioo;g,,.xif~!O!!!mgl!!tl,
VORL.
secondary or early late stages. infectious hepatitis, malaria, htltmt l!IJOltOO!lj. !pU ft!ij
Titers in late stage, even when liltif'l;f~~liw!
leprosy, lupus erythematosus,
untreated. More rapid decline
rheumatoid arthritis, advanced , RPR
Cmjin
with treatment. Becomes ~flll\m.withm~
age, pregnancy.
nonreactive in 1-2 yrs. MO!!. l!miti« lliln iffl iqXW!j
False Negative Technical errors, Low antibody
following successful treatment. Same as Sj?lf!
titers, Prozone phenomenon VDRL

TREPONEMAL ANTIBODY DETECTION
Fluorescent Treponemal Antibody Absorption Test
(FTA-ABS):
• Confirmatory tests
•The FTA-ABS test detects treponemal antibodies by
using a killed suspension of T. pallidum as an antigen and
a fluoresceln-conjugated antlhuman globulin reagent.
TREPONEMAL ANTIBODY DETECTION
Fluorescent Treponemal Antibody Absorption Test
1. A dilution of heat Inactivated patient serum Is Incubated w ith a sorbent
consisting of an extract of nonpathogenlc treponemes !Reiter strain) .
2. Slides used for this test have the Nichols strain of T. pa/1/dum fixed to
them .
3. They are kept frozen until use and then are equilibrated at room
temperature for 30 minutes.
4. Diluted patient samples and controls are measured and applied to
individual wells on the test slide .
5. Slides are then Incubated In a covered moist chamber at 37vc for 30
minutes.
TREPONEMAL ANTIBODY DETECTION
Fluorescent Treponemal Antibody Absorption Test
6. They are rinsed with deionized water and placed in a Coplin jar w ith
phosphate-buffered saline for S minutes.
7. After a second rinsing, the slides are air-dried, and antibody conjugate
(antlhuman immunoglobulin conjugated with fluorescein ) Is added to each
well.
8. Slides are reincubated as before, and a similar washing procedure is
followed .
9. Mounting medium Is applied, and coversllps are placed on the slides.
10. They are examined under a fluorescence microscope as soon as possible.

TREPONEMAL ANTIBODY DETECTION TREPONEMAL ANTIBODY DETECTION

TREPONEMAL ANTIBODY DETECTION
Fluorescent Treponemal Antibody Absorption Test Treponema pallldum Immobilization Test: Treponema pallldum Immobilization Test:
NOTE:
• If specific patient antibody Is present, It will bind to the T. pa/1/dum • Standard test to which other treponemal test are evaluated. • Interpretation:
antigens. The antibody conjugate will, In turn, only bind where patient
• Involves mixing of patient serum with live, actively motile T.
immunoglobulin is present and bound to the spirochetes.
pal/idum extracted from testicular chancre of a rabbit and If 50% or more = Positive
• When slides are read under a fluorescence microscope, the intensity of Doubtful Result
complement. 20%to50%
the green color is reported on a scale of Oto 4+.
•Testis considered positive if:!: SO treponemes are If 20% or less = Negative
• No fluorescence Indicates a negative test.
• A result of 2+ or above is con,idered reactive. immobilized.
• A result of l+means that the specimen was minimally reactive, and the
test must be repeated with a second specimen drawn in 1 to 2 weeks.

