Immunology Lab Manual 2015

IMMUNOLOGY

LAB MANUAL

Course title: Immunology (BT205IU)

Number of credits: 3+1
Prepared by: Phan Ngoc Tien; Nguyen Thi Thu Hoai; Hoang Thi Lan Xuan

-2015-

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 Immunology Lab Manual 2015

CONTENTS

No. Experiment Page No. Remark

01 Cells of the Immune System 3

02 Heamagglutination - Typing of Human Blood 8

03 Immunoprecipitation tests 13

04 Immunoelectrophoresis 21

Enzyme-linked immunosorbent assay (ELISA) for detection of

05 23
hepatitis B surface antigen (HBsAg)

06 Final Practical Exam

Lab1: Cells of the Immune System

-Identify the leucocytes (white cells).
-Determine the relative numbers of the types of leukocytes

Lab2: Heamagglutination
- Demonstrate the specific interaction between antibody and antigen
- Determine ABO blood groups
- Coombs test

Lab 3 : Immunoprecipitation:
Radial immunodiffusion
Immuno-double diffusion

Lab 4: Immunoelectrophoresis
Countercurrent electrophoresis
Rocket electrophoresis

Lab 5: Enzyme-linked immunosorbent assay (ELISA)

Detection of hepatitis B surface antigen (HBsAg)

Lab 6: Examination of immunological labwork

Lab report Due dates

Lab 1 Report
Lab 2 Report
Lab 3 Report
Lab 4 Report
Lab 5 Report

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 Immunology Lab Manual 2015

A. INSTRUCTORS
Lab Instructor: Dr. Nguyen Thi Thu Hoai; Office: A1.707
Teaching Assistant:
Lab Location: A1.702, IU building.

B. GENERAL INSTRUCTIONS

REGULATIONS
1.Lab attendance is compulsory.
2.Must wear a lab coat. Bring a permanent marker.
3.Read lab standard operations procedure before attending lab.
4.Students work in pairs.

EVALUATION
1.The lab is worth 20% of the final course mark; 10% for lab reports and quiz and 10% for lab
examination.
2.You must pass the lab to pass the course (10/20%).
3.The lab reports due dates are listed in the schedule. All lab reports must be handed in on due
date or one mark is subtracted for each class day thereafter until all marks are gone.
4. Lab report value varies depending on the lab, as some lab reports are far more complex than
other lab reports. Total marks will be given on each lab report. The marks are added together and
divided by total to give a final mark out of 10%.
5. The lab exam takes place during scheduled lab period (refer to schedule for date).
6.Lab report marks are final unless an obvious error in addition of marks has been made. If a
student feels they have a legitimate complaint, please direct attention to lab instructor.
7.Approximately two weeks prior to the lab exam, a brief outline of lab exam format and
information content will be available on the website.
8. You must notify the lab instructor no later than two school days after the missed lab. Failure to
comply will result in a zero on your lab exam.
9. Plagiarism (copying another student’s lab report (present or previous year) or copying published
literature without citing is a violation of University regulations. Refer to the STUDENT DISCIPLINE
BY-LAW in your student handbook (rule book) for action taken for plagiarism.

LAB REPORT PRESENTATION

1. All reports must have an Honesty Declaration attached at end of report. Available as a
pdf file on lab website.
2. Lab reports may be done as an individual effort or a group effort by the two students that
carried out the experiment. The decision on the number of reports per group is totally dependent
on members of the group. This decision may be changed any time during the term. Therefore for
each lab report the group has the option to hand in one or two
reports exclusive of what has been done before or after that particular report. Indicate on the cover
page of the report if the report is a group report or an individual report. If
handing in an individual report also include lab partner’s name.
3. A reference file is available in the science library (1 hour reserve).
4. If an experiment requires class data for lab report analysis, it will be available on the website.
5. Lab reports must be typed. Up to 10% of the mark subtracted for reports not typed. No
binders. Stapled left hand corner.

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6. On the front page of the report state:

• Course name and number
• Experiment number and Title
• Group #
• Individual or Group name(s). If handing in an individual report, alsoinclude lab partners name.
• GROUP report or INDIVIDUAL report
• Date
7. Number pages.
8. Lab reports consist of data presentation, data analysis and possibly questions. The
information is to be presented exactly as requested. Number sections the same as the lab
manual.

LAB STANDARD OPERATIONS PROCEDURE

Bench area: Clean bench area before and after use
Personal safety
1. Always wear lab coat and CLOSED shoes.
2. Do not eat, drink or smoke in the laboratory.
3. Regard the bench as a contaminated area. Do not place any object on the laboratory bench
that could later transfer to your mouth. Do not sit on the bench.
4. Practice good aseptic techniques and take care when disposing of class materials.
5. Care must be taken when handling with human blood samples and HBsAg containing
solutions to avoid any infection incident.
6. Do not discard any demonstration slides.
7. Dispose used lancets, broken glass in the “SHARPS” discard containers.
8. At the end of your class, clean up your bench.
9. Before you leave the laboratory, wash your hands thoroughly with soap and water.

