MODULE 1: SERIAL DILUTION

III. TERMS USED IN EVALUATING TEST

OUTLINE
I Purpose of Serological Tests METHODOLOGY
A Preservation of Serum Analytical Sensitivity: ability of a test to detect very small
amounts of a substance
B Inactivation of Serum Sensitivity
Clinical Sensitivity: ability of test to give positive result if
II Immunologic Reactions patient has the disease
III Terms Used in Evaluating Test Methodology Analytical Specificity: ability of test to detect substance
IV Acute Phase Reactants/Proteins without interference from cross-reacting substances
V Basic Immunological Procedures Specificity ● False positive results may occur
VI Classifications of Antigen/Antibody Reactions Clinical Specificity: ability of test to give negative result if
A Primary Phenomenon patient does not have disease
B Secondary Phenomenon refers to the strength of binding between a single antigenic
VII Immune Testing determinant and an individual antibody combining site
Affinity ● The antibody binding to the epitope should be shape-
specific (Ex. if the epitope is triangle-shaped, the end of
the antibody binding to it should also be triangle-shaped)
I. PURPOSE OF SEROLOGICAL TESTS a measure of the overall strength of binding of an antigen
• Serological tests Avidity with many antigenic determinants and multivalent
antibodies
o may be performed for diagnostic purposes when an
infection is suspected
o Serology blood tests help to diagnose patients with
certain immune deficiencies associated with the lack of
antibodies, such as X-linked agammaglobulinemia
▪ Other name: Bruton’s disease
▪ An inherited rare immunodeficiency disorder
▪ There is a gene defect that blocks the growth and
production of normal B cells (it doesn’t mature).
o branch of laboratory medicine that studies blood Like a lock and key fit
serum for evidence of infection and other parameters • Antigen
by evaluating antigen-antibody reactions in vitro (nasa o May have 1 or several epitopes (antigenic
labas) determinants; receptor-like)
• Serology • Antibody: Y-shaped
Estimating the antibody by determining the greatest degree to
o scientific study of blood serum which the serum may be diluted without losing the power to
o In practice, the term usually refers to the diagnostic Dilution
given an observable effect in a mixture with specific antigen
identification of antibodies in the serum. ● Serum undergoes serial dilution
o More on the antigen-antibody reactions ● dilution of antigen or antibody solution to the most favorable
concentration
• Serology prerogative of Microbiology ● To quantitate the specimen (serum)
o The term serology has come to imply almost ● Titer
exclusively the detection of antibodies in serum for ○ most dilute concentration of serum antibody that reacts to
its antigen
antibodies in infectious diseases caused by ○ HIGHEST serum dilution that gives a positive result
microorganisms, and terminology has become the (agglutination)
prerogative of microbiologists. ○ 1:160 (titer of the patient)
Titration
o Regardless of what microorganism, as long as they ○ If the dilution is higher and yet there is still agglutination,
it indicates the severity of the disease or infection - the
have virulence factors that cause an infection, it may patient’s degree of infection is higher.
produce antibodies and trigger our immune system. ● A rise in the titer ratio indicates disease
● Antibody Titer: the amount of antibody in the serum;
concentration of antibodies against a particular antigen
A. PRESERVATION OF SERUM ● Different dilutions of serum are tested in mixture with a
Physical Refrigerate for 72 hrs at 4-6°C constant amount of antigen and greatest reacting dilution is
Add 0.001g Merthiolate powder per ml serum or 5% phenol or taken as the measure or Titer.
Chemical
tricresol at 0.1 ml/ml of serum
• Purpose of preserving serum: for transport
• Purpose of inactivating serum: to destroy the
complement activity in the serum

B. INACTIVATION OF SERUM
Heat serum at 56ºC for 30 minutes (commonly done) or heat
Physical
at 60-62ºC for 3-4 minutes only.
Chemical Add choline chloride
Serial Dilution
● development of detectable specific antibodies to
II. IMMUNOLOGIC REACTIONS microorganisms in the blood serum as a result of
• Antigen-Antibody Reactions Seroconversion infection or immunization (vaccination)
● Usually manifests in 2 or 3 weeks after having the
Primary Secondary Tertiary
disease
combination of biologic reaction
demonstrate Ag-Ab ● opposite of seroconversion
Definition Ag-Ab; non- is detectable; In
reaction; In vitro Seroreversion ● This is when the tests can no longer detect
visible reaction vivo
antibodies or antigens in a patient’s serum
agglutination,
opsonization,
fluorescent tests, complement-
Examples phagocytosis,
immunoassays fixation, IV. ACUTE PHASE REACTANTS/PROTEINS
chemotaxis
precipitation
• Plasma proteins that are synthesized in the liver
• Usually increases in concentration by 25% or more during
inflammation
DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 1
 MODULE 1: SERIAL DILUTION

1. Alpha1-antitrypsin a. Concentration of the reactants

o serine protease inhibitor that inhibits action of specific b. Temperature
proteases that cause lung damage and pulmonary ▪ Should be optimal temp: usually between 40-45℃
inflammation ▪ 60°C and above: proteins start to denature
Heterozygous result in increased risk of liver disease, c. Length of incubation
deficiencies glomerulonephritis, and connective tissue disease
▪ Agglutination: within minutes
Homozygous
deficiencies
result in premature emphysema and liver disease ▪ Precipitation: usually hours or even days
2. Alpha2-macroglobulin d. pH of the test system
o a protease inhibitor that plays a role in the coagulation, ▪ Optimum pH: neutral (7.0)
fibrinolytic, and complement components of
hemostasis VI. CLASSIFICATIONS OF ANTIGEN/ANTIBODY
REACTIONS
3. Ceruloplasmin
o absence or deficiency is associated with Wilson’s A. PRIMARY PHENOMENON
disease • initial binding of a single antibody to a single antigen
▪ Rare inherited disorder • Primary phase: Sensitization
o Copper disorder
• Enzyme immunoassays, radial immunodiffusion
▪ excess storage of copper
• Factors that affect primary phenomenon:
▪ Can be seen in the liver, brain, eyes, or in any other
o Affinity and Avidity
organs
▪ High levels of copper, decreasing levels of
ceruloplasmin B. SECONDARY PHENOMENON
▪ Gives a golden eye discoloration or also called as • Basic antigen/antibody reactions are taken a further step,
“golden brown eye ring discoloration” forming cross links or lattice formation to create large
molecules that are easily detectable
• Determine agglutination and precipitation
• Lattice Formation
o Antibody molecules crosslink the particles forming a
lattice that results in visible clumping or
Kayser-Fleischer rings agglutination
4. Fibrinogen
o plays an active role in wound healing after tissue injury

