Immunology Labwork Report

FULL NAME: …………………………………………………………………………

STUDENT’S ID: …………………………………………………

Spring Semester, 2020-2021

Table of Contents
Labwork 1: Cells of the immune system...................................................................................................2
Labwork 2: Immunoprecipitation tests....................................................................................................6
Labwork 3: Immunoelectrophoresis.......................................................................................................10
Labwork 4: Haemaglutination – Typing of human blood.....................................................................14
Labwork 5: Enzyme-linked immunosorbent assay (ELISA)..................................................................18
Labwork examination..............................................................................................................................22

1
Labwork 1: Cells of the immune system

I/ INTRODUCTION
Brief introduction about the experiment / Objectives of this labwork experiment / Other
techniques used for the same reason.

2
II/ PROCEDURE
Time Materials Activities First observation

3
III/ RESULTS AND CONCLUSION
1/ Compare your experiment with other groups’ experiments, give comments.

2/ Identify the type of these cells:

a) b) c) d)

........................................................................................................................................................................................

e) f) g) h)

........................................................................................................................................................................................

Type of leukocyte Monocytes Lymphocytes Neutrophils Eosinophils Basophils

Number

Percentage

3/ What is your conclusion?

4
IV/ DISCUSSION
1/ Is it possible to tell T lymphocytes from B lymphocytes using this stain? Explain.

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

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

4/ This is the result of WBC analysis from a Vietnamese person using flow cytometry. How many white
blood cells had been observed in 1 mm 3 of blood? Explain why is the reference range in this experiment
different from the previous one?

3
Công thức bạch cầu Kết quả (đơn vị: %) Kết quả (đơn vị: /mm ) Thang đối chiếu
Đa nhân trung tính 49.30 3,313 40.00 - 75.00
Đa nhân ái toan 2.70 181.40 1.00 - 4.00
Đa nhân ái kiềm 0.10 7 0.00 - 1.00
Lympho 42.10 2,829 20.00 - 45.00
Đơn nhân 5.80 389.8 2.00 - 8.00

5/ Keynote and Feedback

5
Labwork 2: Immunoprecipitation tests

I/ INTRODUCTION
Brief introduction about the experiment / Objectives of this labwork experiment / Other
techniques used for the same reason.

6
II/ PROCEDURE
Double immunodiffusion
First
Time Materials Activities observation
The practical Agarose  Prepare 10mL of 1% agarose
process lasted for Antiserum X, Y, Z  Microwave the agarose, wait
45 minutes Antigens X1, X2, Y1, Y2, Z1, Z2 until the solution has cooled
Assay buffer down to around 60oC then
Need 1-2 days to Distilled water pour the agarose solution onto
obtain the results Glass slide the slide
Template  Make 3 wells in the triangle
Petri dish shape form
Wet cotton  Load 10µl of antigens and
Gel puncher antibody
Micropipette and tips  Keep slide in moist petri dish
Cylinder at room temperature for 1-2
Beaker days
Microwave  Observe for opaque precipitin
Incubator lines between the antigen and
antiserum wells
Radial immunodiffusion
First
Time Materials Activities observation
The practical Agarose  Prepare 10mL of 1% agarose
process lasted for Antiserum  Microwave the agarose, wait
45 minutes Standard Antigen A until the solution has cooled
Standard Antigen B down to 50oC then add 50µl
Need 1-2 days to Standard Antigen unknown antiserum and mixed well
obtain the results Test antigen  Pour agarose on slide
Assay buffer  Make 3 wells
Distilled water  Load 10µl of each standard
Glass slide and template antigens and test antigen into
Petri dish each well
Wet cotton  Keep the slide in moist petri
Gel puncher dish at room temperature for 1-
Micropipette and tips 2 days
Cylinder
 Observe for opaque precipitin
Beaker
rings
Microwave
Incubator

III/ RESULTS AND CONCLUSION

1/ Compare your experiment with other groups’ experiments, give comments.

2/ Report the results (stick some pictures from your work)

3/ What is your conclusion?

8
IV/ DISCUSSION
1/ Explain the terms “single diffusion” and “double diffusion”.

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

3/ 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

4/ This is the result from Immuno-double diffusion. The experiment was done with Anti-IgG antibody, and
5 proteins: IgG, IgA, Transferrin, IgG heavy chain, and IgG light chain. Know that Anti-IgG Ab can have
precipitation with both IgA and IgG. From the precipitin lines, identify the proteins were put in 5 wells: A,
B, C, D, and E.

5/ Keynote and Feedback

6/ Application of immunoprecipitation
Isolate the protein of interest with low abundance from tissue or cell samples for detection
Study direct or indirect interactions between proteins of interest
Enrich of low abundant proteins
Identify unknown proteins in a protein complex
Verify protein expression in a specific tissue
Determining the in vivo location of binding sites of various transcription factors, histones, and other proteins
(in ChIP)
7/ Differences between immunoprecipitation and agglutination

Agglutination Immunoprecipitation
Immunoprecipitation is the
process that each antigen can be
Agglutination is the process of bound by more than one antibody
Definition clumping of antigens with their
respective antibodies. to form a precipitation

Antigen size Smaller Larger

Solubility Insoluble antigens Soluble antigens

Sensitivity Agglutination reactions are Immunoprecipitation reactions

more sensitive
Resulting compound Formation of agglutinates
The surface of the antigens
Requirements must be exposed for the
antibody to bind and form
visible clumps
Reaction time Minutes to hours
are less sensitive
Formation of precipitates
The concentration of antigen
and antibody should be equal
Hours to days

Applications Blood grouping Quantitative and qualitative

analysis
Labwork 3: Immunoelectrophoresis

I/ INTRODUCTION
Brief introduction about the experiment / Objectives of this labwork experiment / Other
techniques used for the same reason.