TREPONEMAL ANTIBODY DETECTION Sensitivity of Commonly Used Serological Tests for Syphilis
• Inexpensive, simple to perform, • Usually reactive before reagin
STAGE TEST PRIMARY(%) SECONDARY (Qb) lATENT (%) lATE ('lo) and can yield quantitative results tests In primary syphilis
Hemagglutinatlon:
Nontr,ponn,ial (Rtagin) Ttsts • Screening tool • Confirmatory tests
v.n.r.al Oiotast 11<,nltl, l.abonto<yT,st{WAI) 78 100 !15 11137-941 • Monitoring the progress of the
• Reagent: RBC's Sensitized with Nichol's Strain
R,pij pbwna r,agin c:anl test IRPR) 86 100 91 7l disease
• Determining the outcome of
• Hemmaglutination Treponemal Test for Syphilis (HATTS) Spuific T""°"""'I T.,t,
treatment
• T. pal/idum Hemagglutination Assay (TPHA) fluomctnt brpon<mal antibody ab,oq,tion IFIA-ABSI 84 100 100 9ti
• Congenital Syphilis • Latent Syphilis
lest
• Microhemagglutination Assay for Antibodies to T. pallidum • Neurosyphills
T. pa/lidum microh!!nagglutiwn my IMHA-IPI 76 100 'J7 94
(MHA-TP) • Main disadvantage is that they • Difficult to perform .
- f • ~S.... alllfio<h,l• • - ~i'o,y.,t,4:0onf l»,pmll""llw9'ft'NIJ--fll21.Wl-..,~lllll. are subject to false positives.
l,Mr§l-l,
 Interpretation of Syphilis Test Results
Streptococcal Serology
RESULTS INTERPRETATION
RPR reactive Pos for syphllls
•Streptococcus pyogenes Is a gram(+) coccus responsible for a
FTA reactive human Infections, some of which can have serious sequelae.
RPR reactive Neg for syphilis
FTA nonreactive • The M protein is the major virulence factor for S. pyogenes.
ELISA reactive Pos for syphllls
RPR reactive • Bacterial Toxins: 1. Streptolysln O (SLO)
ELISA reactive Late, latent, or previous syphllls 2. Streptolysln S
RPR nonreactive
FTA-ABS reactive
Streptococcal Serology

Streptococcal Serology Streptococcal Serology Streptococcal Serology

Streptolysln O (SLO) • Is an oxygen-labile enzyme that
causes hemolysis by binding to
cholesterol in the RBC membrane.
• It is antigenic, and the presence of
antibodies to SLO is an Indicator of
recent streptococcal infection.
• Infections and Sequelae:
1. Skin infections caused by S. pyogenes include:
Streptolysin S • Is a non-antigenic, a. Cellulitis b. Impetigo c. Erysipelas
oxygen-stable enzyme. 2. Upper respi ratory tract infections caused by S. pyogenes:
• It causes hemolysis by disrupting a. Sore Throat b. Pharyngeal Edema
the selective permeability of the 3. Scarlet Fever
4. Rheumatic fever (RF)
RBC membrane.
5. Post-streptococcal Glomerulonephritis

Streptococcus pyogenes Streptococcal Serology Streptococcal Serology

Laboratory Diagnosis: Laboratory Diagnosis:
1. Antl-streptolysin O (ASO) Titer
• The ASO titer begins to increase approximately 7 days after
1. Anti-streptolysin O (ASO) Titer infection and peaks after 4 to 6 weeks.
2. Anti-DNAse B Principle: (NEUTRALIZATION) SLO is added to serial
3. Streptozyme Testing dilutions of patient serum, along with group O RBCs as
indicator cells.
ASO titer Is reported as the reciprocal of highest dilution
· ·.· .·.' ..-. " )&
·".t',ti•."' .· '•1! that shows no hemolysls & Is expressed In Todd units.
g, ~- -
 Streptococcal Serology
Laboratory Diagnosis:
1. Antl-streptolysln O (ASO) Titer
Normal Values:
Healthy adults have ASO titers of less than 166
Todd units, with the usual titer decreasing after 50
years of age.
A 30% rise in titer above a previous level is of
greater significance than a single titer.
Streptococcal Serology Streptococcal Serology
Laboratory Diagnosis:
TUBE FO~ ASO TESTING, 14 TEST TUBE 2. Antl-DNAse B (AD-B)
Streptococci produce the enzyme Deoxyribonuclease B
(DNAse B).
The antl-DN-B test Is a neutralization test that can
demonstrate recent streptococcal infection.
Anti-DN-B neutralizes the activity of DNAse B.
Anti-DN-B levels are increased in the 15% to 20% of RF
patients who do not have elevated ASO titers.
+
0 2 drops of the patients serum
n

