Antigen Antibody Reaction

Question 1

What law governs antigen/antibody reactions

Answer 1

The Law of Mass Action

Question 2

Formula for Antigen/Antibody rxn

Answer 2

[Ag] + [Ab] -> [AgAb]
* [AgAb] being the antigen-antibody complex

Question 3

What is the antigen antibody rxn used for in transfusion science

Answer 3

Used in invitro transfusion science testing to determine the presence or absence of antigens or antibodies

Question 3

Traditionally the visualisation of red cell antigen/ antibody rxns in the lab is achieved by what? (2)

Answer 3

1. Haemagglutination
2. Haemolysis of red cells

Question 4

What happens in the first stage of Haemagglutination

Answer 4

Antibodies attach to their corresponding antigen

Question 5

What is the first stage of haemagglutination known as

Answer 5

Sensitisation or binding or association or coating

Question 6

What happens during the second stage of haemagglutination

Answer 6

Intracellular bridges are formed as antibodies that are already bound to a red cell antigen bind to another free antigen site on an adjacent red cell

Question 7

What is the second stage of haemagglutination called

Answer 7

Agglutination

Question 8

Haemagglutination

Is the first stage reversible or irreversible

Answer 8

Reversible

Question 9

Haemagglutination

Is the second stage reversible or irreversible

Answer 9

Irreversible

Question 10

Antibody combining sites are in which region of the antibody

Answer 10

Fab region

Question 11

Describe the antibody combining sites

Answer 11

Complimentary in shape and size to the antigens that they react specifically with

Question 12

In the first stage of the antigen antibody rxn, the Fab region and antigens react specifically to form what?

Answer 12

Reversible bonds

Question 13

What forces and bonds are involved in antibody binding

Answer 13

• Ionic bonds
• Hydrogen bonds
• Hydrophobic bonds
• VDWs forces
• Randomisation of the water

Question 14

What does the second stage of the antigen antibody reaction involve

Answer 14

The formation of intracellular brides

Question 15

What 2 types of forces are involved in the second stage of the Ag Ab rxn

Answer 15

1. Aggregating forces
2. Repulsive forces

Question 16

How are Aggregating forces involved in the second stage of the Ag Ab reaction

Answer 16

Surface tension charges pulling red cells together

Question 17

How are Repulsive forces involved in the second stage of the Ag Ab reaction

Answer 17

• The surface of the red cell has a net electronegative charge
• Due to sialic acid and neuraminic acid on surface of red cell
• Each red cell is equally charged and so there is a repulsive force which prevents red cells from adhering to eachother

Question 18

What is the Zeta potential

Answer 18

The net strength of the forces that exist between red cells when suspended in saline

Question 19

Zeta potential

The antibody must span what distance to bring about direct agglutination

Answer 19

The antibody must span 20nm distance caused by the zeta potential

Question 20

Zeta potential

What distance do IgM antibodies have between binding sites and what does this mean

Answer 20

IgM antibodies have a max distance of 30nm between binding sites. This means that they can readily span the gap and directly agglutinate red cells

Question 21

Zeta potential

What distance do IgG antibodies have between binding sites and what does this mean

Answer 21

IgG antibodies have a max distance of 14 nm between binding sites so usually cannot bring about direct agglutination of red cells

Question 22

The 4 factors affecting Ag Ab rxns

Answer 22

1. Temperature
2. pH
3. Ionic strength
4. Ag and Ab concentrations

Question 22

Zeta potential

Ways to alter effects of zeta potential (2)

Answer 22

1. Reduce the zeta potential and bring red cells into closer proximity to allow agglutination
2. Assist IgG antibodies to bridge the gap

Question 23

Explain how temp affects Ag Ab rxns

Answer 23

* Antibodies react differently at varying temps * ABO antibodies react best at room temp * Rh antibodies react best at 37 degrees C

Question 24

Opt pH range for Ag Ab rxns

Answer 24

pH 6.5 - 6.8

Question 25

Explain how ionic strength affects Ag Ab rxns

Answer 25

In **low ionic strength solutions, ions are able to be more mobile** and this **favours ionic bonding between Ag and Ab**. This speeds up reaction rates and allows shorter incubation periods for some transfusion serology testing

Question 26

Conditions of excess antigen in a rxn mixture (3) (affects of antigen conc on Ag Ab rxns)

Answer 26

1. The use of **too strong a red cell suspension** 2. The addition of **too small a volume of serum** 3. The addition of **too large a volume of red cell suspension**

Question 27

How will excess antigen in the rxn mixture affect the rxn

Answer 27

Antibodies are **distributed so thinly** that the **formation of intracellular bridges will be sparse** and **involve too few cells**

Question 28

Conditions of excess antibody in a rxn mixture (3) (affects of antigen conc on Ag Ab rxns)

Answer 28

1. Use of **too weak a cell suspension** 2. Addition of **too large a volume of serum** 3. Addition of **too small a volume of cell suspension**

Question 29

How will excess antibody in the rxn mixture affect the rxn

Answer 29

* The majority of **antigen sites will be coated by antibodies**. * This leaves **few free antigen sites available for attachment to the free binding sites on antibodies that are already attached to adjacent red cells**. * Therefore there is a **reduction in intracellular bridge formation** and **corresponding agglutination**

Question 30

Describe the method of detection, direct agglutination

Answer 30

* **red cells suspended in saline are kept apart by a distance of 20nm** * direct agglutination of red cells suspended in saline is usually **only observed with IgM antibodies (large enough to span gap between adjacent red cells)** * for this agglutinantion the Ag Ab conc must be optimised -> **3-5% suspension of red cells in saline**

Question 31

# Indirect agg What test detects indirect agglutination

Answer 31

The anti human glubulin (AHG) test

Question 32

What causes indirect agglutination

Answer 32

IgG antibodies that are **too small to cause direct agglutination invitro**. The IgG antibody is **not large enough to form an intracellular bridge between red cells**.

Question 33

What have IgG antibodies been produced as a result of (2)

Answer 33

1. Pregnancy 2. Transfusion

Question 34

How does the AHG test work

Answer 34

* The **AHG test is used to detect indirect agglutintion where the IgG antibody is too small to cause direct agglutination invitro** * Another molecule, called **anti human globulin (AHG) or Coombs reagent, is added to help bridge the gap** * The AHG test was described by Coombs *et al* and was originally called to Coombs test

Question 35

What is the IgG antibosy referred to as

Answer 35

An incomplete antibody