IS LAB P1 MODULES COMPILATION EXPERIMENT 1B Tube Agglutination test
EXPERIMENT 1A Slide Agglutination test
1. Aside from latex particles give examples of
cells that can be used as carrier particles for
agglutination procedures. *
Other cells that can be used as carrier particles
for agglutination procedures are red blood cells.
 
2. Give the factors that influence agglutination
reactions. *
The factors that influence agglutination reactions
are as follows: size and class of antibody, nature
of antigen epitope, temperature, pH, incubation
time, ionic strength of solution, and incubation
time.
 
3. Enumerate the types of agglutination
reactions and briefly discuss each. *
Direct Agglutination – described as antigen-
antibody reaction that happens when antigens are
located on a particle naturally; use of known
bacterial antigens to test for presence of unknown
antibodies in the patient is one of the best
examples of direct agglutination testing.
Passive agglutination – reaction that happens
when antigens are not generally found on the
surfaces of particles such as erythrocytes, latex,
gelatin, and silicates; generally utilized as
screening tools.
Reverse passive agglutination – reaction wherein
an antibody is linked to a carrier particle instead
of an antigen, and this antibody must still be
reactive and be connected in such a way that the
active sites face forward; microbial antigens are
frequently detected using this method.
Hemagglutination inhibition – work on same
principle as agglutination inhibition, except the
indicator particles are red blood cells; antibodies
to viruses such as HBV and influenza have been
detected using this method.
Agglutination inhibition – based on competition
between particulate and soluble antigen for a
limited number of antibody-combining sites, and
absence of agglutination indicates a positive
reaction; can detect very minute amounts of
antigen.
Coagglutination – bacteria serve as inert particles
to which antibodies are bound; although it is
extremely specific, it may not be as sensitive as
latex agglutination in identifying minute amounts
of antigen.
1. This technique of tube agglutination method
may be incubated at what temperature? *
This tube agglutination method can be incubated
at 37 degrees Celsius.
 
2. What class of immunoglobulin is detected
after the 37 °C incubation time? *
IgG class is the immunoglobulin class that is
detected after 37 degrees Celsius incubation
time.
EXPERIMENT 2 Precipitation test
1. What immunoglobulin class is responsible
for the turbidity at 37 °C? and explain why? *
The immunoglobulin class responsible for
turbidity at 37 degrees Celsius is IgG class. The
IgG will react at RT and 37°C (warm agglutinating
antibody) in saline phase, in enzymes, and
antiglobulin phases.
 