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PRACTICAL 1: Cells of the Immune System

I. Introduction
Hematopoiesis is a process of generating all types of blood cells from an original pluripotent
stem cell and via development of lymphoid and myeloid lineages. Lymphoid group is
responsible for making lymphocytes, including T cells, B cells and natural killer cells.
Meanwhile, Myeloid group is to produce other types of blood cells such as basophils,
eosinophils, neutrophils, monocytes, erythrocytes and platelets.
Apart from erythrocytes and platelets, the remaining blood cell members are called white blood
cells or leukocytes, and they play an essential role in protecting the host body against the
invasion of infectious disease or foreign objects. Therefore, analysing the presence level of
these white cells in a blood sample is a good indicator about the health condition. In this
practical, you will have an opportunity to:
- identify different types of leukocytes in the blood;
- determine the relative numbers of the different types of leukocytes by performing a differential
count.
Wright and Giemsa stains are Romanowsky stains used to stain peripheral blood and bone
marrow smears. There are two groups of blood smears, one stained with Wright's stain, the
other with Giemsa. You will be able to tell very little difference between the two. Giemsa stain is
supposed to stain leukocytes a reddish purple but otherwise it is similar to Wright's stain. The
most important components of these stains are oxidized methylene blue, azure B and eosin Y
dyes. The eosin Y dye stains the cytoplasm of cells an orange to pink color. The methylene blue
and azure B dyes stain the nucleus varying shades of blue to purple. There are several items
that you need to keep in mind when identifying the different types of WBCs. The cytoplasm of
both lymphocytes and monocytes stain blue and depending on the intensity of stain, you may
not be able to distinguish between them. However, you can distinguish between them on the
basis of size. The size of lymphocytes is very near that of RBCs, whereas monocytes are
usually twice as large as RBCs. Keep in mind the color of the granules. Neutrophils have
granules, but the granules do not stain intensely; they just have a fairly faint pink color in most of
these slides. Eosinophils have red granules, and basophils, if you find any, will have blue
granules.
(Historical Remarks: Romanowsky staining is a prototypical staining technique that was the
forerunner of several distinct but similar methods, including Giemsa, Jenner, Wright, Field, and
Leishman stains, which are used to differentiate cells in pathologic specimens. It was named
after the Russian Physician, Dmitri Leonidovich Romanowsky (1861 – 1921), who invented it in
1891.)
II.Practical procedures
II.1. Preparation and staining of a blood smear for inspecting white
cells
Materials/ Equipments
Glass microscope slides, Cover slips, Lancets, Pasteur Pipette, Paper towel, 100 % methanol,
Giemsa stain solution, Water, Ethanol and cotton (for disinfection), Timer.
Method
Preparation of a blood smear
1. Use alcohol swab to disinfect your finger before collecting your blood. Using a sterile lancet to
puncture your finger and get a drop of blood. Place that drop of blood at one end of a slide.

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Discard the used lancet in the container indicated by your instructor. Re-disinfect your finger
with alcohol.
2. Position a second slide or a cover slip (“spreader”) near the drop, at approximately 30-40
degree angle (Figure 1).
3. Allow the blood to capillarise between slide/slide or slide/cover slip.
4. Smoothly and quickly push or pull the “spreader” down the base slide to form a smear of
blood.
5. Air dry. “spreader”

Figure 1. Preparation of a blood smear

Staining a blood smear

6. The blood smear slide is fixed by submerging in 100 % methanol for 5 minutes. Drain well by
touching end of slide on kitchen towel and air dry.
7. Dilute Giemsa stain in 1:20 with distilled water and stain the slide with this diluted for 20
minutes.
8. Gently wash with water by dipping the slide repeatedly for 30 sec. Drain well by touching end
of slide on kitchen towel and air dry.
II.2. Differential blood leukocyte count
Materials/ Equipments
Microscope slides with stained blood smear; Microscope; Immersion oil
Method
1. Place the blood smear under the microscope objective, and examine the smear under low
magnification (40X) to identify areas where reasonable numbers of white cells may be found.
When the blood film is stained using this solution (which contains purified eosin and thiazine
dyes), the nucleus and cytoplasm of while blood cells will take on blue or pink color.
2. Turning to high magnification (100x) with oil immersion, examine a number of white cells.
Make a drawing of each different morphological type of white cell that you see and identify, with
reference to Figure 2.
i. polymorphonuclear leukocytes (granulocytes): neutrophils, basophils, eosinophils
ii. mononuclear leukocytes (agranulocytes): lymphocyte, monocytes
3. When you feel confident of recognising different cell types, examine 50 to 100 different white
cells, allotting them to each type and estimate the percentage of each cell type present in the
smear. This is known as a “Differential White Cell Count”.
4. Record your results.

