APPLIED IMMUNOLOGY

NAME: SHUKRI BIN JAFFAR

MATRIX NUMBER: MEB 040045
DATE : 29.01.2008

DIRECT AND INDIRECT AGGLUTINATION ASSAY

OBJECTIVES
• To understand the concepts of direct agglutination.
• To perform blood group determination test, antistreptolysin O (ASO) latex slide
test, rheumatoid factor (RF) latex slide test and rapid plasma regain (RPR) card
test.

PRINCIPLE
Direct agglutination assay involves clumping of cells or particulate antigen by specific
antibody with 2 or more receptors linking the particles in suspension. Such
agglutination reaction requires a minimum cell surface antigen density and access of
antibodies to their complementary cell surface antigens. The most common example of
a direct agglutination reaction is red blood cell aggregation through the addition of
ABO isohemagglutinins.

BLOOD GROUP DETERMINATION

In blood group determination using direct agglutination method, agglutination takes
place when particulate antigen, e.g. red blood cells, is exposed to the appropriate
antibody under suitable conditions. When these antigens combine with their specific
antibody, agglutination of the particle is seen. If one of the reactants is of known
specificity, the clumping may be used for the identification of the corresponding
reactant.

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MATERIALS
Reagents
1. Known group A, B, AB and O human erythrocytes, washed and resuspended in
normal saline to make a 3-4% cell suspension
2. Normal saline (NaCl 0.85%)
3. Anti-A, Anti-B, Anti-AB and anti-Rh serum

Equipments
1. Glass microscopic slides
2. Disposable Pasteur pipettes
3. Glass Pasteur pipettes
4. Glass test tubes
5. Disposable sterile, stainless steel lancets
6. Alcohol and swab
7. Applicator sticks or toothpicks

PROCEDURE
1. A drop of anti-A serum is placed on the left side of the glass slide and a drop of
Anti-B serum on the right side of the slide.
2. A drop of 3-4% cell suspension of known A cells is placed next to each drop of
serum.
3. The 2 drops on each of the slide is mixed with an applicator or toothpick.
4. The slide is rotated gently while examining it macroscopically. The result observed
is recorded.
5. Step 1 to 4 is repeated to observe the reaction of known cells of group B, ANTIBODY
and O in the presence of anti-A and anti-B serum.
6. My own blood group is determined. A subject’s finger is swabbed with alcohol.
Using a disposable, sterile lancet, the swabbed spot is pricked. Several drops of
blood are allowed to flow directly into a tube containing 1ml saline.

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7. The blood suspension is mixed and proceeds as in steps 1 to 4. The results are
recorded.

RESULTS
Group ‘A’ Group ‘B’ Group ‘AB’ Group ‘O’
Anti-A + - + -
Anti-B - + + -
Anti-AB + + + -
Anti-Rh + + + +

• Positive (+) : Presence of coagulation

• Negative (-) : No coagulation
• My blood group is ‘O’

DISCUSSIONS
Humans can be typed for the ABO blood group where there are four principal
types; A, B, AB, and O. There are two antigens and two antibodies that are mostly
responsible for the ABO types. The specific combination of these four components
determines an individual's type in most cases. The table below shows the antigens and
antibodies with the corresponding ABO type.

ABO Blood Antigen A Antigen B Antibody Anti- Anitbody Anti-

type A B
‘A’ + _ _ +
‘B’ - + + _
‘O’ _ _ + +
‘AB’ + + _ _

For example, people with type A blood

will have the A antigen on the surface of their
red cells. As a result, anti-A antibodies will

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not be produced by them because they would cause the destruction of their own blood.
However, if B type blood is injected into their systems, anti-B antibodies in their plasma
will recognize it as alien and burst or agglutinate the introduced red cells in order to
cleanse the blood of alien protein.