Febrile Disease Serology Febrile Disease Serology

C. Tests for febrile diseases include:
A. Febrile diseases are a group of microbial infections characterized by
fever and the production of antibodies known as febrile agglutinins.
1. Widal's Test, which can detect antibodies in typhoid fever,
tularemia and brucellosis. a rapid screening test. The
B. These diseases Include: antigens used in this procedure. include Salmonella 0
1. Brucellosis (Bruce/la abortus) (somatic) and H (flagellar) antigens.
2. Paratyphoid Fever (Salmonella paratyphl) 2. Well-Felix Test, which is an agglutination test based on the
3. Rocky Mountain Spotted Fever (R/cketts/ae) cross-reactivity of rickettsial antibodies with antibodies to
4. Tularemia IFronclsella tularensis) the somatic •o" antigens of the OX-19 and OX-2 strains of
5. Q Fever (Rlckettslae)
Febrile Disease Serology Proteus vu/goris and the OX-K strain of Proteus mlrobi/ls.

Serological Tests for Other Bacterial SEROLOGY OF VIRAL INFECTION

Febrile Disease Serology Infections
Proteus Suspension
Infection 1/dcobacl!!/Tfloti liarni< &duod!llal ulC!lscall!!d Method rt !hoke: EUSA. Mostt!llldet!CllgG.lS'l\ iintit11=
OX-2 OX-19 OX-K lllibcdes bJH./Tf/cri llapidtesll,PCRl'lliabit. llKc!llful bNlm!t1LAbsl!mail b)'!,!1~
Epidemic Typhus + + 0 Pm 1apid test! should be coofinned by ELM Hepatitis Virus
Pfimaryll)'picalpn!IIIIOII~ Most common: EIA. ijso uo t!!lforlgM &lg(i abs. ~mid HIV
Murlne Typhus
Spotted Fever
+
+
+
+
0
0 pt1M1«iat WAM agglutinili>n,fA.Mol!c·
ula1 m!lhods av.aKablt
• utm. wfiich VAS noosp«ifK. Epstein-Barr Virus (EBV)
!ldiolles
ScrubTypus 0 0 +
Rkienllal lypoos, Rocky Mountain spotted Gold st.ndanl: lfA, 111K10- Organism l!)e<ificil!ijl 1eplace Wei-ftil Dengue Virus
QFever 0 0 0 llllibocles fMI,oth1111delllial infe<tioos IF.PCR availlblt. rxn,'llllchwmoospedfi~
Rlclcettslal Pox 0 0 0
 Hepatitis Hepatitis A
Infectious Hepatitis Is caused by the HAV - PICORNAVIRIDAE
"Inflammation of the Liver" 1. Transmission: Fecal Oral Route
CAUSED BY: PRIMARY HEPATITIS VIRUSES 2. Incubation Period: 28 Days
3. Disease Course: Acute and Self-limiting;
CHEMICALS DRUGS There is no carrier state.
AUTOIMMUNE DISEASES 4. Laboratory Diagnosis. Liver Function Test (ALT)Total Bilirubin
Antibodies to HAV can be detected by
Hepatitis Virus Enzyme Immunoassay (EIA) and RIA Methods.

Hepatitis A Hepatitis A Hepatitis B

Infectious Hepatitis is caused by the HAV - PICORNAVIRIDAE Infectious Hepatitis is caused by the HAV - PICORNAVIRIDAE Serum Hepatitis is caused by the HBV- HEPADNAVIRIDAE
formerly known as the Australia or
Prevention: Household and sexual contacts of infected Hepatitis-associated Antigen
• Marker of Acute Hepatitis A
persons should receive Immune globulin • Peak: During First Mons. of Illness 1. Transmission : Sexual Contact, Blood
lgM Anti-HAY
injections within 2 weeks of exposure. • Declined: 6 to 12 Mons. 2. Incubation Period: 60 to 90 Days
• Solid-Phase Antibody Capture ELISA 3. Disease Course: May be acute, chronic, or fulminant, or
• Result of Natural Infection or the patient may be a chronic
lgG Anti-HAY Immunization. asymptomatic carrier. Symptoms are similar to
• Competitive Inhibition ELISA Test those seen in HAV infections.