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Neutrophil Eosinophil Basophil

Lymphocyte Monocyte
Figure 2. Types of leukocytes stained with Giemsa stain
Reference ranges for healthy adults:
Neutrophils: 40-74 %
Eosinophils: 0-7 %
Basophils: 0-1.5 %
Lymphocytes: 19-48 %
Monocytes: 3-9 %
III. Results
Draw each type of white cell you see

Results of your differential count:

Type of Monocytes Lymphocytes Neutrophils Eosinophils Basophils

leukocyte
Number

Percentage
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IV. Discussion
1. From your results, would you say that the blood you used was from a healthy (“normal”)
donor?

2. From your results, what are the relative percent contributions of the innate and adaptive
immune system to the white cells of the blood?

3. Is it possible to tell T lymphocytes from B lymphocytes using this stain? Explain.

4. What are the main components of the blood apart from red cells (erythrocytes) and
leukocytes?

5. Do you expect the composition of the blood (cells and soluble components) to change after
immunisation? Explain your answer.

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PRACTICAL 2: Haemagglutination - Typing of Human Blood

I. Introduction
Particular antigens combine with their specific antibodies to form complexes that usually
aggregate as visible clumps. This is called agglutination. Haemagglutination is the term used
when the particle agglutinated is red blood cells (erythrocytes). This can be either direct
haemagglutination, where the antigen is a surface antigen of erythrocytes, or indirect
haemagglutination, where an antigen of interest is chemically bound to the surface of the
erythrocytes. In 1900, Landsteiner recognised that the erythrocytes of some individuals were
agglutinated by the serum of others. It was established that the agglutination involved two main
antigens, A and B, and their corresponding antibodies. The population could be divided into four
main groups on the basis of the antigen on their erythrocytes – A, B, A and B, or neither A nor
B; and the corresponding blood groups were designated A, B, AB and O, respectively. The
serum of all individuals contains antibodies to the red cell antigens not found on their own
erythrocytes. For example, an antibody that reacts with antigen A is called anti-A antibody and
group A individuals will not have anti-A antibody.
In clinical blood tests, haemaglutination phenomenon is also employed to identify presence of
immunoglobulins such as IgG and IgM, or presence of complement proteins, which have bound
to antigens on the red blood cells. This practical will assist you in understanding specific
interactions between antibody and antigen via two experiments, determining human ABO blood
types and detecting presence of IgG antibodies binding to the red blood cells by Coombs test.
As part of complete blood count, it is noted that evaluating the number of red blood cells can
also provide diagnostic information such as for anaemia and kidney diseases. A healthy adult
usually has 3.8-5.6x109 red blood cells per ml of blood. In this session, you will also practise the
red blood cell count using haemocytometer.

II. Practical Procedures

II.1. ABO blood group identification by direct haemagglutination
Materials/ Equipments
Microscope slides; Lancets; Alcohol; Swabs; Samples of human blood; Blood typing antisera;
Toothpicks; Tissues; Marking pen

Method
1. Put a microscope slide on a sheet of tissue. Clearly number each position to avoid
confusion later.
2. Prick your finger with a clean lancet and allow a small drop of your blood to fall in each of
the three positions on a microscope slide.
Use alcohol swabs to disinfect your finger before and after collecting the blood.
Discard the used lancets in the provided container.
3. Add a drop of anti-A serum to the first drop of blood, a drop of anti-B serum to the second
drop of blood and a drop of anti-A&B serum to the third drop of blood.
4. Mix well and observe any haemagglutination.
Discard the used microscope slides and mixing sticks in the provided container.

II.2. Globulin identification by direct antiglobulin test (DAT)- Coombs Test

Materials/ Equipments
Microscope slides; Human blood samples (reserved in anticoagulant); Coombs-serum reagent
(antihuman globulin); 1.5ml-Centrifuge eppendorf tubes; Pasteur pipettes, Micropipettes and
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tips; Isotonic saline solution (0.9%) or phosphate buffered saline (PBS); Marking pens;
Centrifuge

Method
1. Collect 6 ml blood and divide into three blood-collecting tubes already containing K3 -EDTA.
Close the lid and gently invert to mix blood with the anticoagulant (to avoid blood clotting).
Centrifuge the tubes at 4000 rpm for 10 minutes at room temperature. Transfer plasma into
eppendorf tubes (200µl/tube) and stored at -20°C for subsequent experiments;
2. Gently transfer 2 drops of the blood cells into a 1.5-ml eppendorf tube then add 1 ml 0.9%
saline (or PBS) into the same tube and mix gently. Close the lid and centrifuge the prepared
tubes as above;
3. Carefully remove the supernatant which contains the white blood cells and plasma;
4. Repeat another two wash cycles. It is noted that the wash has to be done as quickly as
possible since the antibodies can detach from the red blood cells if they are kept in the saline
(PBS) for too long;
5. After the third wash, making a 3-5% red blood cell suspension from the washed cells by
adding 1 drop of the packed cells (50 µl) into 1 ml of 0.9% isotonic saline solution (or in PBS);
Mix gently.
6. Add a drop (50 µl) of the red blood cell suspension into a new tube.
7. Pipette 1 drop (50 µl) of the Coombs-serum reagent, which contains anti-IgG (rabbit) and
anti-C3d (monoclonal), into the same tube. Mix well (or shake gently for mixing) then centrifuge
for 20 seconds at 1000g. Gently resuspend the cells by gently flicking the tubes and observe
macroscopically for agglutination. No visible agglutination is indication of no reaction between
the reagent and the red blood cells (i.e. negative result). If agglutination is seen, on the other
hand, the result will be positive.
Note: Discard the used tips into the provided container.

