IS MODULE 10

BASIC CONCEPTS OF SEROLOGICAL TESTS

 Serological Tests
 are tests that uses antigen and antibody interactions as tools for diagnosis
 Analytes
 are substances to be measured
 they can be bacterial antigen, hormones, drugs, tumor markers, specific
immunoglobulins and many other substances.
 these substances can complex with other substances, and would sometimes
known as ligand (molecule that can bind to another molecule)
*Interactions between antigen and antibodies may be used to detect and
measure analytes of interests.
ANTIGEN AND ANTUBODY INTERACTIONS
 Interpretation of in vitro antigen-antibody reactions
 Qualitative-presence/absence of antigen or antibody
-reported as positive/negative o reactive/nonreactive
 Semi-quantitative-increase in antibody titer
-four fold increase in titer from acute sera to convalescent sera

 Titer-is the highest dilution of the biological sample that still results in a
positive result.
 the reciprocal of the highest dilution of the biological sample that still results
positive.
 Dilution Formula: Dilution =Sample vol
Dilution factor or
Dilution=Sample volume
Sample vol+Diluent
Diluents: NSS- 0.9% or 0.85 % v/w
SEROLOGICAL TESTS INCLUDE:
 AGGLUTINATION
 PRECIPITATION
 NUCLEIC ACID TESTS
 LABELED IMMUNASSAY
 NEUTRALIZATION/INHIBITION
 COMPLEMENT FIXATION
AGGLUTINATION REACTIONS
-agglutinin (soluble antibody) forms a lattice with an insoluble (particulate
or cellular) surface antigen
-involves particulate antigens interaction with an antibody
-semi-quantitative but highly sensitive
Stages:
1st Stage: Sensitization-occurs when Antigen-binding sites of the
antibodies become closely associated with antigenic determinants or
epitopes of the antigen
2nd Stage: Lattice Formation-occurs when antibodies on coated cells
forms cross-linkages between cells resulting in VISIBLE clumping
ADVANTAGES:
 Agglutination of insoluble native antigens or antigen-coated particles
simple to read with or without aid of microscope
 Increased degree of sensitivity
 Great variety of detectable substances
 Requirements:
 Availability of stable cell particle suspension
 Presence of one or more antigens close to the surface
 Knowledge that incomplete or nonagglutinating antibodies are not detectable
without modifications
TYPES OF AGGLUTINATION
1. Direct Agglutination
 antigen is naturally attached to a carrier molecule (carrier can be
RBCs or bacteria)
 patient serum is diluted (in tubes or wells) and reacted with
bacterial antigens specific for the suspected disease
 – Examples of Direct Agglutination
Febrile Agglutinin Tests
Widal Test Weil-Felix Test Hemagglutination:
ABO Forward Typing
 rapid screening test for  detects antibodies to  detects antigens (A
typhoid fever (detects Rickettsia and B) which may
antibodies to Salmonella)  Reagent/antigen: be naturally present
 Reagent/Antigen: O, H, OX-2, OX-19, and in the surface of
K/Vi in bacteria OX-K (P. vulgaris and RBCs
 Spx: Serum (antibody P. mirabilis)  Spx: Whole blood(2-
detection)  Spx: Serum 5% RBCS suspension)
 Reagent: Antisera
2. Indirect Agglutination or Passive Agglutination
 antigen are artificially attached to the carrier molecule
 carriers used includes: polysterene latex, bentonite, beads or
charcoal
 carriers are coated with antigen that is NOT normally found on
their surfaces
 used to detect antibodies
Example: ASO Latex Agglutination test
3. Reverse Passive Aglutination
 antibody are artificially attached to a carrier molecule
 carriers used includes: polysterene latex, bentonite, beads, charcoal
 carriers are coated with antibodies that are NOT normally found on
their surfaces
 used to detect antigens
Examples: CRP Latex Agglutination Test
Detects: C-reactive Protein
Reagent: Anti-CRP antibodies
4. Agglutination Inhibition-done in case when antigen is soluble
Results:
Positive result-absence of agglutination
Negative result-presence of agglutination
 often used when antigen is to be detected on biological fluids
 Example: HCG for Pregnancy Testing
 Has two stages:
1.Neutralization of antigen by addition of soluble reagent
antibodies
2. Indicator Phase; addition of antigen-coated particles bind
 Step 1 Step 2 Result
Addition of Addition of
anti- hCG hCG coated
antibodies latex particles