5. Haptoglobin
o plasma protein that binds to free hemoglobin
o Haptoglobin levels increase two-fold to four-fold
following tissue injury
6. Cold-reactive protein (CRP)
o normally present in trace amount in the serum but may
increase 1000 times than normal during inflammation
o FASTEST responding acute phase reactant in our
Test That Involves Antigen-Antibody Reactions
blood Direct detect unknown antigen in specimen using known anti-sera
o If the patient’s situation becomes chronic, CRP detect unknown antibodies in the serum using known
Indirect
disappears. commercial antigen (prepared kits that can be bought)
o Can be used to assess disease severity and monitor
therapy VII. IMMUNE TESTING
o Rapid Latex Agglutination Test • Two general diagnostic ways
▪ Principle: Reverse Passive Agglutination Test o Direct: Use known antibodies to detect antigens
associated with an infectious agent
o Indirect: Use antigens to detect specific antibodies
in a patient’s blood to determine exposure to a specific
pathogen
• Serological tests employ IN VITRO antigen-antibody
reactions to identify such antigens or antibodies
o Tests may be specific or non-specific
▪ If the patient has an antibody against CRP and o Tests may identify the antigen or antibody
positive/has CRP, it will result in the formation of o Test may be modified to enhance the visibility of the
agglutination/clumping. ag-ab reaction
▪ Clinical Significance ▪ Attach the antigen to larger particle (latex)
- Elevated Levels (above the normal 0.6 ▪ Label the antibody (fluorescent or radioactive)
mg/dL) indicates: ▪ Measure the antigen-antibody reaction to
✓ Inflammation nephelometry (turbidity)
✓ Tissue damage
- As seen in:
✓ Bacterial and viral infections REFERENCES
✓ Rheumatic diseases such as RA
✓ Myocardial infarction Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
✓ Burn injuries
✓ Tuberculosis CANVAS Notes
✓ Malignancies
✓ Renal transplantation

V. BASIC IMMUNOLOGICAL PROCEDURES

1. Sensitization
2. Factors that affect antigen/antibody reactions:

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 MODULE 2: PRECIPITATION AND AGGLUTINATION

OUTLINE
I Precipitation Tests and Assays
A Fluid-Phase Precipitation Tests
i Nephelometry
ii Turbidimetry
B Methods in Measuring Precipitation
C Precipitation by Passive Immunodiffusion Tests
i Radial Immunodiffusion (RID)
ii Oudin Test
iii Ouchterlony
ii. TURBIDIMETRY
iv Double Linear Diffusion • Detect the presence of bacterial growth
D Precipitation by Electrophoretic Techniques • Measures the cloudiness / turbidity of a solution
i Immunoelectrophoresis (IEP) • Measures the decrease in light intensity in a solution of
ii Counterimmunoelectrophoresis (CIE)
antibody-antigen complexes
E Precipitation Reactions/Precipitation Curve
II Agglutination Test o The lower the light intensity, the higher the
A Agglutination Tests concentration of complexes
i Hemagglutination Test • Uses transmitted light and the measurement is
ii Direct Agglutination Test (DAT) spectrophotometer
iii Indirect Agglutination Test or Passive Agglutination • Measurements are made at 180 degree from the incident
B Antiglobulin Technique light beam
C Viral Neutralization
i Neutralization
ii Opsonization
D Complement Fixation

• In microbiology — when preparing bacterial suspension —

I. PRECIPITATION TESTS AND ASSAYS once there is a presence of turbidity, it is suggestive that it
• Precipitation: Soluble antigen + antibody in equal and has bacterial growth.
optimal proportions • In antibiotic susceptibility testing, you compare the
o Process of forming insoluble complexes/ solid masses bacterial suspension to McFarland standard through
o Simplest method for the detection of antigen-antibody cloudiness/turbidity.
• Measurement of Precipitation in Fluid Medium
o Principle: B. METHODS IN MEASURING PRECIPITATION
▪ Soluble antigen + antibody (in proper proportions) 1. In Liquid: Ring Test
→ Visible precipitate o Ascoli test
▪ Lattice Formation (antigen binds with Fab sites of 2 ▪ For Bacillus anthracis
antibodies) o Lancefield flocculation
o Soluble antigens interact with antibodies and form a ▪ Slide flocculation: VDRL
lattice that eventually develops into a visible precipitate ▪ Tube flocculation: Kahn test for syphilis; not
o Precipitin ring test is performed in a small tube. performed anymore
2. Gel Diffusion/Immunodiffusion
A. FLUID-PHASE PRECIPITATION TESTS o Oudin
• Measurement of Precipitation by Light Scattering o Oakley-Fulthorpe
(Optical Methods) o Radial
a. Turbidimetry o Ouchterlony
b. Nephelometry o Immunoelectrophoresis
o Electroimmunodiffusion
▪ Rocket
▪ CIE
▪ Laurell’s
o Immunodiffusion is affected by:
▪ Size of particle
- The bigger the particle, the slower it moves
▪ Temperature: 40-45°C
▪ Gel viscosity and hydration
- Gel (Agar): Agarose gel

C. PRECIPITATION BY PASSIVE
IMMUNODIFFUSION TESTS
• Immunodiffusion procedures are carried out in an agar gel
medium
• The precipitate is easily seen in gels yield visible precipitin
i. NEPHELOMETRY lines
• But no visible precipitate forms in regions of Ab or Ag
• Scattered (reflected) light is proportional to number of
insoluble complexes excess.
• Light is passed through suspension
o More light refracted = more sensitive i. RADIAL IMMUNODIFFUSION (RID)
• Deals with measurement of intensity of scattered light; • Single diffusion, double dimension
usually measured at the right angles to the incident light • Gel containing known antibodies with small holes cut as
beam wells.
o Example: o Sample containing the antigen is placed in the well
▪ Complement component concentration (labeled) with the plate in the normal position.
▪ Antibody concentration o Afterwards, the plate is placed in the incubator in its
• Quantitative determination of complement factors and normal position as well.
immunoglobulins ▪ If turned upside down, the antigen will spill.
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 MODULE 2: PRECIPITATION AND AGGLUTINATION

o After 24 hours or depending on what method was • Antibody in the agarose gel and the antigen solution is
used, there will be a zone of inhibition or also known layered over it.
as ring which is the one measured. • If there is an antigen-antibody reaction, the antigen
• Antibody is uniformly distributed in the gel - Agarose gel diffuses towards the agar gel and will form a precipitin
• Line of precipitation forms in a circle around the well band.
(Precipitin line)
• Used to determine the concentration of an antigen in a iii. OUCHTERLONY
biological fluid
• Double diffusion, double dimension
o Diameter (size) of the ring/zone of inhibition is directly
proportional to the concentration of the antigen in the • Also called Double Radial Diffusion
sample • Most widely used
o Example: • Used for comparing antigens
▪ Salmonella typhi test (Widal) • Usually, this is the first generation that was used for
▪ IgM HbsAg.
▪ IgG • Qualitative test
▪ C4 • Wells are cut in gel with multivalent antibody is placed in a
▪ C3 center well and antigen is remaining wells; both antigen
▪ Transferrin and antibody is moving
• Two methods: • Precipitation lines that form will identify relationship
1. Fahey and McKelvey/Kinetic method between antigens
▪ Diameter is proportional to log of the • Example:
concentration; measures diameter before the o Elek’s gel precipitation
completion of reaction o Test for Corynebacterium diphtheriae
▪ Reading time: 18 hours • Diffusion patterns: after 24 hours
▪ Can be performed in multiple measurements o If you are only comparing a few antigens, then you can
- To monitor the activity of the whole test use a small plate. But if there are many, it is better to
2. Mancini/Endpoint method use a big plate.
▪ IgG: 24-48 hours o Agarose gel is always used.
▪ IgM: 50-72 hours a. Identity:
▪ Square of diameter is proportional to the - precipitation lines meet but do not cross, no
concentration/measures diameter after spurs, indicating no relationship between the
completion of reaction (endpoint method) antigens; smooth curve; antigen in the sample
▪ Only one measurement is performed/ measured is same as known antigen
usually at the end of the test - Positive reaction: fusion of two lines
forming an arch meaning the antigen present
have the same epitopes/antigenic determinants
b. Non- Identity
- precipitation lines formed will crisscross, as
an X, indicating no identical epitopes and no
identity between antigens; antigen in the
sample is different from known antigen
- Demonstrates two separate reactions, that’s
why they crossed; different antigens, different
antigenic determinants
• Like Mueller Hinton Agar (sensitivity testing) - Positive Reaction: no formation of arch;
• Can use small or big plate where we will put a hole antigen do not have the same determinant
• Sources of errors: c. Partial Identity
o Overfilling and underfilling the well - precipitation lines formed will appear partially
o Spilling sample outside of the well crossed, as a line with a spur formation on the
o Nicking the well end, indicating cross reactivity between the
▪ there are scratches or the agar is cut antigens
o Improper incubation time or temperature - antigen in the sample has some similarities with
known antigen
ii. OUDIN TEST - Positive result: fusion of lines but with a
• Single diffusion, single dimension spur; meaning the antigen in the sample have
• Most simple immunodiffusion test similarities with the known antigen
- Example: heterophile antigens
• No electrical current used
• Result: forms a precipitin line or band (semi-
quantitative) iv. DOUBLE LINEAR DIFFUSION
• Advantage: • Oakely and Fulthorpe
o Simple and cheaper • Double diffusion – single dimension
• Disadvantage • Agar on slide or plate; ab incorporated in gel
o Long turnaround time • Antigen and antibody in opposing wells
• Incubation time: 24-48 hours • Reactions:
• Example: Quantification of antigens (Factor VII antigen) o react equidistantly and at equivalence
o contamination or impurity present in antigen reacting
with antibody
• Agarose gel with antibody and antigen on top but there is
an intervening layer in between which is a plain agar.
• Positive result: If there is antigen-antibody reaction, the
antigen and antibody will move toward each other through
the plain agar layer, forming a precipitin band.