Individuals with type O blood do not produce ABO antigens. Therefore, their
blood normally will not be rejected when it is given to others with different ABO types.
As a result, type O people are universal donors for transfusions, but they can receive
only type O blood themselves. Those who have type AB blood do not make any ABO
antibodies. Their blood does not discriminate against any other ABO type.
Consequently, they are universal receivers for transfusions, but their blood will be
agglutinated when given to people with every other type because they produce both
kinds of antigens.

Rh blood typing is also important for transfusions. This blood type test
determines whether the Rh-factor (D) is present (Rh-positive) or not present (Rh-
negative) in your blood cells. If blood type tests indicate that you are Rh-negative, you
should always receive Rh-negative blood except if at all possible. Rh-positive patients
can receive Rh-positive or Rh-negative blood.

CONCLUSIONS
Direct agglutination method can be used to determine the ABO blood group system.

ANTISTREPTOLYSIN O (ASO) LATEX SLIDE TEST

INTRODUCTION
The group A β-hemolytic streptococci produces various toxins that can act as antigens.
One of these exotoxins Streptolysin O was discovered by Todd in 1932. A person
infected with group A β-hemolytic streptococci produces specific antibodies against

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these exotoxins, one of which is antistreptolysin O (ASO). The quantity of this antibody
in a patient’s serum will establish the degree of infection due to the β-hemolytic
streptococcal. The usual procedure for the determination of the antistreptolysin titer is
based on the inhibitory effect that the patient’s serum produces on the hemolytic power
of a pretitrated and reduced Streptolysin O. However, the antigen-antibody reaction
occurs independently of the hemolytic activity of Streptolysin O. This property enables
the establishment of a qualitative and quantitative test for the determination of the ASO
by agglutination of latex particles on slide.

PRINCIPLE
ASO test method is based on an immunological reaction between streptococcal
exoenzymes bound to biologically inert latex particles and streptococcal antibodies in
the test sample. The reagent has been adjusted in the way that presence of an ASO titer
of 200 IU/ml or higher in the serum gives a visible agglutination of the latex particles
without previous sample dilution.

MATERIALS
1. ASO Latex Reagent
2. ASO Positive Control
3. ASO Negative Control
4. Glycine-Saline Buffer
5. Reaction Slide
6. Pipette / Stir sticks
7. Timer
8. Test tubes, Rack
9. Serological Pipettes

PROCEDURE
Qualitative test
1. The reagents and specimens are brought to room temperature before use.

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2. One drop (50μl) of ASO positive control is placed on field 1 of the reaction slide. One
drop (50μL) of the ASO negative control is placed on field 2 of the reaction slide.
3. Using pipette/stir stick, 1 drop (50μl) of undiluted test serum sample is drop to field
# 3. The pipette/stir sticks are retained for mixing step.
4. Gently, the ASO Latex reagent is resuspended and one drop is added to each test
field.
5. It is then mixed well with the flat end of the pipette. The slide is gently rock for two
minutes and immediately read under direct light.

Semi-quantitative test
1. At least five test tubes: 1:2, 1:4, 1:8, 1:16, 1:32 etc is set up and the samples are diluted
according to dilution factors on each test tube with diluted saline solution.
Dilutions 1:2 1:4 1:8
Sample serum 100μl - -
Saline 100μl 100μl 100μl
Diluted sample - 100μl 100μl

2. One drop of each of positive and negative controls is placed onto the slide rings.
One drop of each dilution is placed on successive fields of the reaction slides.
3. Gently, the ASO latex reagent is resuspended and one drop is added to each test
field.
4. It is then mixed well with the flat end of the pipette. The slide is gently rock for two
minutes and immediately read under direct light.
RESULTS
Qualitative test
SAMPLE RESULTS
1 Negative
2 Negative
3 Positive
4 Positive

Semi-quantitative test

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 TITER RESULTS
1:2 Positive
1:4 Negative
1:8 Negative
1:16 Negative
1:32 Negative

• Dilution = 1:2
• Reciprocal =2
• IU/ml of sample 3 = 200 X 2
= 400 IU/ml

DISCUSSIONS
Antistreptolysin-O test is performed for determine the streptococcal infection,
where the Streptococcal bacteria produce an exoenzymes, which is known as
Streptolysin-O. This antibody can be detected by qualitative test or semi-quantitative
test. This test can help distinguish the beta-hemolytic Group A Streptococcal rheumatic
fever from acute rheumatic diseases. A normal ASO reference range for adults is <100
Todd units or a 1:99 dilution.

