Life's Blood


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Class  Notes

BLOOD BANK ANTIGENS AND ANTIBODIES

Antigen

Definition:

Antigens are defined as substances recognized by the body as foreign, causing the body to produce an antibody to react specifically with it

Characteristics of antigens: 

In order to be an antigen to you it must be foreign (not found in the host): THE MORE FOREIGN THE BETTER ANTIGEN!

  • Autologous antigens are  your own antigens (not foreign to you)
  • Homologous, or allogenic, antigens are antigens from someone else (within the same species) that are foreign to you

Antigens must be chemically complex. 

  • Proteins and polysaccharides are antigenic due to their complexity.  On the other hand, lipids are antigenic only if coupled to protein or sugar. 
  • Besides being chemically complex, antigens must also be large enough to stimulate antibody production.  Their molecular weight needs to be at least 10,000.
  • Due to the complexity of these molecules there are specific antigenic determinant sites, or epitopes, which are those portions of the antigen that reacts specifically with the antibody.

Factors determining whether an antigen will stimulate an antibody response:

  • Degree of foreignness.  Only human blood is transfused to humans.  
  • Size and complexity.  Although red cells are smaller than white blood cells, they tend to be more antigenic due to the complexity of the antigens on the cell surface.  Some are proteins and others are oligosaccharides.
  • Dose of antigen administered. How much antigen is the individual exposed to and what is the frequency of that exposure.
  • Genetic makeup of host may also dictate whether an antibody is produced.  Some individuals have a greater ability to make antibody and others have the antigen so they would not make the antibody.

Blood group antigens:

  • There are over 300 known blood group antigens
  • Over 1,000,000 different antigen sites on each red blood cell. 
  • These antigens are attached to proteins or lipids on the red cell membrane and are usually complex sugar groups.
  • Some stick out far on the red cell membrane and some are buried within crypts on the membrane surface.

 

Antibodies:

Definition:

Proteins produced by lymphocytes as a result of stimulation by an antigen which can then interact specifically with that particular antigen.

Serum components

  • Human serum can be separated into albumin and globulin components
  • Globulins can be separated into several different parts:
    a. Alpha 1 and alpha 2 globulins
    b.  Beta globulins (serum complement)
    c. Gamma globulins (immunoglobulins or antibodies)

Parts of an antibody:

  1. Heavy chains - made of alpha, gamma, delta, mu, or epsilon chains
  2. Light chains - made of kappa or lambda chains
  3. Disulfide bonds - hold chains together
  4. Hinge region - allows antibody to flex to reach more antigen sites
  5. Fab fragments - contains variable portion of antibody: antigen-binding sites
  6. Fc fragment - contains constant portion of antibody; also site of complement activation

Classes of antibodies:

  1. IgG - provides long-term immunity or protection
     
  2. IgM - first antibody produced in response to an antigenic stimulus
  3. IgA - found in secretions. Protects against infections in urinary, GI, and respiratory tracts
  4. IgE - involved in allergic reactions
  5. IgD - not much known about it. Surface receptor of B lymphocytes
  6. Most important classes of antibodies in blood banking are IgM and IgG, and to a certain extent IgA

Characteristics of IgG and IgM antibodies

  1. Clinical significance

  • Clinical of red cell antibodies in blood bank depend on whether they can cause in vivo hemolysis, which in turn will cause transfusion reactions or hemolytic disease of the newborn.
  • IgG  will frequently cause in vivo hemolysis due to antibody coating the red blood cells.
  • IgM, with a few important exceptions, usually does NOT cause in vivo hemolysis.  The most important of these exceptions are ABO antibodies.
  1. Size of the antibodies

  • IgG is relatively small since it is comprised of only one immunoglobulin subunit. (monomer)
  • IgM is relatively large since it is comprised of 5 immunoglobulin subunits. (pentamer)
  1. Serum concentration

  • IgG is found in the largest concentration of all immunoglobulins in the plasma.
  • IgM is found in relatively small amounts
  • IgG > IgA > IgM
  1. Complement activation

  • IgG = will do it if conditions are optimal
  • IgM = very good complement activator
  1. Placental transfer

  • IgG is small enough to easily cross placenta and is the only immunoglobulin capable of doing so.
  • IgM and the other classes do not cross placenta
  1. Optimum temperature of reactivity

  • a. IgG = 37oC
  • b. IgM = 4 oC (may react at any temperature below 30C)
  1. Number of antigen-binding sites
  • IgG has 2 binding sites
  • IgM has 10 binding sites

Terms used to describe antibodies

Immunoglobulin:
antibody formed as a result of immune stimulus (exposure to foreign antigen)
 
Naturally occurring
antibody formed without prior exposure to foreign antigen
 
Autoantibody:
antibody formed to one's own antigens (abnormal condition)
 
Alloantibody (unexpected, irregular, atypical):
antibody formed to foreign antigens, but within the same species
 
Agglutinin:
antibody capable of causing agglutination when reacting with corresponding antigen
 
Isoagglutinin:
name commonly given to blood group antibodies anti-A and anti-B
 
Saline agglutinin:
antibody capable of causing direct agglutination of antigens suspended in a saline medium without requiring any enhancement techniques
 
Hemolysin:
antibody capable of causing hemolysis when reacting with corresponding antigen
 
Cold antibody (cold agglutinin):
antibody whose optimal temperature of reactivity is less than 30oC
 
Warm antibody:
antibody whose optimal temperature of reactivity is greater than 35oC

Monoclonal antibodies

Monoclonal antibodies react with very specific antigenic determinants  and therefore shows no cross-reactivity.  They are not produced in humans or animals, but harvested from cells in cells grown in tissue culture.  The tissue culture cells made from fusion of a plasma cell, which is the antibody producer and the myeloma cell, which provides longevity and ability to make large amounts of antibody

Monoclonal antibodies used in most reagent antisera today because contain high concentrations of highly specific antibodies and lack infectious disease hazards associated with human-source antiserum.