II.3. Red blood cell count

Materials/ Equipments
Haemocytometers; Human blood cells; Cover slips; P20 micropipettor and yellow tips;
physiological saline solution (0.9% NaCl) or phosphate buffered saline (PBS); Marking pens;
Microscope

Method
1. Gently dilute blood cells (from previous experiment) 500 times with PBS solution: In order to
achieve this, you will perform serial dilutions with 1:10 then 1:100, then finally 1:500;
2. Place a coverslip onto the haemocytometer to cover the squares (you can see quite clearly
by eyes);
3. Pipette 10 ul of cell suspension into each chamber of the haemocytometer and wait for a
minute for the cells to settle.
4. Place the haemocytometer on the microscope and locate the middle big square (Figure 3).
Observe using 40x objective and count the cells present in three different small squares
following the rule for counting cells depositing on the edges of the square, i.e. only counting
cells on the left and bottom lines.
5. Calculate number of red blood cells (RBCs) per ml.

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Hemocytometer grid:
2
red square = 1 mm , 100 nl
2
green square = 0.0625 mm , 6.25 nl
2
yellow square = 0.04 mm , 4 nl
2
blue square = 0.0025 mm , 0.25 nl
at a depth of 0.1 mm.

Figure 3. Red blood cell counting using haemocytometer

(Source: Wikipedia.org)

III. Results
Record the agglutination you see

ABO type determination experiment

Anti-A serum Anti-B serum Anti-A&B serum
Your blood

Coombs test

Positive Negative

What is the result of your blood cell count?

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IV. Discussion
What blood group do you have?

Human group A erythrocytes often react with serum from Group B individuals, and group B
erythrocytes often react with serum from group A individuals. Explain.

From the above, consider the following mixtures and indicate whether agglutination is likely to
occur.
a. Group O red cells + serum from group O person YES/NO
b. Group A red cells + serum from group O person YES/NO
c. Group B red cells + serum from group O person YES/NO
d. Group A red cells + serum from rabbit immunized with group A red cells. YES/NO

Discuss the significance of human blood typing in blood transfusion

For the Antiglobulin test, how can you identify the cause of agglutination is due to red blood cell-
bound antibodies or other complements?

Comment the health condition based on your blood cell count result.

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PRACTICAL 3: IMMUNOPRECIPITATION TESTS

I. Introduction
The interaction of an antibody (Ab) with an antigen (Ag) is the fundamental reaction of
immunology.
Ab + Ag → Ab-Ag
Most antigens are proteins. Antigens and antibodies frequently form complexes that become
insoluble and precipitate from solution. Precipitation occurs because each antibody can bind to
more than one antigen and each antigen can be bound by more than one antibody. As
increasing amount of antigen is added to a constant amount of antibody, the amount of immuno
complex precipitated rises and then falls (Figure 4). Initially, the amount of the antibody in
solution is much greater than the amount of antigen in solution. This is called the antibody-
excess zone. As more antigen is added, the amount of protein precipitated increases until the
antigen and antibody molecules are at an optimal ratio (roughly in equal proportions).This is
called the equivalence zone, or equivalence point, where maximum precipitation occurs. When
the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation
will decrease. This is known as the antigen-excess zone.

Figure 4. The precipitation reaction

This property of antigen-antibody interaction makes it possible to perform qualitative and

quantitative assays on the antibody-antigen system.

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II.Practical Procedures
2.1 Radial immunodiffusion
Radial immunodiffusion or Mancini technique can be used to quantify the amount of antibody or
antigen in a tested sample. To determine concentration of an antigen, its specific antibody is
added to the melted agar, which is cooled just above its set point. The agar solution is then
poured onto a slide and allowed to set. Small holes (wells) of about 1-2 mm are cut into the gel.
Tested samples, which contain the antigen, are added into each well. The gel is stand for about
24 hours. As the antigen diffuses out of the well it reacts with the antibody forming soluble
complexes. A ring of precipitation is formed where concentration of antigen is equivalent to
concentration of the antibody (Figure 5).

Figure 5. Determination of the Ag concentrations using radial immunodiffusion

The area within the precipitin ring, and thus the ring diameter squared, is proportional to the
antigen concentration. A standard curve can be generated using known concentrations of
antigen (Figure 8). The concentration of an antigen in an unknown sample then can be
interpolated from the standard curve.
Based on the same principle, unknown concentration of antibody can be determined using an
antigen-containing gel.