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 MODULE 2: PRECIPITATION AND AGGLUTINATION

D. PRECIPITATION BY ELECTROPHORETIC o Ab migrates to electrode (-)

TECHNIQUES o Ag migrates to electrode (+)

i. IMMUNOELECTROPHORESIS (IEP) E. PRECIPITATION REACTIONS/PRECIPITATION

• Combination of electrophoresis and immunodiffusion CURVE
1. Zone of antibody excess
• precipitation in agar under an electric field
o Prozone Phenomenon
• Expensive and procedure is more complex
o precipitation is inhibited and antibodies not bound to
• analyzes serum proteins antigen can be detected in the supernatant
• Excellent screening test to differentiate serum proteins o Many antibodies (Y-shaped) scattered, few antigens
and to detect abnormalities o Indicates late infection
• Since it is electrophoresis, the antibody is positively 2. Zone of equivalence
charged (+) and the antigen is negative (-). o Window Period
• two-step double diffusion technique which first involves the o maximal precipitation in which antibody and antigen
electrophoretic separation of proteins form large insoluble complexes and neither antibody
• Result: separate precipitation lines between each protein nor antigen can be detected in the supernatant
and its antibody o Area where antigen and antibody are present in
• useful procedures for the ID of monoclonal proteins optimal concentrations or proportion.
(Bence Jones proteins) 3. Zone of antigen excess
• double diffusion + electrophoresis o Post Zone Phenomenon
• Enhances the semiquantitative of antigens o precipitation is inhibited & Ag. not bound to Ab. can be
• Used to treat myelomas and malignant lymphomas detected in the supernatant
o Many antigens (round-shaped), few antibodies
a. ROCKET ELECTROPHORESIS o This is suggestive of early infection
• Also known as: Laurell’s
• One dimension, Electroimmunodiffusion Note: Both prozone and post zone can cause false negative
• Combination of RID + electrophoresis serologic reactions and erroneous titer results.
• The height of the rocket is proportional to the
concentration of the antigen II. AGGLUTINATION TEST
o The higher the rocket, the higher the antigen • Agglutination occurs due to the cross-linking of
• Total distance of antigen migration and precipitation is particulate antigens by antibody molecules.
directly proportional to antigen concentration • visible reaction of antibody with the particulate antigen
• Antibody in gel and antigen pipetted into a cut out well • Disadvantage: mostly qualitative examination (positive or
• Electrical current is applied and resulting precipitation lines negative) only; actual value cannot be obtained
form narrow triangles, like a rocket • Advantages:
• The rocket lines are formed as antigen concentration o Easy to perform
decreases o No need for any complicated equipment
• A quantitative method for measuring serum proteins • Example: Widal test: for typhoid fever
antibody • Principle
• Advantage: The result can be obtained in a few hours o Particulate antigen + antibody –> clumping
o Lattice formation (antigen binds with Fab sites of 2
b. IMMUNOFIXATION ELECTROPHORESIS antibodies forming bridges between antigens)
• Combination of Immunoprecipitation + electrophoresis • Two steps to agglutination:
o Sensitization
• Most sensitive method used to detect, confirm and
▪ occurs when the antibody and specific antigen (on
characterize monoclonal gammopathies, such as multiple
the surface of the particulate) combine thru single
myeloma, Waldenstrom’s macroglobulinemia, monoclonal
epitope
gammopathy of unknown significance
▪ Factors affecting sensitization:
• To replace IEP; similar to IEP except that after
- Size of the antigen and antibody
electrophoresis is performed the antiserum is applied
- Class of antibody
directly to the surface of the gel
- Nature of antigen
• This method is used to detect the M-band. o Lattice formation
o M stands for monoclonal. ▪ involves interactions between antibody and
o Consists of monoclonal proteins which are abnormally multiple antigenic determinants
present in plasma cells
• Types of Agglutination Reactions
• In this test, you are finding out what particular a. Direct agglutination reactions
immunoglobulins (IgG, IgA, IgM, kappa, and lambda) are ▪ Patient is tested immediately
the M proteins present. ▪ For example, in blood typing, you prick, get the
o Kappa and lambda: parts of the heavy and light chain blood, and put antisera.
of the immunoglobulins - If it agglutinates, then it is positive.
• Example: Western blot b. Indirect or passive agglutination tests
o Confirms the HIV proteins ▪ antigen must be bound first to an inert particle
o Also confirms the ELISA before you can detect the antibody
Note: electrophoretic errors can occur when current is too ▪ Inert particles can be:
strong or too weak, current is applied backward, buffer is not - Charcoal
at correct pH, or time to run is too long or too short. - Sheep’s red blood cells
• Capillary tube precipitation (Ring Test) - Red blood cells
- Latex
ii. COUNTERIMMUNOELECTROPHORESIS (CIE) c. Hemagglutination reactions
• Modification of ouchterlony ▪ Type of agglutination reaction wherein the RBCs
• antigen and ab are placed on the well directly opposite to are the carrier.
each other
• Advantage: speeds up the migration of antigen and A. AGGLUTINATION TESTS
antibody

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 MODULE 2: PRECIPITATION AND AGGLUTINATION

i. HEMAGGLUTINATION TEST iii. INDIRECT AGGLUTINATION TEST OR PASSIVE

• Assays that use RBCs as the antigen particulate reacting AGGLUTINATION
with antibody • Soluble antigen is artificially attached to particulate
carriers (inert particles)
ii. DIRECT AGGLUTINATION TEST (DAT) • Antibodies cause visible agglutination of soluble antigens
• Particulate antigen reacts directly with antibodies affixed to latex spheres.
• Antigen found naturally on the surface of the particles • This can be used to test the antibodies against viruses
(cells). such as Rubella, Herpes zoster (shingles), CMV.
• Ex. Blood typing, Widal, Weil-Felix, Cold Agglutinin test