Pregnant Negative
agglutina
tion
Non- Positive
pregnant agglutinat
i on
Interpretation of results:
*agglutination inhibition involves haptens that are complex to proteins;
the hapten-protein conjugate is then attached to a carrier particle
1. Reagent antibody is added to the patient sample
2. If patient antigen is present, antigen-antibody combination results.
3. When antigen-coated latex particles are added, no agglutination
occurs, which is a positive result.
4. If no patient antigen is there, the reagent antibody combines with
latex particles, and agglutination occurs.
5. AHG-mediated Agglutination Reaction
 also known as Antiglobulin test or Coombs test
 uses anti-human globulin (also know ans antiglobulin or Coomb’s
reagent)
 AHG serves as a bridge to connect two non-agglutinating antibodies
5. AHG-MEDIATED AGGLUTINATION
REACTION EXAMPLES: DAT AND IAT
 Direct Antiglobulin Test
 not specific
 detects in vivo sensitization or coating
 patient RBC’s are saline washed and mixed with AHG reagent at room
temperature, then centrifuged, resuspended, and examined for agglutination
Specimen: Red blood cells
Reagent: AHG
Condition Associated:
 Hemolytic Transfusion Reaction-recipient antibody coating donor RBC’s
 Hemolytic Disease of the Newborn-maternal antibody coating fetal RBC’s
 Autoimmune hemolytic Anemia-autoantibody coating RBC’s
 Indirect antiglobulin test (IAT)
 detects in vitro sensitization or coating
 performed after Rh typing if there is no agglutination to confirm if it is
weak D
 used for weak D testing
Specimen: Serum
Applications:
 Crossmatching-detects recipient antibody reacting with donor cells
 Antibody Screen-detects antibody reacting with screening cells
 Antibody panel-detects antibody reacting with panel cells
6. COAGGLUTINATION
name given to systems using bacteria as the inert particles to which antibody is
attached
-S. aureus is most frequently used, because it has protein A which naturally
adsorbs fragment crystallizablen(FC) portion of antibody molecules of IgG
7. OTHER TYPES OF
AGGLUTINATION
›Viral Agglutination-used in viruses that can be quantified based on their ability
to spontaneously agglutinate red blood cells, such as influenza virus
›Hemagglutination Inhibition-similar to agglutination, except that antigen-coated
RBCs are added instead of antigen-coated latex particles; used for detection of
HBs antigen.
 False Negative
FALSE POSITIVE
⦿ Undercentrifugation
⦿ Overcentrifugation
⦿ Contaminated glasswares, slides or ⦿ Inadequate washing of cells, especially in
reagents antiglobulin
⦿ Autoagglutination ⦿ Reagents not active
⦿ Saline Stored in glass bottles ⦿ Delay in testing procedures
⦿ Presence of Cross reactivity
⦿ Incorrect incubation time
⦿ Presence of Rheumatoid Factor
⦿ Prozone phenomenon and Post zone in AHG
⦿ Failure to add antiglobulin reagent
 A. NEPHELOMETRY
b. Turbidimetry

a. Single Immunodiffusion
b. Radial Immunodiffusion
c. Double Immunodiffusion

a. Rocket Immunoelectrophoresis
b. Counterimmunoelectrophoresis
c. Immunfixation Electrophoresis

-precipitation reaction that results more in formation of

discrete flaky masses (floccules)
-applies in syphilis testing
PRECIPITATION REACTIONS
involves soluble antibody (precipitin) that combines with soluble
antigen in two steps:
 a. rapid invisible formation of antigen-antibody aggregates
 b. slowly forming visible complex lattice of interlocking aggregates
-simplest method for detection of antigen-antibody reaction