Materials/ Equipments
Petri dish; P100 and yellow tip; Agarose; PBS; BSA and BSA antibodies; Gel puncher; Water
bath; Microwave; Duran bottle; Aluminum foil and spatula;

Methods

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1. Prepare 15 ml of 1 % agarose in PBS 1X. When the agarose solution is cooled to 50°C, anti-
BSA antibodies are added and mixed well by gentle swirling. Do not add the antibodies earlier
as a higher temperature will cause their denaturation, also do not add them much later, as
agarose will soon start to solidify; briefly mix to ensure even dispersal of the antibodies.
2. Immediately pour the agarose solution containing the antibodies into the petri dish.
3. While waiting for the agarose to set, prepare standard BSA solutions in PBS at
concentrations of 1, 2, 5 and 10 mg/ml.
4. Make wells on solid gel, label the wells to avoid later confusion and add 20 ul of each
standard solution per well. An extra well is made for loading sample with unknown
concentration.
5. Close the lid and keep the plate overnight at 37°C.
6. Observe result and record the diameters of the precipitin rings, from which the graph for
standard concentrations will be plotted and used to deduce the concentration of the unknown
sample.

2.2 Immuno-double diffusion

Immuno-double diffusion is a technique for qualitative analysis of antibody-antigen system. In

this technique, antigen and antibody solutions are placed in separate wells cut in an agarose
gel. Antibody and antigen diffuse from the wells toward each other and form opaque bands of
precipitate, which are called precipitin arcs, where they meet at equivalent proportions.
A single antigen will combine with its homologous antibody to form a single precipitation line.
When two antigens are present, each behaves independently of each other. Thus, the number
of precipitin bands indicates the number of antibody-antigen systems present (Figure 6).

Figure 6. Immuno-double diffusion

Double diffusion in two dimensions is a useful technique for determining the identity of antigens.
If a solution of antigen is placed in two adjacent wells and the homologous antibody is placed in
the center well, the two precipitin bands that form will join at their closest ends and fuse. This is
known as a reaction of identity (Figure 7a).
When unrelated antigens are placed in adjacent wells and the center well is filled with
antibodies for each antigen, the precipitin bands will form independently of each other and will
cross. This is known as a reaction of non-identity (Figure 7b).
If the antigen in one well and the antiserum in the center well comprise a homologous pair, and
the antigen in an adjacent well is a cross-reacting antigen, the precipitin lines will fuse.

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However, an additional spur will form which projects toward the cross-reacting antigen. This is
known as a reaction of partial identity (Figure 7c).

a. Reaction of identity b. Reaction of non-identity c. Reaction of partial identity

Figure 7. Compare the identity of antigens using immuno-double diffusion technique

Materials
Petri dishes; P100 and yellow tips; Agarose; PBS; BSA and anti-BSA antibodies; HBsAg and
serum containing anti-HBsAg antibodies; Gel puncher; Microwave; Aluminum foil and spatula;
Duran bottle

Procedures
1. Prepare 15 ml of 1% agarose in PBS 1X. When the agarose solution is cooled to around
60°C, pour the agarose solution into the petri dish.
2. Once the gel has been set, make 3 wells which form a triangle shape as described in the
Figure 6. Load antigen(s) and antibodies as instructed by the tutor.
3. Close the lid and incubate the plate overnight at 37°C.
4. Observe and record the results.

III. Results
1. Record the results of radial immunoprecipitation.

2. Draw the standard curve for each essay and determine the concentration of the unknown
sample (You may plot the curves on separate pieces of papers and paste them into this report)

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3. Explain “single diffusion” and “double diffusion”

IV. Discussion
If in a radial immunodiffusion test, there are three precipitin rings form around a well of unknown
antigen. Explain this observation.

Draw the precipitin arcs form in the immuno-double-diffusion set up below being known that
antigen 1 and 2 are totally different, antibody 1 and 2 bind specifically to antigen 1 and 2,
respectively. Clearly label the arc with the correspondent pair of antibody-antigen.

Ag 1, Ag 2
Ag 2

Ab 1,
Ab 2

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PRACTICAL 4: IMMUNOELECTROPHORESIS

I. Introduction
The advantage of immunoelectrophoresis in comparison to immunoprecipitation test is that the
former method can help you to save time when performing antigen-antibody interaction studies.
Two types of immunoelectrophoresis are summarized as followed.

1. Countercurrent electrophoresis
In this assay, pH of the agar gel is chosen so that the antibody is positively charged and the
antigen being tested is negatively charged. The selection is based on the isoelectric point (pI).
In proteins the isoelectric point (pI) is defined as the pH at which a protein has no net charge.
When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a
net positive charge. The pI varies for different proteins. In general, antibodies are of alkaline pI
(pI range: 6.5-9) while antigens are of acidic pI (e.g BSA, pI 4.5).
As a voltage is applied across the gel, the antigen and antibody move towards each other and
precipitate (Figure 8). This method is more sensitive and much less time consuming than the
immuno-double diffusion technique.

