Antibody/ Antigen Reactions. Malcolm Needs CSci FIBMS 1

This lecture is designed to give an overview of: What is an antibody and an antigen. The Law that governs antibody/antigen reactions. Malcolm Needs CSci FIBMS

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. Malcolm Needs CSci FIBMS

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. Malcolm Needs CSci FIBMS 4

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. Malcolm Needs CSci FIBMS 5

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. Malcolm Needs CSci FIBMS 6

The Law of Mass Action governs antibody/antigen reactions. Malcolm Needs CSci FIBMS 7

[Ag] + [Ab] [AgAb] k1 k2 Malcolm Needs CSci FIBMS 8

[Ag] + [Ab] [AgAb] k1 k2 Malcolm Needs CSci FIBMS 9

Antigen/antibody reactions are detected in 3 ways in the laboratory. Malcolm Needs CSci FIBMS

Agglutination. Inhibition. Haemolysis (rarely seen these days, as EDTA chelates Ca++, Mg++ and Mn++, all required at the start of the classical complement pathway). Malcolm Needs CSci FIBMS 11

The forces that enable antibodies to bind to antigens include: Malcolm Needs CSci FIBMS

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. Malcolm Needs CSci FIBMS 13

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. Malcolm Needs CSci FIBMS 14

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. Malcolm Needs CSci FIBMS 15

Factors affecting antibody/antigen reactions. Malcolm Needs CSci FIBMS

Number of antigen sites. Temperature. Incubation time. 3x106 Number of antigen sites. Proximity of red cells. Malcolm Needs CSci FIBMS 17

Ionic strength of the medium. pH. Antibody/antigen affinity. Antigen/antibody concentration. Malcolm Needs CSci FIBMS 18

Temperature. Malcolm Needs CSci FIBMS 19

“Warm” and “cold” agglutinins. 37oC 0-4oC “Warm” Antibodies (mostly IgG). “Cold” Antibodies (mostly IgM). Malcolm Needs CSci FIBMS 20

Different Antibodies React Optimally at Different Temperatures. Malcolm Needs CSci FIBMS

ABO antibodies react optimally at room temperature or below. Rh antibodies react optimally at 37oC. Malcolm Needs CSci FIBMS 22

Incubation Time. Malcolm Needs CSci FIBMS 23

Antibody/Antigen Reactions Take Place in Two Distinct Phases. Malcolm Needs CSci FIBMS

Agglutination (in vitro). Sensitisation. Agglutination (in vitro). Malcolm Needs CSci FIBMS 25

Sensitisation, at the optimal temperature, can take place in a matter of microseconds. Sensitisation at a sub-optimal temperature, however, can take many hours. Malcolm Needs CSci FIBMS 26

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. Malcolm Needs CSci FIBMS 27

The Number of Antigen Sites. 1000 7000 3200 47 500 3x106 Malcolm Needs CSci FIBMS 28

The number of antigen sites differs from one specificity to another, by the age of the individual and, even from individual to individual. Malcolm Needs CSci FIBMS 29

There are only 14 000 copies of Kidd antigens per red cell. The adult A1 red cell has between 810 000 and 1 170 000 A1 antigen sites per red cell. There are only 14 000 copies of Kidd antigens per red cell. Malcolm Needs CSci FIBMS 30

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 250 000 and 370 000 copies of the A1 antigen. Malcolm Needs CSci FIBMS 31

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 10 000 copies per red cell. An individual with exulted D (e.g. D--/D--) will carry between 75 000 and 200 000 copies per red cell. Malcolm Needs CSci FIBMS 32

Obviously, the greater the number of antigen sites, the more exposed they are to binding (sensitisation) by an antibody. Malcolm Needs CSci FIBMS 33

Dosage. Malcolm Needs CSci FIBMS

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. Malcolm Needs CSci FIBMS 35

Examples of anti-M frequently show dosage. Malcolm Needs CSci FIBMS 36

Proximity of Red Cells. Malcolm Needs CSci FIBMS 37

This Can Be Brought About in Several Different Ways. Malcolm Needs CSci FIBMS

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. Malcolm Needs CSci FIBMS 39

Anti-human globulin will bring the red cells into closer proximity with each other by immunological means. Malcolm Needs CSci FIBMS 40

The effect of proteolytic enzymes. Malcolm Needs CSci FIBMS

Malcolm Needs CSci FIBMS - + Malcolm Needs CSci FIBMS 42

Malcolm Needs CSci FIBMS - + Malcolm Needs CSci FIBMS 43

The effect of the indirect antiglobulin test. Malcolm Needs CSci FIBMS

Y Y = AHG. Malcolm Needs CSci FIBMS

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 Malcolm Needs CSci FIBMS 46

Ionic Strength of the Medium. Malcolm Needs CSci FIBMS 47

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. Malcolm Needs CSci FIBMS 48

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. Malcolm Needs CSci FIBMS 49

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. Malcolm Needs CSci FIBMS 50

Please note, incidentally, that LISS stands for low ionic strength solution and NOT low ionic strength saline. Malcolm Needs CSci FIBMS 51

pH. 1 7 14 Malcolm Needs CSci FIBMS 52

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. Malcolm Needs CSci FIBMS 53

Certain antibodies, however, notably some examples of anti-M, react optimally at a greatly reduced pH (approximately 4). Malcolm Needs CSci FIBMS 54

Antibody Affinity. Malcolm Needs CSci FIBMS 55

Certain antibodies “fit” their corresponding antigens “better” than others. Malcolm Needs CSci FIBMS 56

This is to do with the electric charge on each antibody and antigen, together with the degree of complementarity. Malcolm Needs CSci FIBMS 57

For example, IgG anti-D usually “fits” the D antigen “better” than IgG anti-Jka “fits” the Jka antigen. Malcolm Needs CSci FIBMS 58

Antigen/Antibody Concentration. Malcolm Needs CSci FIBMS 59

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. Malcolm Needs CSci FIBMS 60

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. Malcolm Needs CSci FIBMS 61