Postzone reaction-antigen in excess, so insufficient reactive sites on

antibody for lattice formation
Prozone reaction-antibody in excess, so insufficient reactive sites on antigen
for lattice formation
Zone of equivalence-point at which the most antibody is precipitated by the
least amount of antigen
TYPES OF PRECIPITATION REACTIONS
1. PRECIPITATION TECHNIQUE BY LIGHT MEASUREMENT/FLUID
MEDIUM
a.
a measure of light scattering which indicates the concentration of the
solution
Application: Quantitation of immunoglobulins
b.
a measure of the reduction of in light intensity (light blocked) using spectrophotometer
or an automated clinical chemistry analyzer
measure cloudiness of the solution
2. PRECIPITATION BY PASSIVE IMMUNODIFFUSION
Purpose: Detection of antigen-antibody complex formed by
precipitation reaction
Reactants are added to the gel, and antigen-antibody formation occurs
by means of diffusion/flowing
Agar and agarose help stabilize the diffusion process and allow
visualization of the precipitin bands.
 When no electrical current is used to speed up the process, it is
known as passive immunodiffusion
 Dependent on:
 Buffer electrolytes, pH, Temperature
 Relative concentrations of antigen and antibody
 Passive Immunodiffusion reactions can be
classified according to the number of reactants
diffusing and the direction of diffusion:
 One dimension(Single) Linear-Oudin
 One Dimension (Single) Radial-Fahey and Mancini
Two Dimension (Double) Linear-Oakley and
Fulthrope
 Two Dimension (Double) Radial-Ouchterlony
 SINGLE IMMUNODIFFUSION
 Only the antigen diffuses; the antibody is incorporated in the gel
 Types:
a. Single Linear Immunodiffusion
 antibody (serum) is in the agar tube; antigen is overlain in the top
 Antigen moves through the gel to form precipitin band
 Example: Oudin-old method
Positive result=precipitin line
 Agar Plate

B. SINGLE RADIAL IMMUNODIFFUSION

 Specimen is placed in wells
 Wells are cut from an agar plate
 Agar plate contains the antibody
 +=formation of precipitin rings
 Application: C3 detection
 Example: Radial Immunodiffusion

 2 types: Mancini and Fahey Mckelvy

 2 TYPES OF SINGLE RADIAL
IMMUNDIFFUSION
1. Fahey-Mckelvey method
Kinetic Method
uses measurement taken before the point of equivalence is
reached; reading are taken after 18 hours
the diameter of the precipitin ring is proportionate to log of
antigen concentration (d=log of concentration)
a graph is drawn on semi-log paper by plotting the antigen
concentration on the log axis and the diameter on the arithmetic
axis
 2. MANCINI METHOD
Endpoint Method
antigen is allowed to diffuse to completion; and when equivalence is
reached, there is no further change in the ring diameter
 occurs between 24 to 48 hours (IgG) and 72 hours(IgM)
the square of the diameter is directly proportional to the
concentration of the antigen (d2=concentration)
a graph is obtained by plotting concentrations of standards on the x-
axis vs the diameter squared on the y axis, and a smooth curve is fit to
the points.
 DOUBLE IMMUNODIFFUSION/TWO DIMENSIONAL
›antigen and antibody are both free to move toward each
other, at rate proportionate to size and concentration,
and to form a precipitate where they meet.
1. Double Linear Immunodiffusion-Oakley and
Fulthorpe Precipitin line
›Agar on slide or in plate
›Antigen and antibody in opposing wells
Ab
Ag

