2022-2023 FALL SEMESTER

CATHOLİC DE LILLE UNIVERSITY

FGES

IMMUNOLOGY LABORATORY REPORT

Experiment 1: AGGLUTINATION REACTIONS

Student ID Name and Surname

1 2021652244 MURAT GENÇ

EXPERIMENT DATE:03.11.2022 SUBMISSION DATE:07.12.2022

INSTRUCTURE: FAROUK ALLOUCHE, PhD.

TABLE OF CONTENTS
LIST OF FIGURES ................................................................................................................. 2

1.INTRODUCTION ................................................................................................................. 1

2.MATERIALS ........................................................................................................................ 3

3.METHODS ............................................................................................................................ 5

4.RESULTS .............................................................................................................................. 6

5.DISCUSSION ...................................................................................................................... 10

6.REFERENCES.................................................................................................................... 10

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LIST OF FIGURES

Figure 1:Table showing blood groups according to the presence of

antigen……………………........................................................................................................... 1

Figure 2: Graph showing pregnancy test result according to hcg hormone level……………..3

Figure 3: photo of blood sample showing only the presence of anti-D…………………………6

Figure 4: photo of blood sample showing only the presence of anti-A…………………………..7

Figure 5: photo of blood sample showing only the presence of anti-B…………………………..7
Figure 6: photo of blood sample that does not show the presence of any antigen……………..8
Figure 7: photo of the example below one showing that she is pregnant……………………….8
Figure 8: The image of the agar petri dish, on which antigen and antibody serums were dripped,
after 48 hours…………………………………………………………………………………….………9

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1. INTRODUCTION
1.1. PURPOSE
The main purpose of the 3 different experiments to be conducted in this report is to
make a determination by the presence of antigen. First of all, the blood test with the A, B,
O system and Rhesus system, followed by the pregnancy test with the presence of HCG
hormone, and finally the presence of Bovine Serum Albumin by using the precipitation
mechanism will be investigated.

1.2. THEORY

Agglutınatıon

Agglutination is the process of clustering together with the complex formed as a result
of the binding of antibodies and antigens in a small position in a liquid medium where the
necessary conditions are met.

Temperature: although some reactions only take place at +4 degrees, high temperature
activates the molecules and increases the probability of encounter, allowing antigens and
antibodies to form bridges more easily.

pH: The optimum pH is usually between 6-8.

Ionic strength: promotes network formation

DETERMINING BLOOD GROUPS

Blood group is a classification system created to indicate the characteristics of blood

by looking at the antibodies in human blood.

Blood groups are determined by the types of proteins found on red blood cells called
red blood cells. These proteins on red blood cells are called antigens.

There are many types of antigens on erythrocytes in the blood. The most important and
potent of these are A-B and Rh antigens. Blood group classifications are evaluated
especially on these two antigen groups.

Blood groups are based on 2 different systems. these ; It is the A, B, O system and the
Rhesus system. It is the blood group classification made according to whether the
erythrocytes in the blood contain A and B antigens. Accordingly, if the person's blood has
A antigen, it is considered as A blood group, if B antigen is present, B blood group, if both
A and B antigens are present, AB blood group, and if it does not contain A and B antigens,
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it is classified as 0 blood group. Likewise, there are around 50 Rh antigens on erythrocytes
in the blood. The most important and dominant of these is the Rh(D) antigen. If a person
has the Rh(D) antigen on the erythrocytes in their blood, they are considered Rh(D)
positive. If this antigen does not contain erythrocytes in the blood, it is considered Rh(D)
negative.

Figure 1: Table showing blood groups according to the presence of antigen

PREGNANCY TEST

Beta HCG is called human chorionic gonadotropin and is a pioneer for early detection
of pregnancy. It is also known as the pregnancy hormone among the people. Produced by
the placenta, this hormone is usually detected in the blood and signals whether pregnancy
has occurred. Beta HCG hormone can be measured in blood pregnancy tests as well as
diagnosed in home pregnancy tests with urine.

In fact, the name of the hormone checked in pregnancy tests is HCG (Human
Chorionic Gonadotropin), but there are 2 types of this hormone, alpha and beta. It is called
Beta HCG because the reference hormone is beta when checking whether you are
pregnant. In a non-pregnant woman, Beta HCG hormone is in the range of 0-10 mlU/ml,
but it is mostly around 0 and 0. Approximately 11 days after fertilization, high values of
the HCG hormone can be detected in the blood sample, and a few days after that, data via
urine test. obtainable. The increase in the value in the blood test provides information
about the week of pregnancy.

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 Figure 2: Graph showing pregnancy test result according to hcg hormone level

If there is hcg hormone in the urine, the dripped hcg antibody will adhere to the
molecules. and they will visibly cluster together.

OUCHTERLONY

Immunoprecıpıtatıon

Immunoprecipitation (IP) is small-scale affinity purification of antigens using a

specific antibody immobilized on a solid support such as magnetic particles or agarose resin.

Antibody 2 should be paratope as only polyclonal sera will give precipitation reactions.
the antigen must also be at least bivalent (2 epitopes), since a hapten will not react in the same
way.

The ouchterlony method; It is a simple and direct method for demonstrating the precipitation
between antigen and antibody by bidirectional diffusion on agar medium.