Hepatitis B HEPATITIS B VIRUS ANTIGEN HEPATITIS B VIRUS ANTIBODIES

Summarizes order of appearance of HBV Markers Australian Antigen. • Indicator of current ;,r receri't ~ fectk i"n'.• -~,
• First Marker to appear. lgM Anti-HBc
Hepatitis B Surface Antigen • Detects the "~re W!ndow Periodu ·
• lndlc<1tor of active lnfectlon
Hci-titis Bsurface antigen (HBsAg) (HBsAg) lgG Anti-HB~ Persists for the lifetime of the individual.
• Important marker in screening blood
Hepatitis Be antigen (HBcAg) __ donor. Anti-HBe 1
•

Hcpatits Bcore aott'body (HBcAb) Hepatitis B Envelope Antigen

Present during active replication of
the virus.
Hepatitis Be antibody (HBcAb) (HBeAg) • Indicates high degree of lnfectlvity.
Hq,Mitis Bsurface antibody (HBsAb)
Hepatitis B Core Antigen Not detected ir i?s~"'" r. .' '7i
__ {HBcAg) • Det: cted;,!2~,o~g~~IJJ1' . Biopsv ..
• Appears during the recovery period of Acute
Hepatitis B, weeks to months afte"r HBsAg
Anti-HBs di~appea r.
• Provide Protective Immunity:
.: 10 mlU/ml of Serum
HEPATITIS PROFILE RESULT & INTERPRETATION HEPATITIS PROFILE RESULT & INTERPRETATION HEPATITIS SEROLOGICAL PROFILE
HlfsAg(-)
ACUTE HEPATITIS A lgM antl-HAV (+) RECOVERY FROM HEPATITIS B 1otal antl-tfBe (+) lgGAntl HBc
Anti-Hes(+)
RECOVERY FROM
Total antl-HAV (+) HBsAg (+) lgMAnti HBc
HEPATITIS A
Total anti-HBC (+) Anti HBs,
HBsAg (+) CHRONIC HEPATITIS 8/ CARRIER Anti HBe
lgM anti-HBc (-) HBsAg -
Total anti-HBC (+)
ACUTE HEPATITIS B Antl-HBs (-) HBeAg -
' ............
lgM antl-HBc (+)
Antl-HBs (-) HBsAg(-)
HBVDNA • , ...
HEPATITIS B IMMUNIZATION Anti-HBc (-) 0 1 2 3 4 6 6 -- 12
Months after- infection
Anti-HBs (+)

Hepatitis B Other Hepatitis Virus

Serum Hepatitis is caused by the HBV- HEPADNAVIRIDAE Hepotilit(
!nt,HC'I Acut,, ,hroolt 01 ptviol!I inl,<tioo Pos should lrrnofrnr,d by r"°'1ffilll imnurobk!I......,
(11181,) Ill' mol,arlM ....hod.
Prevention: Avoidance of high-risk behavior (e.g., Intravenous
HCV IINA Cun!fll inf,aioo UIO<I for viral load toslitq, ~r,rd/organ dooor iottning.HCV
drug abuse and sexual contact with infected g,no~pirg to d~ormino optimal ttoatmont.
persons). HepotitbD
(dolt, ir,pathk) !art, rrr dwonk lnl,rtion HOY har!oft<tiv"iusthltunorly""'rinpMKlolHB'I.
lgManti-HDV
A vaccine against HBV has been available since
lgG anti-HOV - ~ ouhronic inf,aion
1982. In a health care setting, HBV vaccination and the
HOV IINA Cunont lnl.aioo Mail!! of a<tift,ir,I rtplkition. U5td to moritor t!r<npy.
use of universal precautions can greatly reduce the risk Human Immunodeficiency Virus
He,otitisl I011Sar,currontlynotapp,omlbylOA for u1<inU.I.
of occupationally acquired HBV.