B. ANTIGLOBULIN TECHNIQUE
• Antiglobulin
o Used to demonstrate incomplete antibodies
• Identifies reactions of reagent RBC antigen with specific antibody when the reaction occurs in vitro • Applications:
Indirect
Antiglobulin • Detects antibodies that are floating in the serum o Detection of anti-Rh Ab
(Coombs) Test • Detects in vitro sensitization of cells o Autoimmune hemolytic
anemia
•Qualitative agglutination test o Blood group typing
Direct •Detects in vivo sensitization of cells o The identification of viruses.
Antiglobulin Test •Detects antibodies that are stuck or attached to the RBCs o The diagnosis of certain diseases
•Applications: • Practical considerations: Easy & Semi-quantitative test
• Assays based on the principle of no agglutination as a (+) result o Example: GRAVINDEX
Agglutination o If samples are reactive, it will not produce a visible result. If there is no visible result, then it is positive. ▪ Pregnancy test
Inhibition • Serum (containing antigen) is mixed with beads or latex coated with antigen ▪ Uses urine as specimen
• If the agglutination occurs, the antigen is not present - it is negative. to detect HCG
Reverse Passive • Antibodies (known) are bound or attached to • Used for detection of microbial antigens such as Group A and B streptococcus, S.
Agglutination particulate carriers instead of antigen aureus, N. meningitidis, H. influenza, C. neoformans, M. pneumonia and C. albicans
Coagglutination • Carrier: Bacteria • Used to identify Streptococci, Neisseria meningitidis, Neisseria
(Coat) • Most commonly used carrier: Staphylococcus aureus gonorrhoeae and Haemophilus influenzae.
• Form floccules
Flocculation • Seen in VDRL and Kahn test
• Agglutination of colloidal particles (non-treponemal tests)

C. VIRAL NEUTRALIZATION o consist of known target antigen reagents (beef heart

• This test can also be used to test toxins extract, bacterial antigen) and complement.
• Advantages: Sensitive and specific enough to identify • Indicator system
whether an individual has been exposed to a particular o consist of sheep’s rbc sensitized with hemolysin
virus or viral strain • Positive result: no hemolysis
o Wasserman test for syphilis (in the past)
i. NEUTRALIZATION o Certain viral & fungal diseases
• If complement is fixed by specific antigen-antibody
• Neutralization Reactions reaction, it will be unable to combine with indicator system
• After binding, antibody is not available to react in indicator o Complement binds to the antigen-antibody complex
system and is used up.
• Like agglutination inhibition o Complement-fixation can be used to detect very small
• Results: amounts of antibody
o No agglutination/ hemolysis = positive reaction
o Agglutination or hemolysis = negative reaction
• Antibody did not bind to the origin
• Generally, positive control samples used in inhibition or REFERENCES
neutralization tests show no reaction and negative control
samples show a reaction Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
• Example of inhibition: Hemagglutination inhibition test
for rubella carrier: bacteria (S. aureus) seen in VDRL CANVAS Notes
• Example of neutralization: antistreptolysin O test (ASO)
1. Toxin Neutralization
▪ Toxin-Antitoxin reaction
▪ ASO
2. Virus Neutralization

ii. OPSONIZATION
• Antigen is covered with antibodies that enhance its
ingestion and lysis by phagocytic cells
• Coating of pathogens (antigen) with antibodies to increase
phagocytosis (the ingestion of phagocytes)
• Opsonins may be
o Specific antibody alone (IgG1, IgG3)
o Specific antibody plus complement, via the classical
pathway
o Complement alone, via the alternative pathway

D. COMPLEMENT FIXATION
• First, the body needs to produce complement fixing
antibodies to bind to the antigen before forming
complement fixation.
• Test system

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 MODULE 3: LABELLED IMMUNOASSAYS

o Rabies virus
OUTLINE
I Immunoassays
o Scrub and murine typhus
A Constituents of Labeled Assays
B Direct Immunofluorescence Test or Single Layer (DFA) B. DIRECT IMMUNOFLUORESCENCE TEST OR
C Indirect Immunofluorescence Test or Double Layer (IFA) SINGLE LAYER (DFA)
II Immunofluorescence Tests
• The antibody has a fluorescent tag, added directly to
A Flow Cytometry
B Radioimmunoassay (RIA) - Rosalyn Yallow and Berson unknown antigen fixed to slide, after incubation and
III Enzyme Immunoassay (EIA) washing observe for fluorescence
A Enzyme-Linked Immunosorbent Assay • Use of conjugated antibody to detect patient antigen
B Types of ELISA • Uses known fluorescein-labeled antibody
i Direct ELISA • (+) fluorescence
ii Non-Competitive/Indirect ELISA
C Western Blot
• Detects antigen
IV Recombinant Immunoblotting Assays (RIBA)
A Immunochromatography C. INDIRECT IMMUNOFLUORESCENCE TEST OR
B Chemiluminescent Immunoassays DOUBLE LAYER (IFA)
V Fluorescence Polarization Immunoassay (FPIA)
A Fluorescence in Situ Hybridization (FISH)
• React patient serum with known antigen attached to
VI Molecular Techniques solid phase, wash, add antihuman antibody attached to
A Serological Tests fluorescent tag, wash, observe for fluorescence.
i Application of Agglutination Tests in Serology • Identifies unknown antibody (direct) or unknown antigen
(indirect)
• Detects antigen and antibodies
I. IMMUNOASSAYS • Uses known fluorescein-labeled antiglobulin
• More expensive than agglutination & precipitation • (+) fluorescence
• Introduction • Qualitative to Semi-Qualitative
o Used when there is a need for rapid, specific, sensitive
assays (since agglutination and precipitation are
laborious and takes a lot of time to yield results).
o Labeled immunoassays
▪ Some antigen/antibody reactions not detected by
precipitation or agglutination
▪ Measured indirectly using labeled reactant (i.e.,
Enzyme labeled antibody) II. IMMUNOFLUORESCENCE TESTS
- Uses fluorescent dyes, radioisotopes, and
enzymes (labels) A. FLOW CYTOMETRY
▪ Referred to as receptor-ligand assays • Cells in suspension are labeled with fluorescent dyes
▪ Ligand is the substance to be measured and is • Direct or Indirect Fluorescence
defined as a molecule that binds to another • Cells analyzed on a flow cytometer
molecule of a complementary configuration, • Method of choice for the analysis of B & T cells
usually it binds to the substance the test is trying to
detect
▪ The receptor is what binds the specific target
molecule (i.e., Paratope/Fc, epitope).
• Ligand assay: one reactant is labeled so that the amount
of binding can be measured

