MOI UNIVERSITY

COLLEGE OF HEALTH SCIENCES

SCHOOL OF MEDICINE

BIOCHEMISTRY DEPARTMENT

COURSE CODE: MSB 103

COURSE TITLE: IMMUNOLOGY

COORDINATOR: MARY CHEPKWEMOI.

LEMOOGE LENGIDA
MBCH.B/5469/21
JULY 8TH 2021

Signature
LABORATORY SAFETY MEASURES
Proper wear of protective gear must always be adhered to which include lab coat,
fully closed, face mask, goggles, gloves aa well as safe removal of gloves to
minimize cross infection
Use needle and syringe to remove blood then recap the needle safely and dispose it
safely. Vacuteneous needle and vacuteneous holders are also used to remove blood
There are two vacuteneous tube for collection of blood
1) Red top tube (for collection of serum)
2) Purple top tube (for collection of plasma)
Difference between syringe and vacuteneous needle is that in vacuteneous needle
there is no contamination while in syringe contamination may occur
Avoid mouth pipetting instead use pipet tips and pipet aid
Use micropipette for small volumes of 10-1000microlitters
Carry samples in a well labelled biohazards bag plus its lab request form
No eating and drinking in lab
Highly infection specimen is handled in a class 2 safety cabinet

PRACTICAL 1: CELLS OF THE IMMUNE SYSTEM

Introduction
The experiment was carried out in order to observe and identify the different cells of
the immune system that is white blood cells, in a thin blood film under a light
microscope. The different cells that were expected to be observed include:
neutrophils, basophils, eosinophils and lymphocytes. Red blood cells were also
expected to be observed.
Principle
- Absolute methanol is the methanol is used to fix the cells so that they remain as
they were in the body
-A thin smear is used so that many cells can be viewed under the microscope
-Giemsa stain is used to stain the cells and bring about contrast so that the cells can
easily be viewed. Erythrocytes stain pink, platelets show light pale pink, lymphocytes
cytoplasm stains sky blue.
Reagents used
1. Blood smear
2. Absolute methanol
3. Giemsa stain
4. Oil immersion
Procedure
1. Put a drop of blood on one end of a slide
2. Put another slide on top of the first, slide it toward the drop of blood at an
angle then push it forward to obtain a thin film
3. Air dry the slide
4. Immerse the slide in Giemsa stain for 10 minutes
5. Dip the slide in absolute methanol
6. Air dry the slide again
7. Put oil immersion on the slide
8. View it under a microscope

Test results
The following cells were viewed:
Red blood cells, neutrophil, eosinophil, basophil, lymphocyte
Interpretation
The neutrophils have a lobulated nucleus made of 2-5 lobes, the granules stain
yellow, the nucleus stains pink and they make up 90% of immune cells.
The eosinophils have a bilobed nucleus, the granules are more than those of the
neutrophils, the nucleus stains pink and they make up 2-5% of the immune cells.
The basophils have a multilobed nucleus, a lot of granules which stain dark blue, a
higher nucleus cytoplasmic ratio than other granulocytes and they make up 0-2 % of
immune cells.
The monocytes have a pink-staining horse shoe shaped nucleus. The cytoplasm
stains sky blue. They appear to be precursor cells for macrophages. The cells are
granular with a high nucleus /cytoplasm ratio.
The lymphocytes have one nucleus which stains pink in color and the cytoplasm
stains sky blue.
Conclusion
The immune cells of the body are responsible for mediating defense of the body
against infections by parasites, bacteria and viruses. The cells can be identified by
use of their morphology and the surface markers the progenitor cells originate from
the bone marrow after birth

PRACTICAL 2: AGGLUTINATION TESTS

Introduction
Agglutination reaction refers to clumping together of cells due to reaction of an
antibody with its corresponding antigens. Therefore, these tests can be used the test
for the presence of a specific antibody or antigen in a sample of serum or blood. If
the antigen in question or being used is a red blood cell, then the reaction is referred
to as hemagglutination.
The tests conducted were on: blood grouping, Antistreptolysin O Titer(ASOT) for
streptococcus, Venereal Disease Research Laboratory(VDRL) test for syphilis and
the Widal Test for Salmonella and Brucella.
2a) BLOOD GROUPING
Introduction
The human red blood cell membrane contains a variety of blood group antigens
called agglutinogens. Type A individuals have the A antigen, type B have the B
antigen, type AB have both antigens and type O have neither antigens on
membranes of red blood cells. Type D antigen is used to test for Rhesus factor.
Rhesus positive individuals have antigen D while the negative ones lack antigen D
on the membranes of their red blood cells.
Principle
Antigens present on the cell surface of red blood cells react with their corresponding
antibodies in antisera to cause visible agglutination of red blood cells.
Reagents and tools
1. Antisera A, B and D
2. Blood
3. White tiles
4. Droppers
5. Timer
6. Mixing rods
Procedure
1. Label 3 portions of a white tile A, B and D
2. Place 3 drops of blood near each label
3. Add a drop of each antiserum on it corresponding blood drop
4. Mix with a mixing rod
5. Let them stand for 2 minutes them observe for any visible agglutination