Ab Ag

Precipitin arc

Figure 8. Counter current electrophoresis

2. Rocket electrophoresis
Radial immunodiffusion is time consuming since it usually takes at least 24 hours for the antigen
to diffuse into the gel and reach the equivalent point. This limitation is overcome in rocket
electrophoresis, which was developed based on the same principle as radial immunodiffusion.
In rocket electrophoresis, antigen of unknown concentration is added into wells punched in the
antibody-containing gel. pH of the gel must be selected so that antibody and antigen are
neutrally and negatively charged, respectively. For example, at a pH of 8.5, antibodies are
nearly uncharged, and their slow electrophoretic migration is nullified by the EEO flow (check
remark) through the agarose. Most other proteins are highly charged at pH 8.5, and will migrate
through an agarose gel. Thus, when a voltage is applied across the gel, the antibodies immobile
while the antigens move towards the positive electrode and forming precipitin rockets (Figure 9).
As the antigen proteins enter the gel, they form a concentration gradient, which at some point
gives the proper concentration for precipitation with the antibody in the gel. The more
concentrated the antigen, the further it must run to be diluted to precipitable levels. The result is
that each sample gives a “Rocket”, the length of which is proportional to the concentration of
antigen in the sample. Thus unknown amount of antigen can be interpolated from a standard

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curve. This process can be reversed to measure unknown antibody concentration. Precipitates
can be stained with Coomassie Blue R-250 and detection limits are generally sufficient to
quantitate 0.1 - 0.2 g/ml of antigen.
Remark: EEO Effect: Agarose gels have a small number of fixed charges, which cause a
phenomenon known as electroendosmosis (EEO). EEO causes a slow net flow of water through
the gel away from the positive electrode.

Figure 9. Estimation of antigen concentration using rocket electrophoresis

II. Practical Procedures

In this practical session, you will perform both countercurrent and rocket electrophoresis
method.

Materials/ Equipments
Agarose; PBS; BSA and BSA antibodies; HBsAg and its antibodies; Gel puncher; Duran bottle;
Aluminum foil and spatula; Electrophoresis running buffer; Absorbent paper (Filter paper); P100
and yellow tips; Microwave; Agarose gel electrophoresis apparatus (Horizontal Electrophoresis
Apparatus); 0.3 M Tris-HCl pH 8.6 and pH 7.0

Methods
Rocket electrophoresis
Agarose gel preparation:
1% Agarose in 0.06 M Tris-HCl buffer, pH 8.6. This buffer is prepared by diluting 0.3 M Tris-HCL
pH 8.6 20 times. 50 µl of antibody (e.g anti-BSA) is added to 10 ml of agarose then mixed well.
Pour on glass plate to create the gel and wait for it to be solidified. Use gel puncher to make a
well for loading antigen (eg. BSA).
Electrophoresis
1. Place the gel in the tank and do not let the gel submerged in the running buffer (0.3 M Tris-
HCl buffer, pH 8.6).
2. Load 20 µl of antigen/ antibody per well.
3. Apply wicks (made from filter papers) immersed in buffer to form a bridge between the gel
edges and the buffer troughs. Make sure that the gel is in correct orientation with respect to
polarity of the electrophoresis system [the cathode (-) should be next to the sample wells.
4. Close the lid and run the gel at 75V for 1 hour.
5. Observe the results.

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Countercurrent electrophoresis,
Agarose gel preparation:
1% Agarose in 0.06 M Tris-HCl buffer, pH 7. This buffer is prepared by diluting 0.3 M Tris-HCL
pH 7 20 times. No antibody is added to the agarose. Pour on glass plate to create the gel and
wait for it to be solidified. Use gel puncher to make a pair of wells, one well for loading antigen
(e.g BSA), one well for loading antibody (eg. anti-BSA) with approximately 4 cm distance
between wells.

Electrophoresis
1. Place the gel in the tank and do not let the gel submerged in the running buffer (0.3 M Tris-
HCl buffer, pH 7).
2. Load 20 µl of antigen/ antibody per well.
3. Apply wicks (made from filter papers) immersed in buffer to form a bridge between the gel
edges and the buffer troughs. Make sure that the gel is in correct orientation with respect to
polarity of the electrophoresis system [the cathode (-) should be next to the sample wells.
4. Close the lid and run the gel at 75V for 1 hour.
5. Observe the results.

III. Results
Record whether you see the precipitated band(s) or not.

Discussion
1. Explain your results.

2. If you want to determine the concentration of an antigen using rocket electrophoresis

technique. The isoelectric points of this antigen and its specific antibody are 5.9 and 8.2,
respectively. What pH does your gel should have?

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PRACTICAL 5: Enzyme-linked immunosorbent assay (ELISA)

for detection of hepatitis B surface antigen (HBsAg)

I. Introduction
Over the past three decades, hepatitis B virus (HBV) has been tagged as one of infectious
diseases with high rate of spreading. The infection can be easily transmitted among individuals
via common routes of daily activities, including sexual contacts, mother passing to newborn
during pregnancy and exposure incidences to HBV-contaminated blood. Statistical evaluation
estimated that around two billions of people worldwide had been predictably vulnerable to HBV.

