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Experiment 4: Direct Haemagglutination Test Objective: To learn the agglutination theory by using the lectin and red blood

cell. Method 1. 2. 3. 20L PBS was placed into well 2 to well 24. 40L plant lectin was added into well 1. Transfer 20L of lectin was transfered from well 1 into well 2 and mixed well to avoid bubbles. 4. 20L solution was transfered from well 2 into well 3 and mixed well to avoid bubbles. 5. The same procedure was repeated until you reach well 23. 20L solution was discarded from well 23. Well 24 serves as a negative control . 6. 20L sRBC was added into each well and mix well. From the most concentrated wells to the least concentrated wells was added with sRBC and mixed well. 7. The plate is sealed. The plate was incubated at RT and observed the plate every 10 min for a maximum 40 min.


Figure 1.0

The figure 1.0 shows that the mat is appear on all the well except the well 1 and the well 21. Discussion Lectin is a carbohydrate binding proteins for non-immune agglutination which will agglutinate the cells that present of polysaccharides for example red blood cell. Because of this feature the plant lectin was characterized as haemagglutinin which means the capability of the carbohydrate binding protein agglutinate the erythrocyte. For the lectins which are consist of more than one sugar binding site per molecule because of that the lectin able to cross linking with red blood cell to give an efficiency of agglutination. After the agglutination the clumps of the red blood cell will be precipitated. Theoretically the agglutination will be increasing until the optimum and then drop back until no agglutination. The button appear is because the well are appearing in the V-shape so if no cross-link agglutination occur the red blood cell will start to

accumulate at the bottom to appear in button. For the mat that appear is because of the agglutination of red blood cell so it will distributed around and wont accumulate in the bottom and the solution will become cloudy. However in the result cannot determine the amount of the agglutination and for the well 23 and 24 should be no agglutination is appear. The reason that maybe appear for the error are the red blood cell already lysis due to too much of pressure was applied when we pipette the red blood cell onto the well. The lysis red blood cell will not be accumulate so the solution will become cloudy. Agglutination only occurs when the right amount of the antibody and antigen mix together. The reason is the multivalent or two binding site of the antibody they can bound to different antigen particles and resulting a bridge which create by the antibody molecule spanning 2 antigen molecule. Other than that it will also resulting the lattice which are the stable structure where antigen and antibody are suspended in solution by their attachment of each other. For this precise amount of antigen and antibody is called zone of equivalence. For the excess antibody present, the visible aggregates are minimal because the antibody just compete with a few antigen epitopes therefore the antibody will not undergo the cross-linking and resulting no agglutination of the red blood cell is present. The excess antibody also known as prozone phenomenon. The last one is excess antigen present and also called as postzone phenomenon, if too much of the antigen the antibodies will only tends to bind only one antigen particle and again the cross-linking will not be occur and no lattice will form. That will be several physical factors will affect the agglutination of the antibody and antigen, first is the buffer pH which means the H+ ion concentration inside the solution. H+ ion will be disrupting the charge of the antigen and the antibody therefore to having a great affinity for binding the pH has to maintain at physiological pH. Next is the electrostatic interaction between the antibody and antigen. Since the binding between the antigen and antibody is non-covalent interaction therefore the electrostatic interaction play an important role for strengthening the agglutination for example the electrostatic repulsion cause by the similar charge of antigen and antibody will cause very low strength of agglutination. Moreover, the electrolyte also play an important role for agglutination to occur

because highly present of electrolyte will be neutralize the negative charge on the antigen or antibody therefore it will be reduce the electrostatic charge that interfere with lattice formation. Next is the electrostatic interaction between the antibody and antigen. Since the electrostatic charge play the role in the lattice formation. The antigen and antibody are make up from the protein therefore the temperature will play the role in agglutination. If the temperature are not suitable for the antigen and antibody will cause both particle undergo conformation change and will lower down the affinity of agglutination. The biological factor that effect the agglutination is the isotype switching, because the IgM is a pentameric antibody have the most effective in the agglutination but after the infection of antigen for several week our body will undergo isotype switching to other type of antibody which are lower effectiveness in agglutination compare to IgM. The last one is the concentration of the antibody and antigen. The concentration of antibody and antigen must be precise and equivalence only the cross linking will be present which will increase the strength of agglutination. The important of agglutination on our human immunity are precipitate the antigen to engulf by the phagocyte cell and it also help to quarantine the bacteria to prevent the further infection. The agglutination also was using in the blood cross matching which are determine the blood group. The red blood cell will present the antigen on the surface for the blood group A and blood group B so the antibody added to the red blood cell will form an blood precipitate it means that the antibody was agglutinate the red blood cell. The last one is the agglutination it help us to identify the type of pathogen that infect the patient other than that based on the prozone, equivalence and the postzone can determine the number of the antigen infected the patient. Before starting the experiment have to wear glove to prevent the contamination drop in to the solution. The tips of the pipette must immerse into the solution before release the solution transfer to prevent burble. When release the solution into the the well must slowly to prevent the red blood cell lysis.

Conclusion In antigen excess and the antibody excess phase the amount of agglutination are low. The optimum amount of agglutination only occur in the equivalent of antigen and antibody.

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