2.MATERIALS for BLOOD TEST:

• Micropipettes

• ANTI-A Serum

• ANTI-B Serum

• ANTI-D Serum
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• concave slide

• blood samples

• distilled water

• black rack

MATERIALS for BLOOD TEST:

• Micropipettes

• Urine samples (G1,G2)

• HCG LATEX
• plastic stirrer
• concave slide

MATERIALS for OUCHTERLONY:

• Agar
• plasticized test chart
• punch
• filter paper

• Micropipettes

• Anti-BSA

• Positive control antigen solution

• Negative control antigen solution

• Complete bovine serum

• Rabbit whole blood

• Complete donkey serum

• İncubator

• antibody solution

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3.METHODS for Blood test

-The concave slide is placed on a black background.

-30 microliters of blood is dripped into each of the 3 wells with the help of a micropipette.

-Anti-a, anti-b and anti-d serums are dropped into the pits with the help of a micropipette,
respectively.

-The pits are mixed with a small plastic mixer.

-Every sample is observed and the results are transferred to the table.

METHODS for Blood test

-30 microliters of 2 different urine samples are dropped on each of the concave slides.

- 30 microliters of HCG latex solution is added to each well.

-The pits are mixed with a stick mixer.

-The results are observed and noted.

METHODS for OUCHTERLONY

- The test table is placed under the agar-filled petri dish and one of the five central wells is
punched open.

-Each hole opened is defined with a pencil.

-15 microliters of antibody solution is dropped into the central well and 15 microliters of other
Anti-BSA antibody solution Positive control, Negative control, Whole bovine serum, Rabbit
whole blood, Whole donkey serum is dropped into the other wells.

- slightly moistened filter paper is placed on top of the agar.

- and kept in a 30 degree incubator for 48 hours.

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4. RESULTS:

a. Part 1: Blood test

Figure 3: photo of blood sample showing only the presence of anti-D

Figure 3 Agglutinations were observed by dripping 3 different anti-serums into our first blood
sample. Anti-A and anti-B serum did not change in the wells instilled, but agglutination was
observed in the wells instilled with anti-D, indicating that it was negative and positive. The
absence of change in the first two wells indicates that the blood is group O and the presence of
anti-D is also positive.

Individual has 24 0 positive blood group

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 Figure 4: photo of blood sample showing only the presence of anti-A
Figure 4 There was no change in the wells in which anti-B and anti-D serum instilled, but
agglutination was observed in the wells instilled with anti-A. A change in the anti-A well
indicates that the blood is group A and the absence of anti-d indicates negative.

individual has 23 A negative blood group.

Figure 5: photo of blood sample showing only the presence of anti-B

Figure 5: There was no change in the wells in which anti-A and anti-D serum instilled, but
agglutination was observed in the wells instilled with anti-B. A change in the anti-B well
indicates that the blood is group B and the absence of anti-D indicates negative.
individual has 22 B negative blood group.

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 Figure 6: photo of blood sample that does not show the presence of any antigen
Figure 6: Figure 4 No change was observed in the wells in which Anti-A, Anti-B and anti-D
serum were dripped.
The individual has 21 0 negative blood types.

Anti-A Anti-B Anti-Rh Blood Group

Individual 24 - - + 0+
Individual 23 + - - 𝐴−
Individual 22 - + - 𝐵−
Individual 21 - - - 0−
Table 1: table showing blood groups

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 b. Part 2: Pregnancy Test

Figure 7: photo of the example below one showing that she is pregnant

Figure 7 In the first urine sample seen at the top, the HCG hormone could not be attached to
the latex particles because there was not enough or no amount of HCG, but the opposite situation
seen in the bottom sample is agglutination and this indicates that the HGC hormone, which is a
pregnancy indicator, is present in the urine. The individual whose tube 2 urine sample is taken
is pregnant.

Figure 8: The image of the agar petri dish, on which antigen and antibody serums were
dripped, after 48 hours

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5. DISCUSSIONS

The tests performed in this report were the blood group test, the pregnancy test, and the
ouchterlony test. the common purpose of these 3 different tests. It was about antigens and
antibodies reacting and giving us information.
The antibody that meets its antigen agglutates and aggregates visibly. The presence of this
cluster shows us that there is an antibody specific to our antigen in the fluid we tested.
In Table 1, the test results in which 4 different blood samples were examined are noted. the 2
columns here, anti-a, anti-b and andi-rh columns, respectively, give us information about the
presence of antibodies found in the serum. If there is an antibody in the blood, agglutination
is observed in the well with the relevant antigen, and this informed us that that well is positive
as the antibody tested. accordingly, our 24th individual was 0 rh positive. The individual who
was observed to have no a or b antibodies in his blood automatically belonged to the 0 blood
group. The agglutastone observed in the anti-rh well showed that he had rh antibodies in his
blood and we marked it as rh positive. Likewise, the 23rd individual was named as a rh
negative, the 22nd individual as b rh negative, and the 21st individual as 0 rh negative
because no agglutation was observed.
We followed the same path in the pregnancy test. and we searched for the hormone hcg, which
is a pregnancy indicator. Visible agglutination occurred in only one of the samples mixed with
the antigen of this hormone. this means that the individual from whom the second sample is
taken is pregnant and secretes a sufficient amount of hcg hormone.(look Figure 7)

REFERANCES

• Ewald, DR ve Sumner, SC (2016). Kan grubu biyokimyası ve insan hastalığı. Wiley

Disiplinlerarası İncelemeler: Sistem Biyolojisi ve Tıp , 8 (6), 517-535.
• Cole, L. A. (2010). Biological functions of hCG and hCG-related molecules. Reproductive
Biology and Endocrinology, 8(1), 1-14.
• Ouchterlony, Ö. (1953). Antigen-antibody reactions in gels. IV. Types of reactions in
coordinated systems of diffusion. Acta pathologica et microbiologica scandinavica, 32(2), 231-
40.

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