2. What immunoglobulin class is responsible
for the turbidity at refrigeration temperature?
and explain why? *
The immunoglobulin class which is responsible
for the turbidity at refrigeration temperature IgM
class. The IgM class is known to react best at 4
degrees Celsius (cold agglutinating antibody) in
saline phase, as compared to IgG which reacts at
37 degrees Celsius.
EXPERIMENT 3 Serial Dilution
define the word "titer" *
The antibody titer is a test that detects the
presence and measures the amount of antibodies
within a person's blood. The amount and diversity
of antibodies correlates to the strength of the
body's immune response.
If your titers are positive, it may indicate you have
already been exposed to a specific pathogen or
may have received the vaccine for it. That means
that you have adequate immunity to a particular
infectious disease.
If your titers are positive, it may indicate you have
already been exposed to a specific pathogen or
may have received the vaccine for it. That means
that you have adequate immunity to a particular
infectious disease.
If your titers are positive, it may indicate you have
already been exposed to a specific pathogen or
may have received the vaccine for it. That means
that you have adequate immunity to a particular
infectious disease.
define the phrase "serial dilution" *
A serial dilution is the stepwise dilution of a
substance in solution. Usually the dilution factor at
each step is constant, resulting in a geometric
progression of the concentration in a logarithmic
fashion. A ten-fold serial dilution could be 1 M, 0.1
M, 0.01 M, 0.001 M ... Serial dilutions are used to
accurately create highly diluted solutions as well as
solutions for experiments resulting in concentration
curves with a logarithmic scale.
Serial dilution is a common technique used in many
immunologic procedures. A small amount of serum
or solute can be serially diluted by transferring
aliquots to diluent. One of the most common series
doubles the dilution factor with each transfer (1:2,
1:4, 1:8...). These dilutions can be done in
microtiter plates or test tubes depending on the
volumes of sample and diluent used.
define the word "dilution" *
Dilution refers to the process of adding additional
solvent to a solution to decrease its concentration.
This process keeps the amount of solute constant,
but increases the total amount of solution, thereby
decreasing its final concentration.
define the phrase "pro-zone effect" *
Prozone phenomenon is a false negative response
resulting from high antibody titer which interferes
with formation of antigen- antibody lattice,
necessary to visualize a positive flocculation test.
The prozone effect is most often associated with
secondary syphilis, Human immunodeficiency virus
(HIV) co-infection, and pregnancy. The hook effect
or the prozone effect is an immunologic
phenomenon whereby the effectiveness of
antibodies to form immune complexes is
sometimes impaired when concentrations of an
antibody or an antigen are very high. The formation
of immune complexes stops increasing with greater
concentrations and then decreases with extremely
high concentrations, producing a hook shape on a
graph of measurements.
EXPERIMENT 4A WIDAL reaction slide test
(screening test)
Define: febrile agglutinins *
Febrile (warm) agglutinins are active at normal
body temperatures.
Define: Salmonellosis *
Salmonellosis is an infection with a bacteria called
Salmonella, Salmonella live in the intestinal tracts
of animals, including birds. Salmonella are usually
transmitted to humans by eating foods
contaminated with animal feces.
Define: typhoid fever *
Typhoid fever is a bacterial infection that can
spread throughout the body, affecting many
organs. Without prompt treatment, it can cause
serious complications and can be fatal. It's caused
by a bacterium called Salmonella typhi, which is
related to the bacteria that cause salmonella food
poisoning.
Define: rose spots *
Rose spots describe the rash that occurs in up to
30% of people infected with Salmonella enterica
serovar typhi. Characteristically, rose spots are
seen in untreated typhoid fever. Rose spots usually
occur between the second and fourth week of
illness.
Groups of 5–15 pink blanching papules (little
bumps) appear on the anterior trunk. The papules
range in size from 2–8 mm. They are usually
distributed between the level of the nipples and
umbilicus, but can also be found on proximal
extremities and back.
Each lasts 3–5 days.
Differentiate the O and H antigen of Salmonella
typhi. *
S typhi O positive means active infection of typhoid
fever. Active infection indicates the patient has an
infection of Salmonella enterica serovar typhi. S
typhi H positive means there is a past infection or it
shows the result of the immunized person's serum
test report.
EXPERIMENT 4B WIDAL Reaction tube test
(confirmatory test)
1. Aside from the WIDAL test, what are other
methods in determining febrile agglutinins? *
The ICT immunochromatographic test (IgM)
is better alternative to Widal test for early and
accurate diagnosis of typhoid fever. The Dot-EIA
(enzyme immunoassay; typhidot) is a more
sensitive and specific test which is easy to perform
and more reliable compared to the Widal test and
that it is useful in early therapy. ELISA is an
alternative diagnostic methodology for WIDAL.
 
2. Explain the principle of the Widal reaction. *
The main principle of widal test is that if
homologous antibody is present in patients serum,
it will react with respective antigen in the reagent
and gives visible clumping on the test card and
agglutination in the tube. The antigens used in the
test are “H” and “O” antigens of Salmonella Typhi
and “H” antigen of S.
3. Cite the possible disadvantage/s of Widal
test. *
Limitations of Widal Test:
The Widal test may be falsely positive in patients
who have had previous vaccination or infection with
S. Typhi. Besides cross-reactivity with other
Salmonella species, the test cannot distinguish
between a current infection and a previous
infection or vaccination against typhoid.
EXPERIMENT 5A Lateral Flow Chromatographic
Immunoassay Onsite Syphilis Antibody Test –
Dip Strip
What are the antibodies detected in primary and
secondary syphilis? *
Primary syphilis
IgM Ab: expect to be + 2-3 weeks after infection
IgG Ab: expect to be + 4-5 weeks after infection
Secondary syphilis
IgM Ab: should still be + early in stage 2, but may
start to decrease
IgG Ab: expect to be + at HIGH LEVELS
NOTE: all nontreponemal and treponemal tests
should be + during this stage
(RPR, FTA, TP-PA, MHA-TP, VDRL)
There are 2 different types of serologic tests
classified based on the type of antigen the
antibodies are directed against. Treponemal tests
detect antibody to T pallidum proteins.
Nontreponemal tests detect antibodies directed
against lipoidal antigens, damaged host cells, and
possibly from treponemes.
Enumerate the mode of transmission of
syphilis. *
Syphilis is transmitted from person to person by
direct contact with a syphilitic sore, known as a
chancre. Chancres can occur on or around the
external genitals, in the vagina, around the anus ,
or in the rectum, or in or around the mouth.
Transmission of syphilis can occur during vaginal,
anal, or oral sex. In addition, pregnant women with
syphilis can transmit the infection to their unborn
child.
Risk factors - You face an increased risk of
acquiring syphilis if you:
Engage in unprotected sex
Have sex with multiple partners
Are a man who has sex with men
Are infected with HIV, the virus that causes AIDS
EXPERIMENT 5B VDRL [Venereal Disease
Research Laboratory] SLIDE QUANTITATIVE
ON SERUMVDR
Aside from serum, give another specimen in
which VDRL can also be done: *
The VDRL test, which was designed by the
Venereal Disease Research Laboratories, is both a
qualitative and quantitative slide flocculation test for
serum, and there is a modification for use on spinal
fluid.
Antigen for all tests must be prepared
fresh daily and is done so in a highly regulated
fashion.
CSF can also be utilized
Collection Container/Tube: Sterile vial Submission
Container/Tube: Plastic vial
Specimen Volume: 0.5 mL
Remember:
The VDRL is a non-treponemal-specific test;
therefore, a treponemal-specific confirmatory test
should be performed unless prior syphilis infection
has been documented for this patient.
SPECIMEN REQUIREMENTS: Draw blood in a
plain red-top tube(s), serum gel tube(s) is
acceptable. Spin down and send 1 mL
of serum refrigerated in a plastic vial.
Non – treponemal antibodies are reacted with? *
Nontreponemal tests are an indirect method in that
they detect biomarkers that are released during
cellular damage that occurs from the syphilis
spirochete.
Syphilitic infection leads to the production of
nonspecific antibodies that react to cardiolipin.
T he non-Treponemal tests such as RPR or the
VDRL test measure the hosts response to non-
Treponemal antigens such as cardiolipin and
lecithin released from the damaged host cells, as
well as lipoprotein-like material released from the
Treponema
This reaction is the foundation of “nontreponemal”
assays such as the VDRL (Venereal Disease
Research Laboratory) test and Rapid Plasma
Reagin (RPR) test.
What is the purpose of heating the serum at 56
°C? *
 The serum specimens to be tested are
heated at 56ºC for 30 minutes to inactivate
complement. Also the risk if coagulation of serum is
rather slight at temperatures about 56ºC.
EXPERIMENT 5C Rapid Plasma Reagin (RPR) EXPERIMENT 6B Rapid Latex Agglutination
Test Antistreptolysin O Procedure