In this practical, you will learn a method to detect the presence of hepatitis B surface antigen
(HBsAg) in human serum samples using sandwich ELISA. Enzyme-linked immunosorbent
assay (ELISA) technique is widely used in Immunology and in other areas of biological sciences
as a reliable and reproducible method of quantifying specific soluble proteins in a mixture such
as serum or tissue culture supernatants.
The assay relies on the specificity of antibody to identify only one component of a mixture of
proteins. There are two types:

Direct ELISA: Used to measure the amount of antibody to a given antigen present in a
test sample.

Sandwich ELISA: Used to measure the amount of a given antigen present in a test
sample.
In general, both types of assay use multi-well trays as the solid support (matrix). The following
diagrams illustrate the principles of the two types of assays.

Figure 10. Types of ELISA assay

There are several different enzymes that can be linked to the developing antibody. The two
most widely used are Peroxidase (PO) and Alkaline Phosphatase.
The standard protocol for a sandwich ELISA assay can be summarized as following:
1. Coating the microtiter plate with primary antibody:
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Primary antibody is added into wells of the microtiter plate and incubated at 37oC for about 90
min or at room temperature for 24 h. The unbound antibody is removed by washing the plate
several times with washing reagent, which usually is phosphate buffer saline (PBS) containing a
detergent such as Tween 20 (0.05%).
2. Blocking all unbound sites on the surface of the wells to prevent the false positive result:
Plate is incubated with blocking reagent such as fetal bovine serum (FBS), skim milk, or gelatin
at 37 oC for 90 min. Excess blocking reagent is then removed by washing several times with
PBS-Tween.
3. Incubating with tested samples:
Tested sample/serum is added into each well and incubated for about 60 min at 37oC. Proteins
that bind unspecifically to the primary antibody are removed by washing the plate several times
with PBS-Tween.
4. Incubating with second antibody conjugated to an enzyme:
Second antibody conjugated to an enzyme, which catalyzes the conversion of the substrate - a
chromogen - into a colored product, is added into each well. Plate is incubated for 45-90 min at
37oC and then washed many times with PBS-Tween to remove excess antibody.
5. Incubating with substrate:
Substrate is added into each well and plate is incubated at room temperature for 10-30 min.
Enzyme reaction is then terminated by adding stopping reagent.
6. Recording the results by reading the plate on a plate reader.
To determine the concentration of antigen or antibody in an unknown sample, a sample with
known concentration of antigen or antibody, a standard, is required. Serial dilution of the
standard is prepared and treated the same way as for the tested sample. Standard curve is
constructed with concentrations of the standard are plotted on the horizontal axis and optical
densities are plotted on the vertical axis. Concentration of the unknown sample is determined by
interpolating its optical density to the standard curve.
In this practical, you will be using sandwich ELISA technique to detect hepatitis B surface
antigen (HBsAg) in human serum samples.

Figure 11. The oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to 3,3',5,5'-tetramethylbenzidine diimine

under peroxidase catalyzation. In the presence of Horseradish peroxidase (HRP), the TMB and peroxide
contained in the substrate solution react to produce a blue byproduct (Amax = 370nm and 652nm). The
color intensity is proportional to the amount of HRP activity, which in turn is related to the levels of target
analyte in an optimized ELISA procedure. Addition of sulfuric acid stop solution changes the color to
yellow, enabling accurate measurement of the intensity at 450 nm using a spectrophotometer or plate
reader.

II. Procedures
Materials/ Equipments
96-well ELISA plate (microtiter plate) coated with anti-HBsAg antibodies; Micropipettors P200

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Pipette tips; tissue paper; Serum samples; Anti-HBsAg antibody conjugated to peroxidase;
Washing reagent; TMB (3,3’,5,5’-tetramethylbezidine) substrate; Stopping reagent (H2SO4 1N);
Negative control R3 (DMSO); Positive control R4 (purified HBsAg in DMSO)

Methods
1. Collect about 1 ml of blood in a 5 ml test tube containing anti-agglutination reagent (K3-
EDTA).
2. Centrifuge the blood samples at 4000 rpm for 10 min to get the cell-free sera.
Be careful when removing tubes from the centrifuge to avoid disturbing the pellets.
3. Add 100 µl of serum or control reagents (R3, R4) to each well on 96-well ELISA plate that
was pre-coated with anti-HBsAg antibody
4. Add 50 µl of Peroxidase-conjugated anti-HBsAg antibody to each well.
5. Cover the plate with new adhesive film and incubate the plate for 60 minutes at 37oC.
6. Remove liquid from wells by “flicking” the plate. Blot the plate of a sheet of paper towel to
remove remaining liquid.
7. Fill all wells with washing reagent. Flick off and blot plate.
8. Repeat step 5 & 6 four times.
9. Quickly add 100 µl of TMB substrate reagent to each well. Incubate at room temperature (18
– 30 oC) for 10-30 min and in the dark condition. Do not use adhesive film during this incubation.
10. Add 100 µl of stopping reagent to each well to stop the reaction.
11. Observe and record the color change in each well or read your plate on the plate reader (if
available) at 450 nm after have been waiting at least 4 minutes since stopping solution addition.