r,
Human Immunodeficiency Virus Human Immunodeficiency Virus Two Serotype
a. HIV-1
O Member of the Family: Retroviridae
33.2 million people were living Genus: Lenti virus
- Three subtypes
l.M
with HIV infection, 2.5 million - Clades(A,C, D, H, G, K, Fl, F2,J)
\,_;:.~ people became newly infected Etiologic agent of Acquired Immunodeficiency · Clade B (Homosexual)
Syndrome (AIDS)
and 2.1 million people died of
- Clade A, C, E (Asia & Africa)
2. N (Non-M, Non-0)
AIDS - WHO, Diameter of 100-120 nm with a spherical morphology 3. O (Outlier)
2007 b. HIV-2
- Subtypes (A-E)
Viral Genome

ga g
9 200 KB
9 • :9

cnv

po l
p17
,, 2 ...
p9
p7
gp 1 20
g p4' 1

pGG
---- - -
8-i nd !i 'lo j:l<nomk ffN A
-·-
ln , ~:t ?!1-U r'f• ec o f o1t:n'.!c.lop<"
C c:n-< cac,t f o r' nu c t i:-t c achJs
<:o,,.._.-bind ; n g p1ot_el n

B i nd·~ 11.0 C ~4 o n i · ceft.s

1 ,.nn.s.n1.cmb Ya nc prat e.i n
ttS$ od n , cd w1c:h 9 p12'0
S u b unit c, f rcvE:'r'.SC
Viral Replication

CD4
I

gp120
• Key Component for Viral Entry

(gpl20 and gp41-Attach to CD4

Primary Receptor:
-CD4

CORECEPTOR
•
LTR I I t I ,1 II ,. - , I I I I L•·n ~ t os-c:.; d e g r ades
pol o ·n'g i na l H IV RNA -CXCR4
p 5 1 Subunit of' r cvc ~ c
-CCR5
Cell MembrMe
tl"l1 "'11C T ,1p t ft:11.C

HIV-1 p 31 1.,1 c-9 ;e1sc- : n,cdi at"c:s

in t_c.9 ro tto·n o f" HtV D NA
. .:~" •~ .hpst g<"n-~mir: - -
• Terms
Composed of 9 genes encoding 3 structural, 2 envelope, and 6 p lO P.Jotcusc t h at. c lc• ~ 909 a. T - tropic or X 4 strains
regulatory proteins .... , iP u :curso r
Ac-tiv a t c:'S tr n .n ~c r'i p ~ ;; a f H IV b. M - tropic or RS strains
c. Provirus

MODES OF TRANSMISSION SIGNS AND SYMPTOMS Symptoms of opponunlstlc Infections:

- After exposure to HIV, some people have a flu-like illness
Coughing, shortness of breath
that lasts between a week to a month.
1. Intimate sexual contact • Fever
• Fever
• Lack of coordination, forgetfulness,
2. Contact with BLOOD and other body fluid (SEMEN, • Headache
• Vision loss
VAGINAL SECRETION, CSF, SVNOVIAL FLUID, , Enlarged lymph nodes
Persistent diarrhea
PERICARDIAL FLUID, PLEURAL FLUID, PERITONEAL - Several symptoms of occur due to a decreasing CD4 T cell
count including:
• Severe headaches
FLUID,AMNITIOC FLUID AND OTHERS)
Extreme fatigue
• Fatigue, weight loss
3. Perinatally, from infected mother to infant • Nausea, abdominal cramps, vomiting
• Frequent fevers and sweats
• Conjunctivitis, ear infections, tonsillitis (children)
• Persistent skin rashes or yeast infections
• Short-term memory loss

