Antigen-Antibody Interaction Essay

Antigen-Antibody Interaction

Antigens usually bind to the side chain of the antigen binding site of the antibody, which may either be a protein, polypeptide or lipid. Antigens and antibody interactions are mediated by non-covalent bonds such as electrostatic forces, van der Waals forces and hydrophobic forces. Binding mediated by this type of bonds produces a reversible reaction.

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Antigens and antibodies are complementary to each other and each antibody binds to specific kind of antigens. The specific antibody binding site of antigen is called epitope. The epitope is usually made of amino acid residues or monosaccharides. Multivalent antigens have multiple epitopes hence several binding sites of different kind of antibodies. Interaction between multivalent antigens and antibodies is more stabilized and strong. Any binding between antigen and antibody is reversible. As the results of experiment show that saturation curve is never reached and multivalency can cause steric difficulties. Their bimolecular interaction depends on thermodynamic principles.

Ka = [Ab-Ag]
[Ab][Ag]

Ka = [Ab:(c)1] / [Ab] * c

Where Ka = Affinity constant

            Ab = free Antibody

            c= free antigen

            Ab:(c) and Ab-Ag = Antigen and antibody complex

Affinity of antibody with a particular antigen is determined by the affinity constant (Ka). Antigen and antibody complex (in molar concentration) is divided by the product of free antigen and antibody.

The time required to attain equilibrium depends on the affinity of antibody for antigen and also on the diffusion rate. The affinity constant varies depending upon the type of antibody-antigen interaction.

The antigen-antibody interaction can be described using equilibrium constants which describe the strength of the interaction. Another important aspect of this interaction that should be looked into is the dissociation rate which describes the rate at which the unbinding reaction occurs.

Equilibrium constants and the dissociation constants may be determined using a method called equilibrium dialysis. This allows the reaction to reach equilibrium. Upon reaching this state, the concentrations of free antigen, antibody, as well as the antigen-antibody complex are measured. In a given experiment, an amount of antibody was placed in side 1 of the dialysis chamber. Then, side 2 of the chamber was modified by adding radioactive antigen. Upon reaching equilibrium, samples from both sides of the membrane were taken. A scintillation counter was used in obtaining the counts. The specific activity of the radioactive antigen was found to 1015 cpm/mole. Table 1 shows the counts from each side in cpm/ml.

Table 1. Sample Counts from each side of the dialysis chamber.

Side 1
Side 2
39443
50
43880
60
47713
70
51060
80
54007
90
56621
100
72422
200
79891
300
84270
400
87166
500
89236
600
90799
700
92028
800
93025
900
93857
1000

            Using the data obtained, bound antigen was plotted against the free antigen. The graph below shows the saturation curve.

Another way of looking into the antigen-antibody interaction is the use of a scatchard plot. A scatchard analysis employs plotting the concentration of the bound antigen against the ratio of the bound antigen to the free antigen.

The following shows the data transformed using scatchard analysis. Transforming these data involves dividing the number of bound antigen with the number of free antigen found in side 2.

The following is a sample computation.

=           bound antigen

           ———————-

               Free antigen

=          39,443 cpm/mol

          ————————-

                      50 cpm/mol

=         788.86

Bound (cpm/ml)

Bound/Free

The units would then cancel out.

39443.000
788.860
43880.000
731.3333
47713.000
681.6143
51060.000
638.250
54007.000
600.0778
56621.000
566.210
72422.000
362.110
79891.000
266.3033
84270.000
210.675
87166.000
174.332
89236.000
148.7267
90799.000
129.7129
92028.000
115.035
93025.000
103.3611
93857.000
93.857

            From the transformed data above, a scatchard plot may then be constructed with the ratio of the bound over free on the y-axis. The x-axis contains the concentration of bound antigens.

                        The following shows a scatchard plot of the data above.

            The scatchard plot allows for the determination of the affinity constant of the antibody (Ka). By getting the slope of the generated line, a constant is obtained. The negative reciprocal of this constant gives us a value corresponding to Ka.

            By using two points from the line, a slope is obtained using the following computations.

            Slope     =           788.860 – 600.0778

                                     —————————

                                    39443.000 – 54007.000

                          =                   188.78

                                    —————————

                                              14564.00

                        =      – 0.013

            Getting the negative reciprocal of – 0.013 gives us the affinity constant 76.92.

If affinity constant is high it means that affinity of antibody for antigen is also high and more antibodies are attracted towards antigen binding sites. If affinity constant is low it means that affinity of an antibody for an antigen is low and fewer antigen binding sites are occupied by antibodies. Avidity determines the stabilization of the antigen-antibody complex. Cross reactivity is the binding of antibody to the epitope of another antigen. This only happens when the stabilization of the antigen-antibody complex is not very strong or specificity of antibodies is not very strong for particular type of antigens but it can bind with different antigens with somewhat identical epitopes.

References

Antigen antibody interaction. http://www.chemicon.com/resource/ANT101/a1.asp

Antigen-antibody interaction. www.lclark.edu/~reiness/immuno/lectures/Ag-Ab%20Interactions.pdfvv

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