26 Jun An Investigation of ABO Group Antigen Structure
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An Investigation of ABO Group Antigen Structure
The ABO blood group antigen epitopes are carbohydrates present on a cell membrane protein. The basic core carbohydrate (known as H) is present in group O individuals. Group A and group B individuals carry different saccharide additions to the basic core. Group AB individuals carry both the additional saccharides (on different core carbohydrates).
This experiment investigates the structure of the ABO antigens and should allow you to deduce what inherited factors produce these antigens.
Group O red blood cells have been incubated with either UDP-galactose or UDP-N-acetyl galactosamine together with group A, group B or group O serum for six hours at 37oC as follows:
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6
Group O Group O Group O Group O Group O Group O
cells cells cells cells cells cells
UDP-NacG UDP-Gal UDP-NacG UDP-Gal UDP-NacG UDP-Gal
O-serum O-serum B-serum B-serum A-serum A-serum
Mix 10ml of cells from each tube with 10ml group O serum on a microscope slide, and check for agglutination.
Record your results.
Here are the results of the experiment
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No agglutination was observed with tube 1, 2, 3 and 6
Agglutination was observed with tube 4 and 5
The Labororatory report- It should contain:
• The AIM of the experiment
Why you are doing it
• The Experimental Procedure
How you did it
• The Results
What you observed
• The Discussion and Conclusion
What it means
· Include references at the end.
You should consider the following
1. What sugar is added to the basic core carbohydrate to produce
a) the group A epitope?
b) the group B epitope?
2. Why does a group A individual produce the group A epitope while a group O individual does not?
3. What do you think is the inherited factor which determines that an individual is group O, A, B, or AB? (Remember that group O is a recessive characteristic.)
4. Write an equation for the enzyme reaction by which the Group A and Group B saccharide residues are added to the core carbohydrate.
(Hint: look up the mechanism of synthesis of glucogen in a biochemistry textbook.)
5. Look up the structures of the ABO antigen epitopes.
These points will guide you to explain your observations.
Title: An Investigation of ABO Group Antigen Structure
Aim of the Experiment
Investigate the Biochemistry that’s results in the formation of ABO groups
The Experimental Procedures
Group O red blood cells have been incubated with either UDP-galactose or UDP-N-acetyl galactosamine together with group A, group B or group O serum for six hours at 37oC as follows:
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Group O cells
Group O cells
Group O cells
Group O cells
Group O cells
Group O cells
UDP-NacG
UDP-Gal
UDP-NacG
UDP-Gal
UDP-NacG
UDP-Gal
O-Serum
O-Serum
B-Serum
B-Serum
A-Serum
A-Serum
Before you enter the lab, wear Lab coat and put on latex gloves. You will be dealing with blood samples which are biohazards. All lab safety rules should be followed.
When you enter the lab, the following items should be placed onto your desk: Glass slides, white paper or white tile, pipette plus tips.
6 glass slides should be placed onto a white plain piece of paper. This is so that when the samples are placed onto the glass slides the agglutination should be visible. If visibility is poor you may use a microscope to aid you.
A marker pen should be at hand so that you can label each slides. The slides should be labelled from 1 to 6 on the top right hand corner of the slide
You will also require a p20 pipette. The pipette should be adjusted to 100 which will allow you to pipette 10ul accurately. You will be required to pipette 10ul of each sample with 10ul of O-Serum.
You will require small yellow tips which can be attached to the pipettes. You will need to change the tip after each sample tube you use. This is to avoid contamination
When the samples are placed onto the slides with the O-Serum, it is important that you mix the two solutions thoroughly to allow the sample to completely mix with the Serum so that agglutination can take place.
You should avoid dipping the pipette into the bottom of the samples tubes as they may have settled solids which may give show you lumps which may look like agglutination in turn give you false results.
You should see agglutination in some tubes while not in others, this observation should be recorded.
You will then be require to explain why clumping occurs with some and not with others
Need to consider the reaction happening in the tubes and relate to the formation of the blood groups.