From the results obtained, the concentration of antistreptolysin O (ASO) in sample 3 is

400 IU/ml. But, there are some limitations of this test include:
a) Consumption of certain drugs and hemolysis serum may affect the results
obtained.
b) Reading test result within two minutes could probably incorrect because of
timing.

CONCLUSIONS

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Antistreptolysin O (ASO) slide test can be used to detect and measured the
concentration of antistreptolysin O (ASO) antibodies. The concentration of ASO
antibody in sample 3 is 400 IU/ml.

RHEUMATOID FACTOR (RF) LATEX SLIDE TEST

INTRODUCTION
Rheumatoid factors (RF) are antibodies directed against antigenic sites in the Fc
fragment of human and animal IgG. Their frequent occurrence in rheumatoid arthritis
makes them useful for diagnosis and monitoring of the disease. One method used for
rheumatoid factor detection is based on the ability of rheumatoid arthritis sera to
agglutinate sensitized sheep red cells, as observed by Waaler and Rose. A more
sensitive reagent consisting of biologically inert latex beads coated with human gamma

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globulin was later described by Singer and Plotz. The RF kit is based on the principle of
latex agglutionation assay by Singer and Plotz. The major advantage of this method is
rapid performance (3 minute reaction time) and lack of heterophile antibody
interference.

PRINCIPLE
The RF reagent is a suspension of polystyrene latex particles sensitized with specially
prepared human IgG. The reagent is based on an immunological reaction between
human IgG bound to biologically inert latex particles and rheumatoid factors in the test
specimen. When serum containing rheumatoid factors is mixed with the latex reagent,
visible agglutination occurs. The RF latex reagent sensitivity has been adjusted to detect
a minimum of 8 IU/ml of rheumatoid factors according with the WHO International
Standard without previous sample dilution.

MATERIALS
1. RF Latex Reagent
2. RF Positive Control
3. RF Negative Control
4. Glycine-Saline Buffer
5. Reaction Slide
6. Pipette Stir Sticks
7. Timer
8. Test Tubes
9. Serological pipettes

PROCEDURE
Qualitative test
1. The reagents and specimens are brought to room temperature before used.

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2. One drop (50μl) of the RF Positive Control is placed on field 1 of the reaction slide.
One drop (50μl) of the RF Negative Control is placed on field 2. The remaining fields
are used for test specimens.
3. Using pipettes provided, one drop of the undiluted specimens is placed on
successive fields. The Pipette Stir is retained for mixing step.
4. The RF Latex Reagent is gently resuspended and one drop is added to each test
field. The pipette Stir Stick is used to spread reaction mixture over entire test field.
5. The slide is rotated manually or with a mechanical rotor at 80-100 rpm for 2 minutes
and immediately read under direct light.
6. Presence of agglutination of the latex particle is a positive result. Agglutination
indicates a RF concentration of equal or more than 8 IU/ml. Sera with positive
agglutination should be run again with the quantitative test.

Quantitative test
1. The reagents and specimens are brought to room temperature before used.
2. Using the Glycine-Saline Buffer, the specimens are diluted 1:2, 1:4, 1:8, 1:16, 1:32 or
as needed.
3. One drop (50μl) each of negative and positive controls on two slide rings. One drop
(50μl) of each dilution is placed on successive fields of the reaction slide.
4. The RF Latex Reagent is gently resuspended and one drop is added to each test
field. The pipette/Stir stick is used to spread reaction mixture over entire field.
5. The slide is rotated for 2 minutes and read immediately under direct light.