COMPLICATION TREATMENT LABORATORY TETING FOR HIV INFECTION

Kaposi's sarcoma
Cervical cancer HIV SCREENING TEST
• Pneumocystis carinii : pneumonia [IjRETROVIRAL DRUGS 1. ELISA
2. RAPID TEST (IMMMUNOCHOMATOGRAPHIC)
• Toxoplasma gondii, Cryptococcus neoformans
• CMV, HSV, Mycobacterium avium, HIV CONFIRMATORY TEST
1.WB
Candida alb/cans, etc.
• Non-Hodgkin lymphoma
[?lPROPHYLACTIC THERAPY 2.IFA
a. AIDS-related Burkitt lymphoma: L__!_WITH RETROVIRAL DRUG 3.NAAT
TEST STAGE AND MONITOR HIV
chromosome-translocation
1. Viral Load
b. AIDS-related Large cell lymphoma:
c. EBV infection
d. AIDS-related Primary effusion lymphoma: HHV-8 infection
I?lvACCINE 2. CD4 T-CELL COUNT
 Appearance of HIV Markers Appearance of HIV Markers
Screening Test:
HIV-RNA
ELISA Test (Enzyme Linked llMIU COIIMINTS

~,. ,
lmmunosorbent Assay) YnlPM ll!!!aabltwldilndaJ!ofriftdlon.
p24ag Core C01tb nud!ic 1dd, Dtt!Clible in 2-l v.l B«omes undete!11ble II abs ilMl!.l', then detel1lllle ~n in ~tt stages
I -- IS i111111111!1Jll!f11 bils& ~IIJI repliCll!!.
" . Confirmatory Test:
l;,11b llslllllydet!!llblelnHwt. lliffiienl P!ibiniboutl-lv.t,lllllet!dllifeiboii Hwtkt!f.
. .... &.. - Western Blot or

....
,-- - I'¼---' lmmunoflouroscent Assay
lg(iib llde<1l~tsh111t~aftt1lgllGradual f intit!f OY!11t1!11I moot!,, long bltiig. 0 1·0 2·0 3·0 40
Oay-s After Exposure
So

•By 3rd gentwalion ElA. lgM firsl , then lgG.

eb 70

HIV Screening Test False Positives and Negatives with HIV Confirmatory/Supplemental Tests
TEST omm wr.-PERIOD" <Olllllm
1s, _ _
IJA/IUSA
HIV-Antibody ELISA Testing IlS1 orncrs COIIIIEIIS
lgG,l,toHIV-1 Hl..t
lndg,n,,- lgG,btoHN-1/l 6-11..t CAUSES OIJAISE POSllMS CAUSES OF FAISE NEGATMS
Westemblltl\lffl) Ab!IHN llillitiaNlconfvmatmyttstbutnotas1tnslti'lt1S~ ~0!NMT.nt•
J1dgon<ntian lgG &lgM ob to HIY-1/l Hwk
-41hgtMation lg(i&~M,blD HIY-1/!&p24 og l wl
ll!at illa!livalion of serum· Blood dr1W11 brior,serOCOll'lfflion (window p!fiod) pr!litiM ~cmtrll'l!!lial llllmll!lkbmport pos tt II IUII 2of~ lolllMilllJ
tioll. Pl4,g & HIV lb= ,.ubll,h,d ln~C!lon.
umntly""' •PPfD"d ro, "'"";"'-' blood lltpfit!d&ttling/thmlgofserum llypoga11111i1globtlinfflia l bllllsaie prelflt pl4,gp41, gpll0/160. NM! 1tqli!dldlowing neg 01
donOIS. Autoantibodies lmllltllOIUp~, lhe!JPY ~-~Nttoill!!Pf!I.
bpld- lgG&~M1b111HIV 4-11..t lmmunochtomat091aphlc aSQ)'S. Can be p,1- MlltifR 1ngnan<i!s StrainofHIVnotdetertedbJr,181
formtd on wholt blood. serum. o,.I flukt. Li'ffldls!IS! TedmiQ!NrOll lndiect iimarioftuor!l<e!lt AblDHN 5emitility &lfJ!lif'dyool)illbl! to Wtsttm ~ -Not fi~t~ 115ed,
lludeiudd HIVRNA Sday, 11a1 .... -...~1n111011..uing,. MninistrlOOII olim11U1oglobu6ns !llaYIOfA) ~11Jbjel1il!.
1mplltlootian u..dluf,=blooddonon&""""'-(Ab Ainin~ttatian of tr1tain ~dll!!
lmint(IWT) tets,nn'r · inchildrm<l!mondtsai
,gt. Abs from lnft<led molhelQII be prmnt Somtffliignms NAAI HIVIIIA Qlliitmt'51151dbCllffl11Nliln.
""' Wmildlsn'tlnl,md.)