Ab
Ag
2. DOUBLE RADIAL/2 DIMENSIONAL
IMMUNODIFFUSION
aka Ouchterlony, Double Angular Diffusion
Semi-quantitative analysis of concentration of antigen or antibody
 Antigen is placed in outer wells, antibodies are placed in the inner wells
at angle
 Reactions:

a. Identity-indicated by presence of smooth arc

b. Nonidentity-presence of crossed/intersecting line
 c. Partial Identity-presence of single spur, pointing towards the simpler antigen.
PRECIPITATION BY ELECTROPHORECTIC TECHNIQUES
General steps in electrophoresis
1. Electrophoresis/Separation
2. Staining-to visualize bands
3. Densitometry-to quantify the bands after staining

Electrophoretic techniques intended for separation

1. Zone electrophoresis-for 5 bands seen only
2.High resolution electrophoresis-better, visualizes 12
bands that includes interzones
🞄
for separation of substances
TYPES OF ELECTROPHORESIS
A. Immunoelectrophoresis
Principle: Combination of electrophoresis and double
immunoduffusion
serum electrophoresed to separate its constituents according to
electrophoretic mobility
trough on each side and parallel to line of electrophoresis are filled
with antiserum
incubation allows immune diffusion to take place
each antibody diffuses outward, perpendicular to its through
each serum proteins diffuses outward from its point of electrophoresis
-Uses:
1.Distinguished ployclonal (2 or more immunoglobulins) from
monoclonal (single immunoglobulin) elevations in gamma globulin
2.Observe decreased or absent immunoglobulins in immunodeficiency
disorders
3.Identify proteins in body fluids (e.g light chains of Bence Jones protein
in urine)
B. Electroimmmunodiffusion

Principle: Directed toward movement of antigen and

antibody to form antigen-antibody complexes in semi-solid
medium
TYPES OF ELECTROIMMUNODIFFUSION
1. One stage electrophoresis/One dimensional single
immunodiffusion
a. Rocket electrophoresis or Laurell Technique
also known as Rocket immunoelectrophoresis or voltage facilitated single
immunodiffusion or one dimension electroimmunodiffusion.
antibody is incorporated in the gel ; only the antigen diffuses with the aid of
electric current
positive result: formation of precipitin rockets (bullet shaped)
Rocket pattern occurs because of precipitation along lateral margins converge to
form a sharp point
total distance of antigen migration (length of rocket) proportional to antigen
concentration
quantitation of antigens other than immunoglobulins
b. Counterimmunoelectrophoresis
-aka countercurrent immunoelectrophoresis or electroprecipitation
-antigen moves toward the + electrode
-antibody moved toward the – electrode
-precipitation occurs at intermediate point
Positive result: formation of precipitin lines
Use: Detection and semiquantitative measure of bacterial/fungal antigens in
the body fluids
Advantages:
1.Visible precipitation lines within 30 minutes (24 hours for double linear
diffusion)
2. Sensitivity is 10x greater than standard (linear) double diffusion
2. Two Stage Electrophoresis
Two steps processes
1st Stage: electrophoretic separation of proteins in the
sample
2nd Stage-either passive immunodiffusion or
electrophoresis
Examples: Trough
1. Classic Immunoelectrophoresis
 Electrophoresis+Immunodiffusion
YYYYYYYY
 Ab is placed in trough
 Positive result: Precipitin Arc
Ag
2. Immunofixation electrophoresis
 Electrophoresis+Immunodiffusion
 Used to identify a monoclonal immunoglobulin (Bence Jones
Protein-monoclonal light chain)
 Urine may be examined by this method for the presence of BJP
 Ab is overlain in the surface of gel
 Positive result: precipitin bands
3. Crossed (two dimensional) immunoelectrophoresis
 Electrophoresis+electrophoresis
 Ab is incorporated in the gel
 Positive result: Precipitin rockets
2. Immunofixation electrophoresis
 Electrophoresis+Immunodiffusion
 Used to identify a monoclonal
immunoglobulin (Bence Jones Ab is overlain in the
surface of the gel
Protein-monoclonal light chain)
Y Y YY YYY Y YY YY
 Urine may be examined by this
method for the presence of BJP
 Ab is overlain in the surface of gel Ag

 Positive result: precipitin bands

 Crossed/Two dimensional electrophoresis

Ab

YYY Y Y

Ag