RESULTS
Qualitative test
SAMPLE RESULTS
A Positive
B Negative
C Negative
D Negative

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Quantitative test
TITER RESULTS
1:2 Positive
1:4 Positive
1:8 Positive
1:16 Negative
1:32 Negative

• Dilution = 1:8
• Reciprocal =8
• IU/ml of sample A =8X8
= 64 IU/ml

DISCUSSIONS

A rheumatoid factor (RF) latex slide test measures the amount of the RF antibody
present in the blood. Normally, antibodies are produced by the immune system to help
destroy and eliminate invading bacteria and viruses that can cause disease. This method
is best used as a first-time screening test for rheumatoid arthritis.

However, the RF antibody can attach to normal body tissue, resulting in damage. A
high level of rheumatoid factor can be caused by several autoimmune diseases
(including rheumatoid arthritis) and some severe infections. Occasionally an elevated
level of RF is present in healthy people.

In this experiment, sample A

showed a positive sample with
means presence of autoantibody in
serum. The concentration of

POSITIVE NEGATIVE

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rheumatoid factor for this sample is 64 IU/ml. Nevertheless, there are some limitations
for this test :
a) High level of rheumatoid factor could also due to other diseases such as
infectious mononucleosis, sarcodosis, lupus erythematosus and Sjogren’s
syndrome.
b) Certain patient with rheumatoid arthritis is negative of rheumatoid factor on
their serum.
c) Taken the reading results after 2 minutes could be inaccurate because can formed
alike positive of clumping.

CONCLUSIONS
The Rheumatoid factor (RF) latex slide test is standard test for first-time screening test
for rheumatoid arthritis, as to detect the presence of autoantibody (rheumatoid factor)
in patient serum. Experiment on four type sample have showed patient A with 64
IU/ml.

RAPID PLASMA REAGIN (RPR) CARD TEST

PRINCIPLE
Treponema pallidum, the etiological agent of syphilis, produces at least two types of
antibodies in human infections: treponemal antibodies that can be detected by
Fluorescent Treponemal Antibody- Absorption (FTA-ABS) test and non-treponemal
antibody (reagin) that can be detected by RPR antigen card test.

The RPR card test is a non-treponemal test for the serological detection of syphilis.
Either clear unhemolsyed serum or plasma from EDTA anticoagulated blood may be

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tested. The antigen suspension consists of carbon particles and a cardiolipin-lecithin
extract of beef heart. This non-specific antigen detects regain, an IgM or IgG
immunoglobulin present in the plasma or serum of patients with syphilis and
occasionally present in the serum or plasma of individuals with other acute or chronic
conditions.

If reagin is present it binds to the cardiolipin antigen resulting in flocculation. The

carbon particles become trapped in the flocculation and appear agglutinated or as black
clumps against a white background on the test card. The agglutination can be seen
macroscopically. Specimens that are nonreactive, that is, do not contain reagin, have a
uniform light-gray color and even particle distribution (no clumping).

MATERIALS
1. Timer
2. Rotator (100 ± 2 rpm)
3. Saline (0.9%)
4. Test Tubes
5. Pipettes

PROCEDURE
Qualitative test
1. The RPR carbon antigen suspension controls and samples are brought to room
temperature.
2. The pipette stirrer is hold in vertical position and one drop of serum or plasma
sample is dispensed onto a separate circle on the test card with the disposable stirrer
pipettes supplied. A fresh pipette stirrer is used for each sample.
3. The flat end of he stirrer pipettes is used to spread the sample over the entire area of
the test circle. The pipette stirrer is disposed. The procedure is repeated for a
number of specimens tested.

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4. The carbon antigen suspension dispensing bottle is shaken prior to use. One drop
(20μl) of free falling antigen suspension is placed onto test circle containing the
sample.
5. The test card is placed on an automatic rotator and rotated for 8 minutes at 100 rpm.
6. The results are read macroscopically under a high intensity incandescent lamp or
strong daylight.