Brief introduction
Immunoelectrophoresis is the subject of the second practice session. Immunoelectrophoresis is a common
term for a range of biochemical approaches that use electrophoresis and antibody reactions to isolate and
classify proteins. When compared to immunoprecipitation tests, the benefit of immunoelectrophoresis is
that it will help you save time when conducting antigen-antibody interaction studies. In this lab work, we
have a chance to perform antibody-antigen interactions experiment by applying two kinds of
immunoelectrophoresis known as Countercurrent electrophoresis and Rocket electrophoresis

Experiment objective:
Regarding to the objectives of this lab work, students will learn how to use immunoelectrophoresis to
examine the specificity of the antigen-antibody interaction. We should also completely comprehend and
perform the techniques associated with these two kinds of methods.
Some different methods of immunoelectrophoresis:
1. The immunoelectrophoretic analysis ad modum Grabar: is the classical method of
immunoelectrophoresis. Electrophoresis is used to isolate proteins, in which antibodies are added in a
trough next to the separated proteins, and immunoprecipitates are produced after a process of
diffusion of the separated proteins and antibodies against each other.
2. Counter Current Immunoelectrophoresis is a type of immunoelectrophoresis in which antigen and
antibody pass in opposite directions and meet in optimal proportions to form precipitates in the region
where they meet.
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, regarding to Isoelectric point (pI): is 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
In general, antibodies are of alkaline pI (pI range: 6.5-9) while antigens are of acidic pI (e.g BSA, pI
4.5).

3. Rocket immunoelectrophoresis is one-dimensional quantitative immunoelectrophoresis. Unknown

concentration antigen is added into wells punched in the antibody-containing gel, similar to the radial
 immunodiffusion principle. The pH of the gel should be set such that the antibody is neutral and the
antigen is negative, thus when the voltage is applied, the antibody remains immobile and the antigens
move to the positive electrode, forming precipitin rockets.

4. Fused rocket immunoelectrophoresis is a one-dimensional quantitative immunoelectrophoresis

modification that is used to measure proteins in fractions from protein separation experiments in great
detail.
5. Affinity immunoelectrophoresis: this method had been applied as a way to estimate binding
constants. It is dependent on the interaction or formation of proteins with other macromolecules.
II/ PROCEDURE

11
III/ RESULTS AND CONCLUSION
1/ Compare your experiment with other groups’ experiments, give comments.

2/ Report the results (stick some pictures from your work)

3/ What is your conclusion?

12
IV/ DISCUSSION
1/ 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?

pH of the gel must be selected so that antibody and antigen are neutrally and negatively charged,
respectively
The pI of antigen is 5.9 and pI of antibody is 8.2, since we want to make a neutrally charged antibody, the
pH of the gel should match the pI of the antibody, which is 8.2. Furthermore, when the pH of the gel is 8.2,
the antigen's pI is lower, resulting in the antigen becoming negatively charged.
Thus, the gel should have pH = 8.2

2/ These are results from two Countercurrent electrophoresis tests, each test had 3 times of running with
different parameters. What is your conclusion in each test? What is your suggestion for the next trial?

Test 1:

Test 2:

3/ Keynote and Feedback

13
Labwork 4: Haemaglutination – Typing of human blood

I/ INTRODUCTION
Brief introduction about the experiment / Objectives of this labwork experiment /
Other techniques used for the same reason.

14
II/ PROCEDURE
Time Materials Activities First observation

15
III/ RESULTS AND CONCLUSION
1/ Compare your experiment with other groups’ experiments, give comments.

2/ Report the results (stick some pictures from your work)

3/ What is your conclusion?

16
IV/ DISCUSSION
1/ Explain the importance of identify blood group before blood transfusion. ABO is the popular system of
blood types, is there any other system of blood types?

2/ Consider the following mixtures and indicate whether agglutination is likely to occur.
• Group O red cells + serum from group O person YES/NO
• Group A red cells + serum from group O person YES/NO
• Group B red cells + serum from group O person YES/NO
• Group A red cells + serum from rabbit immunized with group A red cells. YES/NO

3/ Fill in the blank:

Blood Antigen on Antibody in - is the receiver of - is the donor to
Genotype
type ......................... ............................... blood type blood type

AB

4/ Why can the AB-type people receive the blood transfusion with the blood from A-type people, but
not the vice versa?

5/ Identify the blood type of these samples

a) b) c)

........................................................................................................................................................................................

d) e) f)

........................................................................................................................................................................................

6/ Keynote and Feedback

17
Labwork 5: Enzyme-linked immunosorbent assay (ELISA)

I/ INTRODUCTION
Brief introduction about the experiment / Objectives of this labwork experiment / Other
techniques used for the same reason.

18
II/ PROCEDURE
Time Materials Activities First observation

19
III/ RESULTS AND CONCLUSION
1/ Compare your experiment with other groups’ experiments, give comments.

2/ Report the results (stick some pictures from your work)

3/ What is your conclusion?

20
V/ DISCUSSION
1/ Explain the use of positive and negative controls. If they did not work, what could be the reasons?

2/ What is/are the most important step(s) in this assay?

3/ Keynote and Feedback

21
Labwork examination
Date of examination: ................................................................

Your partner (if yes): ..............................................................................................................................................

.................................................................................................................................................................................

Experimental job: ...................................................................................................................................................

Your work and results Comments

Number of theorical questions: .............................................................................................................................

Your answer Comments

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Your answer Comments

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