Step by Step Process in lab
1. Label slide
2. Pipette 10ul of tube one and place onto centre of slide. Dispose of pipette.
3. Change tip and Pipette 10ul of O Serum and place on to slide
4. Mix the two solution thoroughly with the tip of the pipette and dispose of the tip
5. Observe results and record
6. Repeat steps for the next 5 tubes
When you are done, dispose of the glass slides into the biohazard jar for glass. All used pipette tips should be disposed into the designated waste tip jar.
The Results
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Agglutination
–
–
–
+
+
–
No agglutination was observed with tube 1, 2, 3 and 6
Agglutination was observed with tube 4 and 5
The Discussion
Agglutination is the clumping of cells such as red blood cells in the presence of antibody. The antibody binds to multiple cells creating a large complex. When red blood cells agglutinate it is called hemagglutination. This occurs when a blood group is mixed in with an incompatible blood serum. For example group O has no antigen and therefore can be donated to any group as agglutination will not occur. However a group O individual will have anti A and Anti B in their blood plasma. This means that agglutination will occur if they receive blood from a group A or B.
The table below shows when agglutination will occur with the different blood type.
Donor
Recipient
ABO
O
A
B
A,B
O
+
–
–
–
A
+
+
–
–
B
+
–
+
–
A,B
+
+
+
+
Carbohydrates found on red blood cell membrane form epitopes. These are distinctive structures fround on an antigen which can be detected by the immune system. All blood groups contain a basic carbohydrate structure known as H. the H carbohydrate core is a precursor and is not an epitope. That is why O blood groups do not have any antigen. Group A and B are formed by the addition of either UDP-galactose or UDP-N-acetyl galactosamine. This leads to the formation of an epitope which can be detected by antibody. Group A have a UDP-N-acetyl galactosamine and group B have a UDP-galactose epitope. The production of A,B and H controlled by the action of transferase. These are enzymes which control the addition of the sugars onto the carbohydrate chain. H , A and B have different transferase which add different specific sugars. H genes cause L fucose to be added to form the H antigen. The A gene can then add NacG to form A group and B genes add D-gal to form B group.
The transferase are coded by the genes. The phenotype of each type of blood group are coded by allele obtained from both parents. This table shows what allele is required from each parent for each type of blood group to form.
Phenotype
Genotype
A
IAIA or IAi
B
IBIB or Ibi
A, B
IAIB
O
ii
All group O cells contain Anti A and Anti B. this means that if A or B cell Groups are present they will be agglutinated.
Group
Antigen on cell
Antibody in Plasma
O
None
Anti-A, Anti-B
Group O is a universal donor however can only accept blood from group O. Blood from any other group will cause agglutination.
The Conclusion
For any group to be formed a[part from group O it requires the correct transferring. Tube 4 contains D-gal and B serum which is the correct transferring therefore B group if formed. However because Anti B is present in the serum agglutination occurs.
Tube 5 contains UDP-NacG and A serum. This contains the correct transferrin and therefore Group A blood type if formed. The Anti A present in the serum also cause the agglutination of the Group A blood
Tube 1 and 2 have no agglutination because O serum has no transferrin and therefore when incubated, the group O cell remain the same. As Group O blood has no antigen present no agglutination will take place.
Tube 3 contains UDP-Gal and Serum B. this is an incorrect combination and therefore Group O cells remain the same.
Tube 6 contains UDP Gal and A serum. This is also an incorrect combination and the group O cells remain the same.
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
Anti A, Anti B
Anti A, Anti B
Anti A, Anti B
Anti A, Anti B
Anti A, Anti B
Anti A, Anti B
O cell
O cell
O cell
B Antigen
A Antigen
O cell
No affect
No affect
No affect
Agglutination
Agglutination
No affect
The Reference
M.Roitt, J Brostoff, D.K.Male.1996. immunology, Gower Medical Publishing LTD
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