International University, Vietnam National University HCM

IMMUNOLOGY LABORATORY
VIETNAMESE NATIONAL UNIVERSITY – HCMC INTERNATIONAL
UNIVERSITY
SCHOOL OF BIOTECHNOLOGY

IIMMUNOLOGY LABORATORY
Course’s ID: BTBT18IU11
Instructor: PhD. TRAN THI HAI YEN
Class: TUESDAY 13:00 PM - 17:00 PM

REPORT WEEK 2
Labwork 3: Immunoelectrophoresis

Group 4:
Nguyễn Nguyên Khang BTBTIU18098
Đỗ Thụy Hồng Nhung BTBTIU18185
Nguyễn Thị Mỹ Yến BTBTIU18355
Hoàng Nguyễn Minh Châu BTBTIU18374

Tuesday Afternoon Class

Semester II, 2020 - 2021
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Abbreviations
Ab, Antibody
Ag, Antigen

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I/ INTRODUCTION
1/ Introduction
Immunoelectrophoresis is the method based on the immunodiffusion that we already discussed the
previous lab work, which means Immunoelectrophoresis is the method for quantification and
qualification of antigen-antibody interaction. This method refers to precipitation in agar under an
electric field. It is a process of a combination of immuno-diffusion and electrophoresis In this
labwork, we performed 2 types of immunoelectrophoresis: Countercurrent electrophoresis and
rocket electrophoresis.
Countercurrent electrophoresis
Counter Current Immunoelectrophoresis is a modification of immunoelectrophoresis in which
antigen and antibody move in opposite directions and form precipitates in the area where they meet
in concentrations of optimal proportions. It is also referred to as countercurrent or crossed-over
immunoelectrophoresis. In this assay, we choose positively charged antibody and negatively
charged antigen, and tests on agar gel played as the environment.

Rocket electrophoresis
Rocket Immunoelectrophoresis is an adaptation of radial immunodiffusion developed by Laurell. It
is also known as electroimmunoassay or electroimmunodiffusion. It is called “rocket
electrophoresis” due to the appearance of the precipitin bands in the shape of cone-like structures
(rocket appearance) at the end of the reaction. In rocket immunoelectrophoresis, antigen migrates in
an electric field in a layer of agarose containing an appropriate antibody. The migration of the
antigen toward the anode gives rise to rocket-shaped patterns of precipitation. The area under the
rocket is proportional to antigen concentration.

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2/ Objectives
Counter-current immunoelectrophoresis
Counter-current immunoelectrophoresis depends on the movement of antigen towards the anode and
of antibody towards the cathode through the agar under the electric field. The test is performed on a
glass slide in the agarose gel of high electro-endosmotic flow. A pair of wells is punched out where
one well is filled with antigen and the other with the antibody. Electric current is then passed
through the gel. The migration of antigen and antibody is greatly facilitated under the electric field,
and the line of precipitation as precipitin arcs is made visible in 30–60 minutes, which indicates a
positive reaction.
Rocket immunoelectrophoresis
Rocket immunoelectrophoresis is a quantitative one-dimensional single electro-immunodiffusion
technique. In this method, the antibody is incorporated in the gel at a pH value at which the
antibodies remain essentially immobile. The antigen is placed in wells cut in the gel. Electric current
is then passed through the gel, which facilitates the migration of negatively charged antigens into the
agar. As the antigen moves out of the well and enters the agarose gel, it combines with the antibody
to form an immune complex that becomes visible. During the initial phase, there is considerable
antigen excess over antibody and no visible precipitation occurs. However, as the antigen sample
migrates further through the agarose gel, more antibody molecules are encountered that interact with
the antigen to form an immune complex. This results in the formation of a precipitin line that is
conical in shape, resembling a rocket.
The greater the amount of antigen-loaded in a well, the further the antigen will have to travel
through the gel before it can interact with sufficient antibody to form a precipitate. Thus, the height
of the rocket, measured from the well to the apex and area is directly proportional to the amount of
antigen in the sample.
3/ Other techniques
Fused rocket immunoelectrophoresis is a modification of one-dimensional quantitative
immunoelectrophoresis used for detailed measurement of proteins in fractions from protein
separation experiments.
Two-dimensional immunoelectrophoresis is a variant of rocket electrophoresis. The test is a two-
stage procedure. In the first stage, antigens in solution are separated by electrophoresis. In the
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second stage, electrophoresis is carried out again, but perpendicular to that of the first stage to obtain
rocket-like precipitation.