III. Results
Record the color change you see on the template provided below. Clearly indicate the name of
samples used. Grade the intensity of color developed in each well with ++++ for the most
intensive samples and (-) for the least intensive samples.

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IV. Discussion
Explain the use of positive and negative controls.

If the positive control did not work, what could be the reasons?

If the negative control did not work, what could be the reasons?

From your results, what is your conclusion for each test sample?

In practice, to conclude whether a test sample positive or negative for HBsAg, the plate must be
read by a plate reader at 450 nm. A cut-off OD is determined. Samples with optical density (OD)
≥ cut-off OD are regarded as positive. Those with OD < cut-off OD are regarded as negative.
With the ELISA system we use for this practical, the cut-off OD is calculated as follow:
Cut-off OD = Average OD of R3 + 0.050
(R3 is the negative control provided by the manufacturer)

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APPENDIX
Buffer recipes
1. Phosphate buffer saline (PBS)
Reagents/ Amount to add Final concentration Amount to add Final concentration
1L (for 1X solution) (1X) (for 10X stock) (10X)

NaCl 8g 137 mM 80 g 1.37 M

KCl 0.2 g 2.7 mM 2g 27 mM
Na2HPO4 1.44 g 10 mM 14.4 g 100 mM
KH2PO4 0.24 g 1.8 mM 2.4 g 18 mM
If necessary, PBS may be supplemented with the following:
CaCl2•2H2O 0.133 g 1 mM 1.33 g 10 mM
MgCl2•6H2O 0.10 g 0.5 mM 1.0 g 5 mM
PBS can be made as a 1X solution or as a 10X stock. To prepare 1 L of either 1X or 10X PBS,
dissolve the reagents listed above in 800 mL of H2O. Adjust the pH to 7.4 (or 7.2, if required)
with HCl, and then add H2O to 1 L.
Dispense the solution into aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05
kg/cm2) on liquid cycle or by filter sterilization. Store PBS at room temperature.
2. 0.3 M Tris-HCl pH 8.6
1X Tris-HCl (0.3M Tris Base, pH8.6):
Trizma Base ---------------------------------- 3.63 g
Distilled water ------------------------------- 1000 ml
Adjust pH 8.6 using concentrated HCl
Store this solution at room temperature.

Instruction for use of micropipettors

Micropipettors (Figure 10) are used to accurately transfer small amounts (< 1 mL) of liquids.
They are very expensive and delicate instruments which should be used properly to avoid
damage. Different micropipettors are used for different volume ranges, but each has upper and
lower limits that should not be exceeded, and require matching plastic tips. The three common
sizes are listed below. Selection of appropriate size is critical for accurate measurement.
Micropipettor Volume range (µL) Type of pipette tip
P20 2 – 20 White
P200 20 – 200 Yellow
P1000 200 - 1000 Blue

Figure A.1 Structure of a micropipettor

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1. Set the desired volume by turning the centrally located rings clockwise to increase volume or
counterclockwise to decrease volume.
2. Place a tip on the discharge end of the pipettor. NOTE: If sterile conditions are necessary do
not allow the pipet tip to touch any object (including your hands).
3. The plunger will stop at two different positions when it is depressed. The first of these
stopping points is the point of initial resistance and is the level of depression that will result in
the desired volume of solution being transferred. Because this first stopping point is dependent
on the volume that is being transferred, the distance you have to push the plunger to reach the
point of initial resistance will change depending on the volume being pipetted. The second
stopping point can be found when the plunger is depressed beyond the initial resistance until it
is in contact with the body of the pipettor. At this point the plunger cannot be further depressed.
This second stopping point is used for the complete discharging of solutions from the plastic tip.
You should not reach this second stop when drawing liquid into the pipettor, only when expelling
the last drop. Before continuing, attempt to depress the plunger to each of these stopping points
until you can easily distinguish between these points.
4. Depress the plunger until you feel the initial resistance and insert tip into the solution, just
barely below the surface of the liquid and not as deep as possible.

Figure A.2 Structure of a micropipettor

5. Carefully and slowly release plunger. NOTE: If the solution you are pipetting is viscous, allow
the pipet tip to fill to final volume before removing it from solution to avoid the presence of
bubbles in the plastic tip which will result in an inaccurate volume. If you release the plunger too
quickly, it will suck liquid up into the pipettor and damage it.
6. Discharge the solution into the appropriate container by depressing plunger. This time,
depress the plunger to the point of initial resistance, wait one second, and then continue
pressing the plunger as far as it will go in order to discharge the entire volume of solution.
7. Remove tip by pressing down on the tip discarder.

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