 

ANTIGEN-ANTIBODY REACTIONS IN GENERAL

Rules of Thumb For in vivo Antigen-Antibody Reactions

  1. If a person's cell have the antigen, the antibody should NOT be present in that person's serum
  2. If an antibody to a blood group antigen is present in the serum of a person, his or her cells should lack that antigen
  3. The antigens are on the cells and the antibodies are in the serum

Stages of Antigen-Antibody Interaction

The first stage is sensitization.  Sensitization occurs when antibodies react with antigens on the cells and coat the cells.

 

 

 

 

 

The second stage of the reaction is agglutination.  Agglutination occurs when antibodies on coated cells form cross-linkages between cells resulting in visible clumping.

FACTORS AFFECTING SENSITIZATION (In vivo or in vitro)

  1. Specificity depends on the spatial and chemical "fit" between antigen and antibody

  1. Since the immunoglobulins and the red cell membranes both have an electrical charge, there is an optimum pH.  pH differences cause differences in chemical structures of antigens/antibodies, affecting the "fit".

 

  1. The optimum temperature depends on the type of antibody involved.  IgG antibodies react best at 37oC; IgM react best at 4oC.

  1. Optimum incubation time:  you need to incubate long enough to reach equilibrium, but not too long

  1. The antigen's accessibility is also important since the antibodies must be able to reach antigens.  Those antigens, like the ABO antigens, are on the surface of the red cell while others may be hidden in the crypts of the cell membrane.

FACTORS AFFECTING AGGLUTINATION IN VITRO

Number of Antigen Sites

The number of antigen sites on the red cell is important since the more antigen sites result in more antibodies being attached and forming cross-linkages.  These cross-linkages result in agglutination

Size and Structure of the Antibody

The larger antibodies (IgM) can reach between more antigen sites on different red cells and therefore causing stronger agglutination reactions.  IgM antibodies also have more binding sites to react with antigens and potentially causing cross-linkages between 5 different cells.

Distance between Cells

Centrifugation of the cells attempts to bring the red blood cells closer together, but even then the smaller IgG antibodies usually can not reach between two cells.  The larger antibodies, IgM, can reach between cells that are further apart and cause agglutination. 

  • The concept Zeta potential is important to understand why the cells will maintain a certain distance from each other.  Zeta potential refers to the repulsion between the red blood cells.It is due to an electric charge surrounding cells suspended in saline. 
  • It is cause by sialic acid groups on the red blood cell membrane which gives the cells a negative charge. 
  • The positive ions in saline attracted to the negatively charged red blood cells. 
  • The net positive charge surrounding cells in saline keeps them far apart due to repulsion from electric charges
  • Smaller antibodies (IgG) cannot cause agglutination when zeta potential exists
  • To overcome zeta potential techniques need to neutralize these charges One of the common techniques is:
    1) Add albumin to test mixture
    2) OH- groups of albumin neutralize positive charge

Antigen-Antibody Ratio

The optimum ratio is 80 parts antibody to 1 part antigen.  There are specific terms for variations in this ratio.

Prozone - antibody excess:
Antibodies saturating all antigen sites; no antibodies forming cross-linkages between cells; no agglutination
Zone of equivalence:
antibodies and antigens present in optimum ratio, agglutination formed
Zone of antigen excess (Post-zone):
too many antigens - any agglutination is hidden by masses of unagglutinated antigens

In order to get optimum antigen-antiboy concentration in Blood Banking we make washed 3% saline suspension of red cells to mix with our reagents.

 

OBJECTIVES - ANTIGENS AND ANTIBODIES IN BLOOD BANKING

  1. Define the following terms:
    Antigens
    Antigenic determinant
    Autologous antigen
    Homologous (allogeneic) antigen
    Antibody
    Immunoglobulin
  2. Describe the characteristics common to all antigens
  3. List the factors that determine whether an antibody will be formed in response to an antigen stimulus
  4. Describe the characteristics of blood group antigens
  5. Identify the parts of an antibody, and state the purpose of each part
  6. Differentiate between:
    an autoantibody and an alloantibody
    an immune and naturally-occurring antibody
    an agglutinin and a hemolysin
    a warm and a cold antibody
  7. Explain what is meant by "clinically significant"
  8. List the classes of immunoglobulins
  9. State the differences between IgG and IgM antibodies regarding:
    Size
    Number of binding sites
    Serum concentration
    Ability to cross placenta
    Ability to fix complement
    Ability to cause direct agglutination
    Optimum temperature of reaction
  10. Briefly describe how monoclonal antibodies are made
  11. State the main characteristics of monoclonal antibodies
  12. Differentiate between sensitization and agglutination
  13. List five factors that affect sensitization
  14. List five factors that affect agglutination
  15. Define specificity in antigen-antibody reactions, and explain how pH may affect it
  16. Explain how length of incubation affects antigen-antibody reactions
  17. Explain how accessibility affects antigen-antibody reactions
  18. Explain how the number of antigen sites on the red cells affect agglutination
  19. Explain how the size and structure of antibodies affects agglutination of red cells
  20. Explain what the zeta potential is, and how this may be overcome to promote agglutination
  21. Differentiate between prozone, zone of equivalence, and zone of antigen excess
  22. State the optimum ratio of antigen to antibody to promote agglutination

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Clinical Microbiology Syllabus