II/ PROCEDURE
1/ Rocket electrophoresis
Time activities Material Experiment notes

- Dissolving agarose in TBE

14:28 solution by mixing them
20ml TBE
solution,
- Putting it into the microwave for 0.2g agarose
14:31 completely dissolved
(1 minute)

14:35 - Adding 200uL antiserum to form Completely Before mixing up antiserum and
distribution of antibody dissolved agarose solution, we must cool down
agarose the solution till 55-60oC in order to
- Pouring quickly solution on the solution, glass don’t destroy the antiserum by high
14:35
slide slides, gel heat.
puncher and Pouring quickly and let the gel spread
- Using a gel puncher to create 3
antiserum all space we need but do not let the gel
14:51 wells on a straight line
spread outside the slides

- Labeling each well on the other

sides of slides
14:59
- Labeling information about 2 groups will
samples prepare 2
different slides
- Loading 10µL of antigen and (4 slides in
15:00- antiserum in each well following to total):
15:04 the labels - standard
antigen
- Putting the slides in to the - sample
electrophoresis tank
15:07 - Pouring 1X TBE buffer to cover
the gel slide

15:10- - Electropheres at 100volts _ 60mA Fill the buffer side by side to balance,
15:40 - Till the blue dye travels 3-4 cm then slowly let them flow into the
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from well middle

Don’t pour directly into the middle -> if
pour it directly, the buffer will take up
space in wells

- Observe the appearance and shape

15:45 of the “rocket” precipitation on the
slides after incubation
TABLE 1: Rocket electrophoresis’s procedure

2. Countercurrent electrophoresis
Time Activities Material Experiment notes

- Dissolving agarose in buffer solution

14:29 by mixing them
30ml buffer solution,
0.45g agarose
- Putting it into the microwave for
14:32
completely dissolved

- Adding 200uL antiserum to form
14:36
distribution of antibody
- Pouring quickly solution on the slide, slides, and gel
14:37
- Using a gel puncher to create 4 wells
that form a straight line and 1cm-
14:52
distancing between them
Completely dissolved
agarose solution,
Antibody, glass
puncher
Pouring quickly and let the
gel spread all space we need
but do not let the gel spread
outside the slides
When using gel puncher, do
quickly or the shape of wells
become messy because we
did not pump all the gel out.

- Labeling each well on the other sides 2 groups will prepare

15:00 of slides. 2 different slides that
contain:
- Loading 10uL of antigen sample to - Standard Antigen
15:02 the well that would be placed toward - Standard Antibody
the negative electrode. - Antiserum
- Test antigen
- Loading 10uL of antiserum samples (unknown
15:03
to the well towards the positive concentration)

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electrode.

- Putting the slides in to the Gel slides Fill the buffer side by side to
electrophoresis tank 1X Electrophoresis balance, then slowly let
- Pouring 1X Electrophoresis buffer to Buffer them flow into the middle
15:05 cover the gel slide Electrophoresis tank Don’t pour directly into the
middle -> if pour it directly,
the buffer will take up space
in wells

15:05- - Electropherese at 100volts. Till the

15:35 precipitate lines are observed

15:37 - Observe the precipitin lines

TABLE 2: Countercurrent electrophoresis’s procedure

III/ RESULTS AND CONCLUSION

 
1/ Compare your experiment with other groups’ experiments, give comments.
A- Countercurrent electrophoresis:
Grou Picture Comments
p

1 The precipitin lines of Ab control and Ag are

formed.
The precipitin lines of Ab test and Ag are
formed.

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2 The precipitin lines of Ab test and Ag are

formed.
The precipitin lines of Ab control and Ag are
not formed.

5 The precipitin lines of Ab test and Ag are

formed.
The precipitin lines of Ab control and Ag are
not formed.

Comment: The result from group 1 appear the precipitin lines in the Ab control, and the Ab test
with Ag did not form the precipitin lines, which means the Paratopr of Ab- and Epitope of Ag are
not specific. The result from group 2,3 did not appear in the precipitin lines in Ab control, and the
Ab test with the Ag form precipitin lines. That may be due to the misunderstanding of group 2,3
while marking the order of the Ab+ and Ab test, or they added the wrong well when pipetting.

B- Rocket Electrophoresis:

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Group Picture Comment

3 The antigen
(Standard Antigen C- concentration of well
0.47 mg/ml) C: very high (1.4 cm)
D: low (0.5 cm)
Unknown: high (1.25
cm)

4 The antigen
(Standard Antigen D- concentration of well
0.23 mg/ml) C: very high (1.4 cm)
D: low (0.5 cm)
Unknown: high ( 1.35
cm)

Comment: The results of two group are similar. The height of precipitin rocket C is the highest,
followed by U and D.
2/ Report the result (stick some pictures from your work)
Day Result (picture) Comment
16/03/2021 - This slide after electropheres at
100volts _ 60mA in 30 minutes:
- The rocket precipitaion is formed
but not clearly. Especially rocket
precipitation of Standard Antigen C
(0.47mg/ml), the top of C rocket is
blurry
- The rocket precipitation of Standard
Antigen C (0.47mg/ml) and
unknown concentration sample are
much longer than precipitation of
Standard Antigen D (0.23mg/ml)
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- This is the slide after 3 days; we can

observe the shape of rocket
precipitation clearly as well as
measure the length these
- The final record as:
- Standard Antigen C (0.47mg/ml):
1.35cm
19/3/2021 - Standard Antigen D (0.23mg/ml):
0.5cm
- Unknown concentration sample:
1.4cm