Western Blot Tests to Stage and Monitor HIV

W@slafn Bio,
l"\lnom
Tm COIIMOOS
-gp190
-gpl10 unri.dope poto.i
C04 T-all COtlll HNinffdl(l)t!!ls..ldms-asd'w!tpi!IJ!!l!l.<tOOl~llmsAIOSmlingtoCOCAlso
ll!ld toimM«~- PtnOOtl!fflJ J-6 mo. flow (Jlolnt1Jy is gokl IOOJid
- ~ - ap41 ·~-pfc,ID,n HW-lvialloadasiays Quantitative NAA! to determine p!Ma HN RNA. ll!td to p!!G<I diseal! piog1!11iln, ilftennil! wll!n
-a,,:.u -.1ug1-.i
to nift antir!lromal t!ttrapy, &monitor l!!pOll!t to th!!apy. Test l-8 wlafte11tart of th!lav, &then
Ntfj l-4 mo. lamussay shoo!! b! ll!ld in onle1to asse11 chMtges.
- p2 A cou , ptOl-1
Epstein-Barr Virus
 Epstein-Barr Virus (EBV) Epstein-Barr Virus (EBV)
Infectious Mononucleosis (IM)
Causative agent of: Burkitt's Lymphoma • is an acute, self-limiting disease typically seen In young adults.
Nasopharyngealcarcinoma • The disease is characterized by fever, sore throat, cervical
Infectious Mononucleosis (IM) lymphadenopathy, splenomegaly, and mild hepatitis.
• The WBC count is elevated, and reactive lymphocytes are seen
• The virus is ubiquitous; 80% to 90% of healthy adults have EBV
In the peripheral blood.
antibodies. EBV infects B lymphocytes. • There is a relative and absolute lymphocytosis.
• The average incubation period is approximately 2 to 8 weeks.
Epstein-Barr Virus (EBV)
Antigens and Antibodies:
1. VIRAL CAPSID ANTIGEN (VCA) is found in the cytoplasm of
EBV-infected lymphocytes. lgM antibodies against VCA are
detectable early In the infections, but disappear within 2 to 4
months. lgG antibodies against VCA develop within 1 week after
infection and can persist for life.

Epstein-Barr Virus (EBV) Epstein-Barr Virus (EBV) Epstein-Barr Virus (EBV)

Antigens and Antibodies:
2. Early antigen-diffuse (EA·D) and early antigen-restricted (EA·R)
antigens are found in the cytoplasm of infected B lymph()cytes.
EA-D is also found in the nucleus. lgG antibodies to EA-D can
be indicators of active disease. lgG antibodies to EA-R are
sometimes seen in young children who have active IM infection,
but not in infected young adults.
Antigens and Antibodies:
Antigens and Antibodies:
4. Heterophlle antibodies are stimulated by one antigen and will
3. Epstein-Barr nuclear antigen (EBNA) is found in the nuclei of all react with unrelated antigens from different mammalian
infected cells. lgG antibodies to EBNA develop slowly but can species. The heterophile antibodies of IM are lgM antibodies
remain detectable throughout life. and are seen in 50% to 70'¼ of patients with IM. They persist
for 4 to 8 weeks after infection.