What is the purpose of chlorine chloride in the Cite some causes of false-positive reactions in
RPR test? * the test involved. *
chlorine chloride stabilizes the antigen and False positive ASO titers can be caused by
inactivate complement so that serum does not increased levels of serum beta-lipoprotein
have to be heat-inactivated before use produced in liver disease and by contamination of
the serum with Bacillus cereus and Pseudomonas.
Aside from RPR and VDRL tests, give another ASO titers are elevated in 85% of patients with
example of the reagin test for syphilis. * rheumatic fever but may not be elevated in cases
toluidine red unheated serum test (TRUST), involving skin or renal sequelae.
and reagin screen test (RST)  
Compare Streptolysin O from Streptolysin S. *
Streptolysin O is oxygen-labile whereas
streptolysin S is oxygen-stable. This means that
EXPERIMENT 6A anti-Streptolysin O (ASO) streptolysin S is stable in the presence of
Titration atmospheric oxygen. In blood agar, the streptolysin
S forms a zone of β hemolysis around the colonies
What are the complications of S. pyogenes of streptococci grown under routine aerobic
infection? * conditions.
Complications of S. pyogenes infections
can be divided into suppurative and non- Key Difference – Streptolysin O vs Streptolysin S
suppurative complications.
Streptolysin is considered as a
Suppurative complications include streptococcal hemolytic exotoxin. In simple terms, it
peritonsillar abscess, peritonsillar cellulitis, is the hemolysin produced by Streptococcus
retropharyngeal abscess, otitis media and sinusitis, bacteria. Hemolysins are compounds that could be
uvulitis, cervical lymphadenitis, meningitis, and lipids or proteins that have the potential to cause
brain abscess, arthritis, endocarditis, osteomyelitis, the destruction of red blood cells through lysis.
and liver abscess. There are different streptolysin types such as
streptolysin O (SLO), streptolysin S (SLS), etc.
Non-suppurative complications are Streptolysin O is active only during conditions of
rheumatic fever, post-streptococcal reversibly reduced states and Streptolysin S is
glomerulonephritis, PANDAS (pediatric stable during conditions where oxygen is present at
autoimmune neuropsychiatric disorders associated higher concentrations. Therefore, streptolysin O is
with streptococcal infections), Sydenham chorea, considered as oxygen labile and streptolysin S is
and other autoimmune movement disorders considered as oxygen stable. This is the key
difference between Streptolysin O and Streptolysin
https://www.ncbi.nlm.nih.gov/books/NBK554528/ S.
 
Enumerate some diseases or conditions
wherein the ASO titer is increased. *
positive test can indicate current but more recent
group A, C, and G streptococcal infection and may
support the diagnosis of poststreptococcal infection

streptococcal infections (acute rheumatic

fever, glomerulonephritis)