After 30 minutes of diffusion, there are three rocket precipitations with different length
+ Standard Antigen C ( 0.47 mg/ml)
+ Standard Antigen D ( 0.23 mg/ml )
+ Unknown: tested antigen
The size (diameters) of standard Ag C, Standard Ag D, and Unknown Ag are 1.35cm, 0.5cm, and
1.4cm, respectively.
Concentration (mg/ml) Rocket length (cm)
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0.47 1.35
0.23 0.5

From the results, we construct a standard curve regarding length(cm) and concentration of
antigens(mg/ml). However, there were only two points taking into account, so the equation and
regression coefficient are not accurate. Hence, we proximately determine the concentration of the
test antigen (the caculation may not correct exactly).
We have the equation: y = 0.2824x + 0.0888
 1.4 = 0.2824x +0.0888
 x = 4.64 mg/ml
 As a result, the approximate antigen concentration of the unknown is 4.64 mg/ml
3. Conclusion:
Counter current immunoelectrophoresis: rapidly check any antisera for the presence and specificity
of Ab for a particular Antigen. Rocket Electrophoresis can be reserved to measure unknown
Antibody concentration. During the experiment, the amount of Antigen, the thickness of gel or the
distance between the well and qualitative antigen and antibodies can affect to the experiment results.
In conclusion, some of the results are not as obvious and accurate as we expected. This may be due
to either of the following reasons: (1) Using gel to load antiserum and antigens may contaminate the
samples; (2) Hot agarose solution may destroy structure of antibodies when applying them; (3) In
rocket electrophoresis, poor technique is considerable when we poured the liquid agarose. Since the
solidification of the agarose may be not at appropriate state, the agarose gel was still watery that the
tension surface did not have the ability to keep. The gel, therefore, was quite thin so it could not
hold high amount of antigen solutions when pouring TBE buffer before electrophoresis; (4) When
constructing a standard curve, more than two standard antigens are needed in order to get more

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accurate results; (5) In countercurrent electrophoresis, distance between the antibodies and antigens
is important since it determines when and how much the precipitin lines are formed.

IV/ DISCUSSION
1. If you want to determine the concentration of an antigen using the 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 gel must be selected so that antibody and antigen are neutrally and negatively charged,
respectively. Also, the pH of the solution must be higher than the pI of the antigen to make the
antigen-negative, but this pH must also be equal to the pI of the antibody to keep it neutral. So, the
pH of the gel should be 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 on each test? What is your
suggestion for the next trial?
Test 1:

- Slide (1): Antigen and antibody have the same direction movement => wrong pH value.
- Slide (2): Antigen is neutrally charged =>pI of antigen=6.0. It doesn’t have precipitin line
because the gel has the wrong pH value.
- Slide (3): Antigen and antibody go towards each other but they have not created a precipitin
line yet=>not enough time for running.
Thus, we will increase the time for testing and the pH of the gel should be 5.5 so that it will
create a precipitin line after a specific time (increase running time up to 60-90 mins).

Test 2:

- Slide (1): Antigen and antibody directions are opposite with each other => wrong side
electrode both Ab and Ag.
- Slide (2): Antigen and antibody directions are opposite with each other => wrong side
electrode both Ab and Ag => Ab is at cathode and Ag is at the anode. The pH of the gel is
7.0 which nearly to the pI of Ab makes it nearly neutral, consequently, Ab cannot move
further.
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- Slide (3): Antigen and antibody have the same direction movement => pI of Ab and Ag <9.0
=> Ab and Ag has the same charge => move to only one direction. Additionally, Ag moves
much further than the two previous slides due to the pH of gel (9.0) much larger than the pI
of Ag, which has acidic properties (positive charge) and the gel now has alkaline properties
(negative charge). As result, Ag will move faster because the force in this slide now is the
strongest
Thus, consider whether the test has an appropriate side electrode, and the pH of the gel should be 5.0
to get a precipitin line
3/ Your questions about this labwork (if any)

V/ Homework

When pH > pI , there is an excess amount of OH- in solution. The excess OH− is attracted to the
positively charged amine group resulting in the removal of an H+ ion to form. The amine group has
a neutral charge leaving only a negative charge on the carboxylate group. Overall, the amino acid
will have a charge of −1
When pH < pI, there is an excess amount of H+ in solution. The excess H+ is attracted to the
negatively charged carboxylate ion resulting in its protonation. The carbohydrate ion is protonated,
making it neutral, leaving only a positive charge on the amine group. Overall, the amino acid will
have a charge of +1

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