A. CONSTITUENTS OF LABELED ASSAYS • Principle

• Labels which may be used include o Incubate specimen with 1 or 2 monoclonal antibodies
o Fluorescent tagged with fluorochrome
o Radioactive o Single cells pass through incident light of instrument
o Chemiluminescent and enzymes (laser) which excites fluorochrome and results in
• Fluorescent Antibody Test - Albert Coons emitted light of different wavelength
o Uses fluorescent dyes called o Intensity of fluorescence measured to detect cells
Fluorochrome/fluorophores possessing surface markers for the specic monoclonal
▪ Dyes, chemically linked to an antibody without antibodies that were employed
affecting antibody’s ability to bind antigen o Forward light scatter indicates cell size or volume
▪ Glows bright green when exposed to fluorescent o 90° side-scattered light indicated granula
light • Common uses Flow cytometry
▪ They have markers that have the ability to absorb o DNA analysis
energy & they will convert it into light that is why it o Reticulocyte counts
will glow o Leukemia
• Fluorescence: time to do it is only a few minutes to hours o CD 4 cell estimations in AIDS/HIV patients
• Fluorescent probes used: • Advantages of fluorescent techniques
o Fluorescein isothiocyanate: green fluorescence a. Methods fairly simple; sensitive and specific
o Rhodamine: red-orange fluorescence b. No hazardous reagents to use or discard.
o Phyocyanin: red fluorescence c. Increased sensitivity over radiolabeled and enzyme
o Texas red: red fluorescence reactions.
• Fluorescein-labeled antibodies used in 2 types of • Disadvantages
tests: a. Fluorescent compounds are sensitive to environmental
o Direct fluorescent antibody test changes.
o Indirect fluorescent antibody tests b. Stability of labels
• Example: c. Nonspecific binding can diminish signal.
o Herpes virus IgM d. Bilirubin or hemoglobin can absorb the excitation or
o Dengue virus emission energy.
DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 7
 MODULE 3: LABELLED IMMUNOASSAYS

e. Requires expensive dedicated instrumentation 7. Can be used for qualitative or quantitative procedures
8. Sensitivity similar to RIA (Radio immunoassay) without
B. RADIOIMMUNOASSAY (RIA) - ROSALYN health hazards and reagent disposal problems
YALLOW AND BERSON 9. Expensive equipment not necessary,
spectrophotometers.
• Radioactive isotopes (Carbon 14, Iodine 125)
• Disadvantages:
• Check sample for presence of antigen.
1. Some samples may have natural inhibitors.
• Known amounts of radioactively labeled antigen, known
2. Size of enzyme label limiting factor in designing some
antibody and unknown sample mixed together.
assays.
• If sample has high level of antigen, it will compete with
3. Nonspecific protein binding may occur.
labeled antigen and little radioactive antigen will bind.
4. Enzyme reactions very sensitive to temperature
• If sample has low level of antigen, it will not be able to
• Enzymes chosen as labels according to the following:
compete with labeled antigen and much radioactive
1. Number of substrate molecules converted per
antigen will bind
molecule of enzyme
• If there is no antigen in the sample then there will be a 2. Purity
high level of radiation since the radiolabeled antigen can 3. Sensitivity
bind to all antibody sites. 4. Ease and speed of detection
• Radioactivity (radioactive decay) is measured by Gamma 5. Stability
Scintillation Counter 6. Absence of interfering substances
o Also known as: Competitive RIA 7. Availability
o Results: relationship is inversely proportional 8. Cost
• One of the most sensitive techniques for measuring • Typical enzymes used include:
hormones, drugs, & vitamins at conc. Of <0.001 ㎍/㎖- 1. Green: Horseradish peroxidase (most common)
The principle involves competitive binding of radiolabeled 2. Glucose oxidase
Ag and unlabeled Ag to the limited supply of a high affinity 3. glucose-6-phosphate dehydrogenase (uses
Ab. fluorimetric means)
• Results: 4. Yellow: Alkaline phosphatase
o Non-reactive = High radioactivity 5. Beta-D-galactosidase
o Reactive = Low radioactivity Peroxidase
B- Alkaline
• Advantages galactosidase Phosphatase
Source Horseradish E. coli Bovine intestine
o Immune reactions highly are specific Method for
o Immune reactions are highly sensitive enzyme
Periodate oxidation Dimaleimide Glutaraldehyde
(Nakane Method) method method
o Can detect even small quantities labeling
• Disadvantages • Labels:
o Radiation hazards (radioactive) o Alkaline phosphatase
o Requires specially trained persons o Horseradish peroxidase (color green)
o Time: 1 day or 24 hours o Glucose oxidase
o expensive o Beta-galactosidase
o Labs require special license to handle radioactive Note: Colorimetric reaction: The darker the color, the higher
material the concentration
o Requires special arrangements for
▪ Requisition, storage of radioactive material A. ENZYME-LINKED IMMUNOSORBENT ASSAY
▪ Radioactive waste disposal • Also called ELISA, enzyme immunoassay or EIA
• Radioallergosorbent Test
• Involves colorimetric reaction
• Used to identify which allergen is causing the allergy by
detecting the specific antibody (IgE) that binds to it. • A biochemical technique used mainly in immunology to
(Allergic reaction) detect the presence of an antibody or an antigen in a
RAST
• Non-competitive solid phase immunoassay (old sample.
method) • The ELISA has been used as a diagnostic tool in medicine
• Use of enzyme or fluorescent labels rather than • ELISA can be performed to evaluate either the presence
radioactivity of antigen or the presence of antibody in a sample
• Radioimmunosorbent Test
• Because the ELISA can be performed to evaluate either
• Competitive RIA (old method - Used to quantitate the
amount of IgE generated from the allergic reaction) the presence of antigen or the presence of antibody in a
RIST sample
• Serves as a screening test to determine if more specific
allergy testing is indicated • Time to perform ELISA is about 1 day.
• 1st test developed to measure total serum IgE • The darker the color, the ↑ (higher) the concentration of
the analyte
• Screening method for HIV
• Principle of ELISA:
o Uses an immune reaction lie RIA (Radioactive
immunoassay), differs in detection method
o Detection based on Enzyme catalyzed reaction of
fluorescent probe
o ELISA techniques use antibodies linked to an enzyme
or horseradish peroxidase or alkaline phosphatase
Radioisotopes Enzymes
125I Horseradish peroxidase
3H Beta-D-galactopyronoside
14C Alkaline phosphatase
III. ENZYME IMMUNOASSAY (EIA) Fluorochromes Chemiluminescent molecules
• Advantages of enzyme immunoassay Fluorescein Luminol
1. Labels cheap and plentiful Rhodamine Acridium esters
Dioxetane phosphate
2. Labels have a long shelf life
3. Easily adapted to automation • Fluorochrome absorb energy from the light source and
4. reaction measured using inexpensive equipment convert it to light of a longer wavelength and lower energy
5. Very sensitive • Chemiluminescent molecule produce light energy from a
6. No health hazards associated with reagents chemical reaction

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 8

 MODULE 3: LABELLED IMMUNOASSAYS

B. TYPES OF ELISA

i. DIRECT ELISA
• used for antigens with multiple epitopes/antigenic
determinants
A. Sandwich or Capture Assays
o Antibody to one epitope fluid, antibody to second
epitope fixed.
o Enzyme label used to detect reaction
o Excess antibody attached to solid phase, allowed to
• Applications of Immunoassays
combine with test sample.
o [RIA & ELISA]
o After incubation, enzyme labeled antibody is added.
▪ Analysis of hormones, vitamins, metabolites,
o Second antibody may recognize same or different
diagnostic markers
epitope than solid phase antibody.
- Example: ACTH, FSH, T3, T4, Glucagon,
o Enzyme activity directly related to concentration of
Insulin, Testosterone, vitamin B12,
antigen.
Prostaglandins, glucocorticoids
o Epitope must be unique to the organism and present
▪ Therapeutic drug monitoring:
in all strains.
- Barbiturates
o Major use of this technique is for measurement of
- Morphine
immunoglobulins, the immunoglobulin is the antigen
- Digoxin
and an anti-antibody is added to the test system.
▪ Diagnostic procedures for detecting infection
o Disadvantage:
- HIV
▪ Linear relationship does not exist
- Hepatitis A
▪ Must prepare standard curve
- Hepatitis B, etc.
▪ Test conditions must be carefully controlled
o Results: • Advantages of ELISA
▪ Reactive: Change in color o Sensitive: Nanogram levels or lower
▪ Non-reactive: No change in color o Minimal reagents
o Qualitative & Quantitative
▪ Qualitative assays: HIV testing
▪ Qualitative assays: Therapeutic drug monitoring
o Greater scope: Wells can be coated with Antigens or
Antibodies
o Suitable for automation