Serological Responses of Patients with

Epstein-Barr Virus-Associated Diseases
Epstein-Barr Virus (EBV) Epstein-Barr Virus (EBV)
ANll-VCA ANTI-EA Serological Testing:
HETEROPHILE Serological Testing:
CONDITION ~M 196 lgA EA-0 EA-R ANT!-EBNA ANTIBODY(l9M)
The DAVIDSOHN DIFFERENTIAL TEST can distinguish heterophile
-
.

.
\Minl,rtol
The Paul-Bunnell Test sheep cell agglutinins in human serum caused by IM, serum
IM
Coml"""IIM
+ H

•
!
1 .
·- --·--~-·---- Can detect only the presence or absence of sickness, and Forssman antigen.
l'.nlinJtrtioo lM • ·----
I·
heterophlle antibodies.
! °""1lc: ,ctj,. '"'" '"" 1M
+I + ! H
It cannot determine the specificity of the antibodies. MONOSPOT TEST is based on the principle that horse RBCs are
1111!-lmffll)lml agglutinated by the heterophile antibodies of IM.
l!mpllojJWli/tr.>tllt di!a!t H . t + !

~..........,
Bllrlin\ .,,._ tt+ ! t+ t

+++ + H +
 Davidson Differential Test Davidson Differential Test
Adsorption Pattern Agglutination with Sheep RBC's after Adsorption
I, i.';s~j! '-•L' ~• ,t s,,J. > r••i"· ij-,h,,l,•L~ r
J
Absoo,tion by Beef
$1i:1d!W. RBCs -~ · ·i i
·l
' Type of .
Heterophile ''
,Absorption by Beef
, RBCs [; ,
11 F~ffljjht,·· rt 1~"':";ti f I · Antibody . ',
Antibodies In IM + Antibodies In IM +
Forssman + Forssman +
·Serum Sickness + + Serum Sickness
Serologic Test for Diagnosis of Recent Infection

Serologic Test for Diagnosis of Recent Infection Serologic Test for Diagnosis of Recent Infection Serologic Test for Diagnosis of Recent Infection
Clinically Clinically Clinically
Organism Test
Significant Result
Organism Test Significant Organism Test Significant
~,a - - 11.(l
Result Result
Anti-DNAse B 1:240
Indirect IFA :?: 1:64 Cold Arudutinins 1:128
Anti-Hvaluronidase 1:512
M. Pneumonlae Complement Fixation 1:32
WidalTest B. burgdorferi EIA (+)
EIA (+\
lndlrect,fFA- Western Blot·lgG :?: 4 of 9 bands\ R. rickettsl Indirect IFA 1:64
RPR
E. cha/feensls Indirect IFA 1:64
IT. pa/1/dum VDRL
B. henselae Indirect IFA 1:128
FTA-ABS Western Blot lgM 2 of 9 bands
u -··•-·= C:11\ I ' \

Serologic Test for Diagnosis of Recent Infection Serologic Test for Diagnosis of Recent Infection
Clinically Clinically
Organism Test Significant Organism Test Significant
Result Result
. hlsto/ytlca Indirect Hemagglutination <! 1:256 lmmunodiffusion (+)
(+) Aspergillus sp.
EIA Complement Fixation <! 1:32
Indirect IFA <! 1:64 lmmunodiffusion (+)
(+) B. dermatltldls
EIA EIA <! 1:32
Indirect Hemagglutination <! 1:128 lmmunodiffusion (+)
EIA (+l Candldasp.
Latex Particle Agglutination <! 1:80
Bentonlte Flocculatlon <! 1:5 lmmunodiffusion (+}
EIA (+) H. capsulatum
Complement Fixation <! 1:32
r:aro sp. EIA ;,: 1:32