Test results
There was visible agglutination with all antisera
Interpretation
The patient’s red blood cells contain antigens A, B and D that reacted with all
antisera to cause agglutination.
Conclusion
The blood type of the patient is AB+

2b) ANTISTREPTOLYSIN O TITER (ASOT)

Principle
Antigens specific to streptococcus bacteria react with their corresponding antibodies
to form visible agglutination reaction.
Latex is used as a carrier for the specific antigen
Reagents and tools
1. Latex
2. Serum
3. Positive control
4. Negative control
5. Black Tile
6. Dropper
7. Timer
8. Mixing rods
Procedure
1. Take a black tile with 3 wells for the positive control, negative control and serum
2. Place drops of each reagent in its well
3. Add a drop of latex to each well
4. Mix with a mixing rod
5. Let them stand for 2 minutes them observe for any visible agglutination

Results
The serum and latex mixture resembled that of the negative control and latex.
Interpretation
The patient had no antibodies corresponding to streptococcus antigens
Conclusion
The patient was negative for the streptococcus bacteria

2c) VENEREAL DISEASE RESEARCH LABORATORY(VDRL) TEST

Principle
Antigens specific to Treponema pallidum react with their corresponding antibodies to
form visible agglutination reaction.
Carbon is used as a carrier for the specific antigen
A white tile is used since carbon is black in color
Reagents and tools
1. Carbon
2. Serum
3. Positive control
4. Negative control
5. White tile
6. Dropper
7. Timer
8. Mixing rods
Procedure
1. Take a white tile with 3 wells for the positive control, negative control and serum
2. Place drops of each reagent in its well
3. Add a drop of carbon to each well
4. Mix with a mixing rod
5. Let them stand for 8 minutes them observe for any visible agglutination
Results
The serum and carbon mixture resembled that of the negative control and carbon.
Conclusion
The patient was negative for the Treponema pallidum
Interpretation
The patient had no antibodies corresponding to streptococcus antigens

2d) WIDAL TEST

Principle
Antigens specific to Salmonella or Brucella bacteria react with their corresponding
antibodies to form visible agglutination reaction.
Reagents
1. Bentonite
Both TH(specific for bacteria with flagella) and TO( specific for somatic bacteria)
reagents
2. Serum
3. Positive control
4. Negative control
5. White tile
6. Dropper
7. Timer
8. Mixing rods
Procedure
1. Take a white tile with 6 wells, three on top for TH and three at the bottom
For TO and each of their wells for positive control, negative control and serum
2. Place a drops of the positive control serum and in its well for both the top and
bottom 3 wells
3. Add a drop of TH reagent to each well on the top row and a drop of TO to each
well on the bottom row
4. Mix with mixing rods
5. Let them stand for 2 minutes them observe for any visible agglutination

Results
The mixtures of serum with both TH and TO resembled that of the negative control
with the two reagents.
Conclusion
The patient was negative for Salmonella or Brucella bacteria of both the somatic and
flagella types
Interpretation
The patient had no antibodies corresponding to Salmonella or Brucella antigens
PRACTICAL 3: PRECIPITATION TESTS
Introduction
Precipitation tests are used to detect the presence of antibodies or antigens in
solution form. Corresponding antibodies and antigens react to form antigen-antibody
complexes that are visible as precipitates. The tests are carried out in a supportive
gel made from agarose powder. The types of tests that were performed were: double
immunodiffusion/ Ouchterlony and single immunodiffusion/radial/mancini.
3a) Double Immunodiffusion/ ouchterlony
Introduction
Double immunodiffusion (or agar gel immunodiffusion) is a simple method which is
considered to be the standard for detection of extractable nuclear antigens. It is also
one of the simplest techniques used to check antisera for the presence of antibodies
for a particular antigen and to determine its titre. The method is called "double" since
in this procedure, antigen and antibody are allowed to migrate towards each other in
a gel and a line of precipitation is formed where the two reactants meet. This
precipitation reaction is highly specific.
It is used to evaluate purity of isolated serum protein and in qualitative analysis to
detect presence of antigen and antibody or the complex formed by the two. The test
is also applied in simple diagnosis of several infections such as hydatid disease. It is
performed under room temperature in a moist chamber.
Principle
In double diffusion, both antigen and antibody are allowed to diffuse into the gel. The
antigen and antibody will diffuse in the gel in all directions. This technique can be
used to test the similarity between antigens. Antigens from different species are
loaded into two wells and the known antibody is loaded in a third well located
between and slightly below the antigen wells to form a triangle. The part where they
form an immune complex is represented by a white precipitate. Depending on the
similarity between the antigens, different geometrical patterns are produced between
the antigen and antiserum wells. The pattern of lines that form can be interpreted to
determine whether the antigens are same or different. For a reaction to be efficient,
the Ag and Ab must be specific and the antibody must be in a solution.