C. WESTERN BLOT
B. Competitive ELISA • Specimens used:
o Has the same principle as RIA, only difference is that o Serum
RIA uses radioisotopes, while ELISA uses enzymes o Urine
o Add known quantities of patient sample containing o saliva
antigen + antigen labelled with enzyme • Steps
o Enzyme + Substrate → Product → Measure color o Antigen mixture separated by gel electrophoresis
o Color inversely related to antigen in patient sample o Blotted onto filter paper
1. Enzyme labeled ligand competes with unlabeled o Serum sample applied to filter
patient ligand for binding sites on antibody o Labels used to interpret results
molecules attached to solid phase. o Used to confirm HIV and a positive ELISA test
2. After reacting, a washing step is performed
3. Enzyme activity is determined
4. Enzyme activity is inversely proportional the
concentration of the test ligand
5. Sensitivity in nanograms (10 g/mL) can be
achieved.
IV. RECOMBINANT IMMUNOBLOTTING ASSAYS
ii. INDIRECT ELISA (RIBA)
A. Non-competitive ELISA • Uses recombinant proteins
• Detects specific antibodies
• Antigen is bound to solid phase; unlabeled patient A. IMMUNOCHROMATOGRAPHY
antibody is added. • Similar to drugstore-bought pregnancy tests
• After incubation a wash step is performed and an enzyme • If antigen is present, some labeled antibody will be trapped
labeled substrate is added on the test line
• The amount of enzyme label detected is directly • Single-use
proportional to the amount of antibody in the specimen. • Commercially available
• Indirect ELISAs are very sensitive • Performed about an hour
• Disadvantage: • Excess-labeled antibody is trapped on the control line
o More manipulation of the test

• Advantages
o Rapid test
o Easy to perform
o Can detect antigen or antibody
• Disadvantages
DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 9
 MODULE 3: LABELLED IMMUNOASSAYS

o Costly • Detecting and locating a specific DNA sequence on a

o Concern validated data chromosome
• Can be used in metaphase chromosomes and interphase
B. CHEMILUMINESCENT IMMUNOASSAYS nuclei
• Uses chemiluminescing triphenylmethane dyes
1. Chemiluminescence is the production of light energy
due to a chemical reaction
2. Most common substances used are:
▪ Luminol
▪ Acridium
▪ Esters • Summary:
▪ Dioxetane phosphate 1. Labeled assays are used to measure antigen or
3. The substance is oxidized, usually with hydrogen antibody
peroxide and an enzyme for a catalyst 2. Different labels can be used.
4. Energy given off in the form of light 3. Two classification of enzyme assays:
5. Heterogeneous and homogenous assays available. ▪ Heterogenous: requires a step to physically
• Disadvantages: separate bound ligand from free.
a. False results may be obtained if lack of precision in ▪ Homogeneous assays: require no separation
addition of hydrogen peroxide step
b. Some biologic materials cause quenching. 4. Immunoassay labels must be very specific and
c. Need dedicated instrumentation have high affinity.
5. Radioactive labels
• Advantage
▪ RIA is COMPETITIVE: known antibody added in
a. Reactive stability of label
specific amount to unknown sample, competes
b. Speed of detection
with patient antibody to attach. Indirect
c. Sensitivity comparable to RIA and EIA
measurement.
d. Low cost
6. Enzymes can be used as labels, results in a color
reaction
▪ Most assays today are non-competitive
▪ Sandwich technique is very popular
▪ Homogeneous enzyme assays: no separation
necessary, antibody binding causes antigen
labeled with enzyme to lose activity.
7. Fluorescence
▪ Direct immunofluorescence detect antigen directly
by using labeled antibody
V. FLUORESCENCE POLARIZATION ▪ Indirect immunofluorescence utilizes sandwich
IMMUNOASSAY (FPIA) technique
• Uses fluorescent dyes
• Polarization VI. MOLECULAR TECHNIQUES
• Principle • Polymerase Chain Reaction (PCR)/Nucleic Acid
o Add reagent antibody and fluorescent-tagged antigen amplification Technique
to patient serum o Makes copies of target DNA by repeated cycles of
o Degree of fluorescence polarization is inversely denaturation, annealing, and extension
proportional to concentration of analyte a. Template: DNA region to be amplified; from
o Utilizes three concepts to measure specific analytes in patient’s genomic or mitochondrial DNA or from
homogenous format fluorescence, rotation of miccroorganisms
molecules in solution and polarized light; uses b. Taq polymerase: heat-stable DNA polymerase
fluorescein as fluorescent label absorbs light energy at from the bacterium used to catalyze the synthesis
490 nm and releases this energy at wavelength is 520 of DNA; can withstand high temperature required
nm as fluorescent light. for denaturation
c. Primers: Short segments of DNA designed to
hybridize to template strands
d. Deoxynucleotide triphosphates: building blocks
from which DNA polymerase synthesizes new
strands of DNA (dATP, dTTP, dGTP)
e. Reverse transcriptase: enzyme used to make
DNA from RNA
• Southern Blot
o Specific DNA
• Northern Blot
• Positive Test o Specific RNA
o Antigen present in patient serum binds to reagent • Western Blot
leaving most tagged antigen unbound o Detection of proteins (antibodies) to specific epitopes
o Unbound labeled antigens rotate quickly reducing of antigen;
amount of polarized light produced • Fluorescent in Situ Hybridization (FISH)
• Negative Test o Used of fluorescent-labeled probe to detect target
o If no antigen present in patient serum, tagged antigen nucleic acid detected in intact cells
binds to reagent antibody o Cytogenetics
o Tagged antigen-antibody complexes rotate slowly • Restriction Fragment Length Polymorphisms (RFLPs)
giving off increased polarized light o Detection of genes associated with specific diseases
o forensics
A. FLUORESCENCE IN SITU HYBRIDIZATION o Paternity Testing
(FISH)
• Cytogenetic studies A. SEROLOGICAL TESTS
DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 10
 MODULE 3: LABELLED IMMUNOASSAYS

• Agglutination: Particulate antigens

• Hemagglutination: Agglutination of RBCs
• Precipitation: Soluble antigens
• Fluorescent-antibody technique: Antibodies linked to
fluorescent dye
• Complement fixation: RBCs are indicator
• Neutralization: Inactivates toxin or virus
• ELISA: Peroxidase enzyme is the indicator
Test Use
Immunodiffusion Dx of SY, Pneumococcal pneumonia
Assay production of particular classes of
Immunoelectrophoresis
antibodies
Blood typing, pregnancy, Dx of salmonellosis,
Agglutination Brucellosis, gonorrhoea, Ricketsial infection,
Yeast Infection, Mycoplasmal Infections
Viral Neutralization Dx of infections by specific strains of viruses
Viral hemagglutination Dx of viral infections such as influenza,
inhibition measles, mumps, Rubella, mononucleosis
Dx of measles, influenza A, SY, Rubella
Complement Fixation rickettsial infection, Scarlet fever, Rheumatic
fever, RSV, Coxiella
Dx of Rabies, Infections of Group A
DFA
streptococcus
IFA Dx of Sy, mononucleosis
Pregnancy, drugs in urine, Dx of Hepatitis A,
ELISA
Hepatitis B, Rubella, Initial Dx of HIV infection
Verification of Infection with HIV, Dx of lyme
Western Blot
disease

