1. Antibody/ Antigen Reactions.
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2. This lecture is designed to give an overview of:
What is an antibody and an antigen.
The Law that governs antibody/antigen reactions.
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3. This lecture is designed to give an overview of:
The forces that allow antibodies to bind to antigens.
The different factors that affect antibody/antigen reactions, with examples.
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4. An Antigen.
An antigen can be defined as a substance that, when introduced into the circulation of an individual lacking that antigen, can stimulate the production of a specific antibody.
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5. An Antigen.
The antigenic determinant, which is more correctly termed an epitope, is the antibody-binding portion of the antigen, which is in the region that is complementary to the combining site of the antibody.
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6. An Antibody.
An antibody can be defined as a serum protein (i.e. an immunoglobulin with specific antigen binding sites) produced as a result of the introduction of a foreign antigen, that has the ability to combine with (and, in many cases, destroy) the cells carrying the antigen that stimulated its production.
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7. The Law of Mass Action governs antibody/antigen reactions.
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8. [Ag] + [Ab]
[AgAb] k1 k2
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9. [Ag] + [Ab]
[AgAb] k1 k2
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10. Antigen/antibody reactions are detected in 3 ways in the laboratory.
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11. Agglutination. Inhibition.
Haemolysis (rarely seen these days, as EDTA chelates Ca++, Mg++ and Mn++, all required at the start of the classical complement pathway).
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12. The forces that enable antibodies to bind to antigens include:
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13. Ionic Bonds. Hydrogen Bonds.
Electrostatic attraction of positive and negative charges.
Hydrogen Bonds.
Weak bonds that form between a proton donor or proton acceptor on the antigen and the opposite amino acids on the antibody. These two groups share a hydrogen atom.
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14. van der Waal’s forces.
Polarisation of external clouds of electrons in an atom. When the electrons that spin about the nucleus of an atom swing to one side, a positive charge is created on the opposite side. This temporary positive charge attracts the electrons of a nearby atom.
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15. Changes to the ordered hydration of the membrane.
Stabilising antigen/antibody complexes by the release of H2O, causing hydrophobic amino acids to associate more closely.
Reduction in membrane H2O enhances the ability of fat-soluble immunoglobulin to associate with some antigens.
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16. Factors affecting antibody/antigen reactions.
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17. Number of antigen sites.
Temperature.
Incubation time.
3x106
Number of antigen sites.
Proximity of red cells.
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18. Ionic strength of the medium.
pH.
Antibody/antigen affinity.
Antigen/antibody concentration.
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19. Temperature.
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20. “Warm” and “cold” agglutinins.
37oC
0-4oC
“Warm” Antibodies
(mostly IgG).
“Cold” Antibodies
(mostly IgM).
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21. Different Antibodies React Optimally at Different Temperatures.
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22. ABO antibodies react optimally at room temperature or below.
Rh antibodies react optimally at 37oC.
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23. Incubation Time.
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24. Antibody/Antigen Reactions Take Place in Two Distinct Phases.
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25. Agglutination (in vitro).
Sensitisation.
Agglutination (in vitro).
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26. Sensitisation, at the optimal temperature, can take place in a matter of microseconds. Sensitisation at a sub-optimal temperature, however, can take many hours.
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27. It is usual, for example, to incubate tube indirect antiglobulin tests at 37oC for 15 minutes when the red cells are suspended in low ionic strength solution.
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28. The Number of Antigen Sites.
1000
7000
3200 47
500
3x106
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29. The number of antigen sites differs from one specificity to another, by the age of the individual and, even from individual to individual.
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30. There are only 14 000 copies of Kidd antigens per red cell.
The adult A1 red cell has between and A1 antigen sites per red cell.
There are only copies of Kidd antigens per red cell.
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31. The adult A1 red cell has between 810 000 and 1 170 000 antigen sites per red cell.
The red cells of a newborn will only carry between and copies of the A1 antigen.
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32. The normal D+ red cell will carry 10 000 to 33 000 copies of the D antigen.
An individual with a weak D phenotype will only carry 200 to copies per red cell.
An individual with exulted D (e.g. D--/D--) will carry between and copies per red cell.
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33. Obviously, the greater the number of antigen sites, the more exposed they are to binding (sensitisation) by an antibody.
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34. Dosage.
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35. Many antigens are expressed on the red cell more frequently if the genes are inherited as a double dose (homozygous).
Examples of such specificities may react more strongly, or readily, or only with red cells showing homozygous expression.
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36. Examples of anti-M frequently show dosage.
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37. Proximity of Red Cells.
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38. This Can Be Brought About in Several Different Ways.
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39. Centrifugation will increase gravitational force and bring the red cells into closer proximity with each other by mechanical means.
Proteolytic enzymes will bring the red cells into closer proximity with each other by chemical means.
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40. Anti-human globulin will bring the red cells into closer proximity with each other by immunological means.
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41. The effect of proteolytic enzymes.
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42. Malcolm Needs CSci FIBMS- +
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43. Malcolm Needs CSci FIBMS- +
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44. The effect of the indirect antiglobulin test.
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45. Y
Y = AHG.
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46. Presentation of the Antigen.
Ankyrin
4.2
4.1
P55
Actin
Band 3
Diego
GPA
MNS
GPB
Rh Polypeptide
Rh-associated
Glycoprotein
Lipid
bilayer
Spectrin tetramer
12.5nm
Anti-M
Anti-D
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47. Ionic Strength of the Medium.
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48. The greater the ionic strength of the medium, the smaller the zeta potential, and greater the density of the ionic cloud.
The lower the ionic strength, the larger the zeta potential, and the lower the density of the ionic cloud.
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49. Lower ionic strengths increase the likelihood of a successful approach of a positively charged immunoglobulin to the antigen on the red cell surface, as the density of the positively charged ionic cloud is reduced. Thus, the rate of association is increased.
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50. The rate of association of antibody with antigen may be enormously increased by lowering ionic strength.
The initial rate of association between Anti-D and the D antigen is increased 1000 fold by reducing the ionic strength from 0.17 to 0.03.
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51. Please note, incidentally, that LISS stands for low ionic strength solution and NOT low ionic strength saline.
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52. pH. 1 7 14
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53. The equilibrium constant for most antibodies is highest between pH 6
The equilibrium constant for most antibodies is highest between pH 6.5 and 7.
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54. Certain antibodies, however, notably some examples of anti-M, react optimally at a greatly reduced pH (approximately 4).
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55. Antibody Affinity.
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56. Certain antibodies “fit” their corresponding antigens “better” than others.
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57. This is to do with the electric charge on each antibody and antigen, together with the degree of complementarity.
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58. For example, IgG anti-D usually “fits” the D antigen “better” than IgG anti-Jka “fits” the Jka antigen.
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59. Antigen/Antibody Concentration.
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60. If the number of antigens greatly exceeds the number of antibody molecules present, there will not be sufficient antibody molecules to make an effective lattice between the antigens on different red cells, and agglutination cannot take place.
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61. If the number of antibody molecules present greatly exceeds the number of antigen molecules present, the antibody molecules will swamp all the antigen sites, and, again, agglutination cannot take place.
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