Quantitative test
1. The RPR carbon antigen suspension controls and samples are brought to room
temperature.
2. 50μl of specimen to be tested is placed onto the test circle 1.
3. For each specimen to be tested, 50μl of 0.9% saline is placed into test circles
numbered 2 to 5.
4. 50μl of specimen is placed onto test circle 2. The mixture is mixed up and down the
pipette at least 8 times. Formation of bubbles is avoided. 50μl of mixed sample is
transferred from circle 2 to 3. This serial dilution procedure is repeated in circle 4,
then 5. 50μl from circle 5 is disposed after mixing. Circles 1 to 5 now represent a
dilution series as follows:

Circle 1 2 3 4 5
Dilution 1/1 1/2 1/4 1/8 1/16

5. Using a new pipette stirrer for each specimen, start at highest dilution of serum
(circle 5) and the serum is spread over the entire area of test circle. Then, proceed to
circles 4, 3, 2 and 1.
6. The carbon antigen suspension dispensing bottle is shaken prior to use. One drop
(20μl) of free falling antigen suspension is placed onto test circle containing the
sample.
7. Test card is placed on an automatic rotator and rotate for 8 minutes at 100 rpm.

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8. The results are read macroscopically under a high intensity incandescent lamp or
strong daylight.
9. If the sample is positive in the 1/16 dilution, then the dilution series should be
extended as follows:
a) A 1/50 dilution of non-reactive serum is prepare in 0.9% saline. This is to be used
for making 1/32 and higher dilutions of specimens to be tested. 0.05ml of this
diluents solution is dispensed onto circles numbered 2 to 5.
b) A 1/16 dilution of test specimen is prepared by adding 0.1ml of undiluted serum
to 1.5ml of 0.9% saline. It is nixed thoroughly. 0.05ml of 1/16 dilution of test
specimen is dispensed onto circles 1 and 2.
c) On circle 2, the tip of an automatic 0.05ml pipette is inserted into the resulting
mixture (sample and diluents) and mix by drawing the mixture up and down the
pipette approximately 8 times. Bubble formation is avoided. 0.05ml of mixed
sample is transferred to the next circle. The mixing procedure is repeated. This
serial dilution is continued to circle 5 and 0.05ml is discarded from this last circle.
Circles 1 to 5 now represent a dilution series as follows:
Circle 1 2 3 4 5
Dilution 1/16 1/32 1/64 1/128 1/256

d) The test procedure described under steps 5, 6 and 7 is proceed under Semi-
Quantitative Card Test.
e) The dilution is continued until an end-point titer is reached.

RESULTS
Qualitative test
SAMPLE RESULTS
W Negative
X Negative
Y Negative
Z Negative

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DISCUSSIONS
The RPR test is a
nontreponemal testing procedure for
the serologic detection of syphilis. The
RPR card antigen suspension is a
carbon- particle cardiolipin antigen
REACTIVE NON REACTIVE
that detects reagin, an antibodylike
substance present in the sera from syphilitic persons and occasionally in sera of persons
with other acute or chronic conditions. When a specimen contains antibody,
flocculation occurs with a coagglutination of the carbon particles of the RPR card
antigen, which appear as black clumps against the white background of the plastic-
coated card. Nonreactive specimen appears to have an even light-gray color.

From this experiment, all four samples are negative for RPR test which means no
presence of reagin antibody. Even though there are some limitations for this test which
includes:
a) A false positive results occurs due to pregnancy, drug addiction, and advanced
age. Other diseases also have noted a false positive such as lupus erythematosus,
mononucleosis, leprosy, viral pneumonia, after smallpox vaccinations or
hepatitis C vaccination and influenza.
b) Hemolysed serum also could occurs a false positive which alike of flocculation.
c) Temperature of the reagents and incubation time may also affect the result.
d) 48 hours of plasma sample are not reliable.

CONCLUSIONS
Rapid plasma reagin (RPR) test are easy and primarily test for routine screening in
patient of syphilis. Four samples tested have not presense of any positive of RPR test.

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