Comparison of Precipitation Techniques

Technique Application Principles
Igs,
Light that is scattered at an
Complement,
angle is measured,
Nephelometry CRP, other
indicating the amount of
serum
antigen or antibody present
proteins
Antigen diffuses out into gel
that is infused with antibody. REFERENCES
Igs,
Radial Immunodiffusion Measurement of the radius
Complement
indicates concentration of
the antigen Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
Both antigen and antibody
Complex diffuses out from wells in
CANVAS Notes
Ouchterlony Double antigens such gel.
Diffusion as fungal Lines of precipitate formed
antigens indicate the relationship of
antigens
Differentiation Electrophoresis of serum
Immunoelectrophoresis of serum followed by diffusion of
proteins antibody from wells
Over-or- Electrophoresis of serum
Immunofixation under followed by direct
electrophoresis production of application of antibody to the
antibody gel

Comparison of Agglutination Reaction

Type Of Reaction Principle Result
Agglutination indicates
Direct Antigen is naturally
the presence of patient
Agglutination found on a particle
antibody
Particles coated with Agglutination indicates
Indirect (Passive)
antigens not normally the presence of patient
Agglutination
found on their surfaces antibody
Agglutination indicates
Particles are coated with
Reverse-Passive the presence of patient
reagent antibody
antibody
Haptens attached to
carrier particle. Lack of agglutination is a
Agglutination Particles compete with positive test, indicating
Inhibition patient antigens for a the presence of patient
limited number of antigen
antibody sites
Red blood cells Lag of agglutination is a
Hemagglutination spontaneously positive test, indicating
Inhibition agglutinate if viral the presence of patient
particles are present antigen

i. APPLICATION OF AGGLUTINATION TESTS IN

SEROLOGY

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 11

 MODULE 4: SEROLOGIC TEST FOR SYPHILIS

• Test for Syphilis

OUTLINE
I Syphilis 1. Non-specific or non-treponemal serological tests
A Antigens ▪ For screening of syphilis
B Antibodies ▪ Serological tests that detect reagin
C Spinal Fluid Tests in Syphilis - Reagin: substance present in the serum of patients with certain diseases
i Biological False Positive Antibody (BFP) Reagin Antibody including syphilis
▪ Become positive 1-4 weeks after the appearance of initial symptoms
▪ Other Non-treponemal tests:
I. SYPHILIS - Toluidine Red Unheated Serum Test (TRUST)
• Causative agent: Treponema pallidum - Unheated Serum Reagin (USR)
• Gram (-) negative spirochetes, obligate intracellular parasite - Reagin Screen Test (RST)
• Venereal disease 2. Specific Treponemal antibody tests
• Two types of transmission: ▪ Do this after tested (+) positive in Non-specific or non-treponemal serological tests
1. Acquired: transmission through body fluids (VDRL or RPR)
▪ Sexual contact ▪ Confirmatory test
▪ Sharing of contaminated needles (skin lesion) ▪ Uses Treponema pallidum
▪ Direct contact - Antigen
2. Congenital: Mother to baby transmission - To detect specific antibodies produced by the patients suspected with syphilis
▪ Transplacental ▪ Other Serological Tests For Syphilis
▪ During Childbirth - Anti-Treponemal antibody (ATA)
• Stages of Syphilis - Anti-Treponemal ABs group detected by Reiter Protein Complement Fixation
1. Primary Stage: Hard Chancre/Hunterian Chancre Test (RPCFT)
2. Secondary Stage: Condylomata lata, may involve CNS, eyes, bones, liver ✓ Appears later than specific ABs
3. Latent Stage: No sign asymptomatic/non-reactive ✓ Some syphilis patient do not produce the form of ABs
4. Tertiary Stage: Gummas, involve deep organs ✓ Use is limited
Non-specific or Non-treponemal Serological Tests
Venereal Disease Research Laboratory (VDRL) Rapid Plasma Reagin (RPR)
Old method Modified version
• Read Microscopically using Microscope • Read Macroscopically
○ Cannot be read macroscopically because it will be hard to see the flocculation due o due to charcoal
to its color (White) o Clumping of cells is seen immediately Non-reactive Weakly reactive Strongly reactive
Non-reactive Reactive
Examination

From Left to Right: Reactive (with flocculation), non-reactive (without flocculation)

• Heated serum (common) - heated at 56C for 30 minutes • Unheated Serum
Specimen
• CSF (only if the baby is suspected with neurosyphilis) *Not recommended for CSF specimens
Method Microflocculation method Macroflocculation method
composed of rings which are 15mm in diameter and 1.75mm deep
Slide

• 0.03% Cardiolipin: serves as antigen • Original VDRL reagent • Phosphate: enhances solution
Reagent • 0.21% Lecithin: enhances sensitivity of the cardiolipin • Disodium EDTA: enhances the suspension • Thimerosal: preservative
• 0.90% Cholesterol: enhances the whole reaction • Charcoal: visualization • Choline Chloride: eliminates heating process
1. Add 1 drop of VDRL reagent using an
• Methods:
appropriate needle gauge depending on the
o Qualitative: 18-gauge needle • Add 1 drop of each reagent
Procedure method used (refer below)
o Quantitative: 19-gauge needle • Place in the ROTATOR at 100 rpm for 8 mins
2. Place it in ROTATOR at 180 rpm for 4 mins
o CSF: 22/23-gauge needle
o >4 mins = cannot be read
• Reactive (+): with flocculation • Reactive (+) o HIV • Weakly Reactive
Manner of Reporting/Result
• Non-Reactive (-): without flocculation o Syphilis: w/ warts o HPV: if w/ warts • Non-Reactive (-)
• False (+) positive result o Rheumatic fever o Malaria • False (+) positive result ▪ TB o Pregnancy
False result o SLE o Infectious Mononucleosis (IM) o Pregnancy o Collagen disease ▪ Chickenpox o Old age
▪ Arthritis ▪ Hepatitis • False (-) negative result

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 12

 MODULE 4: SEROLOGIC TEST FOR SYPHILIS

▪ Lupus Erythematosus ▪ Measles o Technical errors

o Infections ▪ Leprosy ▪ wrong specs of agitation
▪ Infectious Mononucleosis (IM) o Autoimmune disease ▪ expired reagents
▪ Malaria ▪ Gonorrhea o Low antibody titer
▪ Prozone