Requirements
1. Agarose powder
2. Water
3. Double immunodiffusion templates
4. Ruler
5. Patient serum
6. Dropper
7. Bunsen burner
8. IgM in solution form
Procedure
1. Mix 1.2 g of agarose powder with 100ml of phosphate buffered saline
2. Heat the mixture until it dissolves and forms molten jell
3. Pour the jell on a slide
4. Let it solidify
5. Make 3 wells on the slide
6. Put known immunoglobulin(IgA) or antigen in one well
7. In other wells put patient serum
8. Observe for precipitates after 24hrs
Results
An identical pattern was formed

Interpretation
The presence of an opaque precipitant line between the antiserum and antigen wells
indicates antigen-antibody interaction. Absence of precipitant line suggests the
absence of reaction. Cross-linking and lattice formation will only occur when antigen
and antibody concentrations are optimal. An increasing amount of antigen is added
to a constant amount of antibody in solution. This is called the antibody-excess zone
(Prozone phenomenon).
Conclusion
A full identity (i.e. a continuous line): Line of precipitation at their junction forming an
arc was formed representing serologic identity or the presence of a common epitope
in antigens.
3b) Single Radial Immunodiffusion/ Mancini test
Introduction
This test is used in the clinical lab for the determination of the immunoglobulin levels
in the patient’s sample. It is used to detect the concentration of antigens or
antibodies present.
If measured, the diameter of the ring is proportional to the log of the concentration of
the antigen since the amount of antibody remains constant. By running different
concentrations of a standard antigen, a standard curve is generated which can be
used to quantitate the amount of antigen present.
The Mancini test is useful in the detection and measurement of immunoglobulin and
complement levels in body fluids. The presence of high molecular weight immune
complexes in cryoglobulinemia and rheumatoid arthiritis conditions due to
aggregation of immunoglobulins particularly IgG tend to give false low values of
immunoglobulins.
Principle
The test operates on the principle of diffusion. It also implements the principle of the
precipitin curve which states that antigen-antibody interact forming visible cross-
linked precipitate when the proper ratio of antigen to antibody is present.
In the test, antibody is incorporated into agar and poured into a glass plate to form a
uniform layer. Circular wells are cut into the agar and antigen is introduced into the
wells. Specific antigens to the impregnated antibodies diffuse through the agar in all
directions from the well and react with the antibody present forming visible
precipitate or a precipitin ring. Ring shaped bands of precipitates form concentrically
around the well indicating reaction. The diameter of the precipitate ring formed
corresponds to the amount of antigen in the solution.
Reagents required
- Agarose powder
- Water
- Double immunodiffusion templates
- Ruler
- Patient serum
- Dropper
- Bunsen burner
- IgM in solution form
Procedure
1. Weigh 1.2g of agarose powder.
2. Mix with 100ml of phosphate buffered saline PBS.
3. Heat the mixture using a Bunsen burner to dissolve the agarose powder. When
dissolved, it forms a molten gel.
4. Transfer the molten gel onto a microscopic slide.
5. Leave it on the bench to solidify.
6. Make five wells using the Pasteur pipette in a horizontal pattern.
7. A series of standards containing known concentration of antigen are placed in
separate wells, while control and ‘unknown’ sampled are placed in other remaining
wells.
8. Put the slide on a flat surface at room temperature in a moist chamber.
9. Check the results after 24-48 hours.

Test results
The ring diameters of the various wells was measured and noted, and following
standard curve was prepared using the ring diameters:
As the antigen diffuses radially, a ring of precipitate will form in the area of the
optimal antigen-antibody concentration.
The ring diameters are measured and noted.
A standard curve is prepared using the ring diameters of the standards versus their
concentrations. This curve is then used to determine the concentration of the control
and unknown samples.
Interpretation
The presence of a precipitin ring around the antigen wells indicate specific antigen-
antibody interaction. The absence of a precipitin ring suggests the absence of a
reaction.
The greater the amount of antigen in the well, the farther the ring will form the well.
Conclusion
Applications of radial immunodiffusion include: the determination of the quantity or
concentration of an antigen in a sample, the estimation of immunoglobulin classes in
sera, the estimation of IgG, IgM antibodies in sera to influenza viruses, determination
of relative concentrations of antibodies in serum, estimation of serum transferrin and
alpha-fetoprotein, comparison of the properties of two different antigens,
determination of the relative purity of an antigen preparation, for disease diagnosis
and for serological surveys.
REFERENCES
1. Moi University Lab Report Writing Manual.
2. A.K. Chemutai. Textbook of Immunology.
3. Subhash Chandra Parija. Texbook of Microbiology and Immunology, 2nd edition.
4. Murphy, Kenneth & Weaver. Janeway’s Immunobiology, 9 th edition.