Specific Treponemal Antibody Tests

Treponema Pallidum Immobilization Fluorescence Treponemal Antibody Absorption Treponema Pallidum Microhemagglutination T. Enzyme Linked
Test
Test (TPI) Test (FTA-ABS) Hemagglutination Test (TPHA) Pallidum Test (MHA-TP) Immunosorbent Assay (ELISA)
• In Other books: Most specific is FTA-ABS • Less sensitive
o If there are TPI and FTA-ABS in multiple choices, • Disadvantage: Less reliable in
Description Most specific test for syphilis
answer FTA-ABS diagnosing primary syphilis (causes
o UPDATED false (+) results)
• Neutralization
o Specific T. pallidum antibodies from • Indirect fluorescence
Principle Indirect agglutination Hemagglutination Indirect ELISA
the patient are mixed with actively o Increase the specificity of the reiter strain
motile T. pallidum (Nichols strain)
• T. pallidum reiter strain • T. pallidum antigen
• T. pallidum nio\chols strain • Glutaraldehyde stabilized either with • Enzyme-labeled antibodies
• Nichols strain: live actively motile T.
pallidum organism extracted from • Fluorescent-labeled antibodies animal RBC (refer below) coated with
Tanned formalin sheep RBC
o Enzyme-labeled used:
Reagent and Composition tissues/lesions of infected rabbits o Composition: Dead T. pallidum. Dried and fixed treponemal antigen
coated with treponemal
Alkaline phosphatase
o Absorbent: Reiter teponemes o Sheep RBC o Substrate used: Para-
• Complement: Guinea pig o Chicken RBC
antigen
o Conjugate: Fluorescent-labeled AHG nitrophenyl phosphate
complement
▪ Fluorescein isothiocyanate: Dye used • Turkey RBC ▪ White turns to yellow when
o Dilution: 1:15 alkaline PO is added
1. Patient serum is diluted and mixed with reiter strain 1. Tanned sheep RBCs are coated with
to remove nonspecific T. pallidum antibodies T. pallidum antigen from Nichol’s strain
Procedure 2. The absorbed serum is then added on a slide 2. If specific T. pallidum antigen is
x\containing nichols strain of T. pallidum present in the sample, agglutination
3. Fluorescent-labeled antibodies are then added will occur
(+) Positive: Fluorescence
• (+) Positive: immobilization of
treponemas
(+) Positive:
Result • Reactive (+): >50% (+) Positive: hemagglutination
hemagglutination
• Doubtful: 20-50%
• Negative: <20%

Antigens Antibodies Tests

Non-Treponemal or Non-specific Wasserman antigen (CARDIOLIPIN) REAGIN and anti-cardiolipin 1. VDRL 2. RPR
1. Treponemal group antigens - REITER CHON 1. Group Abs 2. Specific T. pallidum Abs 1. TPI 3. MHA-TP
Treponemal or Specific
2. Specific T. pallidum Antigens (pathogenic) ○ Anti-reiter CHON ○ Anti T. pallidum abs 2. FTA-ABS 4. TPHA

A. ANTIGENS • Treponemal Antibodies

• Wasserman Antigens o Directed against pathogenic T. pallidum and closely related strains
o Cardiolipin (Wasserman Ag) o Group Antibodies: directed group antigens
▪ A hapten and must be bound to a suitable carrier to be antigenic o Specific Treponemal Antibodies: specific for each treponemal antigen
▪ A phospholipid
▪ Microbial Cell Treponemes: serves as the foreign carrier C. SPINAL FLUID TESTS IN SYPHILIS
▪ Bound Phospholipid (cardiolipin): serves as the antigenic determinant
• Treponemal Antigens • Testing of Cerebrospinal Fluid (CSF) is an important part of patient monitoring as well
o Reiter CHON as diagnostic test.
▪ A protein found in most treponemes (both in saprophytic and pathogenic • Neurosyphilis
treponemes) o Abnormally high WBC count
▪ Specific Treponemal Antigens: specific for each treponeme species o Elevated protein levels in the CSF
o (+) VDRL result
B. ANTIBODIES Note: FTA abs IgG and IgM detection continues to be a confirmatory test in diagnosis of
• Non-Treponemal Antibodies syphilis
o Reagin: an anti-cardiolipin Ags o Currently, most hospitals use ELISA, Western blot and PCR

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 13

 MODULE 4: SEROLOGIC TEST FOR SYPHILIS
▪ Methods that are being studied as an additional diagnostic test especially for o Pneumonia
congenital syphilis and neurosyphilis o Vaccination with live attenuated viruses
o Malaria
i. BIOLOGICAL FALSE POSITIVE ANTIBODY (BFP) REAGIN ANTIBODY o Pregnancy
• Chronic
• Associated with other diseases (BFP)
o Leprosy: reagin titer lowers immediately after treatment
• Acute
Comparison of Precipitation Techniques
Technique Application Principle
Nephelometry Igs, Complement, CRP, other serum proteins Light that is scattered at an angle is measured, indicating the amount of antigen or antibody present
Antigen diffuses out into gel that is infused with antibody.
Radial Immunodiffusion Igs, Complement
Measurement of the radius indicates concentration of the antigen
Both antigen and antibody diffuse out from wells in a gel.
Diffusion Complex antigens such as fungal antigens
Lines of precipitate formed indicate the relationship of antigens
Immunoelectrophoresis Differentiation of serum proteins Electrophoresis of serum followed by diffusion of antibody from wells
Immunofixation Electrophoresis Over – or – under production of antibody Electrophoresis of serum followed by direct application of antibody to the gel

Comparison of Agglutination Reactions

Type Of Reaction Principle Results
Direct Agglutination Antigen is naturally found on a particle
Indirect (Passive) Agglutination Particles coated with antigens not normally found on their surfaces Agglutination indicates the presence of patient antibody
Reverse-Passive Particles are coated with reagent antibody
Haptens attached to carrier particle.
Agglutination Inhibition Lack of agglutination is a positive test, indicating the presence of
Particles compete with patient antigens for a limited number of antibody sites
patient antigen
Hemagglutination Inhibition Red blood cells spontaneously agglutinate if viral particles are present

Comparison of Tests Used for the Diagnosis of Syphilis

Type of Reaction Principle Results Comments
Direct Agglutination Antigen is naturally found on a particle
MICROSCOPY
Indirect (Passive)
Particles coated with antigens not normally found on their surfaces Agglutination indicates the Requires active lesion. Must have good specimen, experienced technologist;
Agglutination
presence of patient antibody inexpensive
Requires active lesion.
Reverse-Passive Particles are coated with reagent antibody
More specific than darkfield; specimen does not have to be live
Haptens attached to carrier particle.
Agglutination Inhibition
Particles compete with patient antigens for a limited number of antibody sites Lack of agglutination is a positive test,
indicating the presence of patient antigen Flocculation; good for screening tests, treatment monitoring, spinal fluid testing;
Hemagglutination Inhibition Red blood cells spontaneously agglutinate if viral particles are present
false positives
RPR Modified VDRL with charcoal particles, more sensitive than VDRL in primary SY
Cardiolipin Reagin
TRUST Uses red particles to visualize the reaction; similar to RPR
Treponemal
FTA-ABS Nichol’s strain of T. pallidum Confirmatory; specific, sensitive, may be negative in primary stage
EIA Treponemal or recombinant Anti-treponemal Simple to perform; can be automated; not as sensitive as FTA-ABS
MHA-TP or SERODIA TP-PA Sheep’s RBCs or gel particles sensitized with T. pallidum sonicate Anti-treponemal Not as sensitive as FTA-ABS
PCR Non-treponemal DNA in patient sample is amplified None Highest sensitivity is in primary stage of SY; availability is limited

Characteristics of Acute Phase Reactants

Protein Response Time (hr) Normal Conc. (mg/dl) Increase Function
CRP 6-10 0.5 1000x Opsonization, complement activation
Serum Amyloid A 24 3.0 1000x Removal of cholesterol
Alpha-Antitrypsin 24 200-400 2-5x Protease inhibitor
Fibrinogen 24 110-400 2-5x Clot formation
Haptoglobin 24 40-200 2-10x Binds hemoglobin
Ceruloplasmin 48-72 20-40 2x Binds copper, oxidizes iron
C3 48-72 60-140 2x Opsonization, lysis
Mannose-Binding Protein 0.15-1.0 Complement activation

• CRP, Serum Amyloid: 1000x increase in inflammation

REFERENCES
• Procalcitonin: biomarker of sepsis Notes from the discussion by Mrs. Maria Redora R. Esteban, RMT
• Albumin, Transferrin, Prealbumin: negative acute-phase reactants CANVAS Notes

DARIO, GRANADA, LAGUD, PRIETO, TRESIANA 14