Antibodies and Antigens In the blood bank 1
Nice game!! http://nobelprize.org/ 2
Karl Landsteiner discovered blood groups in 1901. Awarded Nobel Prize for Physiology or Medicine in 1930 3
Why we study RBC Ag & Ab? Patient Donor blood Antibody Serum Antigen May contain Ag on RBC membrane (eg. A or B Ag) RBC 4
Antigen-Antibody reaction Safe blood transfusion requires delivering compatible donor cells to the patient to avoid the immunological reaction between antigens on donor red cells and the naturally occurring antibodies present in patient serum. Ab Ag + => 5
Antibodies Antibodies are Proteins that recognize and bind to a particular antigen with very high specificity. Antibodies are produced in response to exposure to an antigen. Antibodies Belong to a group of serum proteins called immunoglobulins (Ig). They are found in all body fluids e.g. plasma saliva, tears, etc.. There are five classes of antibodies Individuals have all of the five classes The five classes of antibodies are all prefixed with Ig which stands for immunoglobulin. Each antibody has at least two identical sites that recognise and bind to an antigen: Antigen binding sites or (paratopes). 10/22/2009 6
Antibody Structure Disulphide bonds 7
Antibody Structure All classes of antibodies have a common biochemical structure The basic structure unit is composed of four polypeptide chains: two identical light chains and two identical heavy chains Covalent disulfide bonding holds the light and heavy chains together Variable Regions: Two sections at the end of Y s arms. Contain the antigen binding sites (Fab). Identical on the same antibody, but vary from one antibody to another. Constant Regions: Stem of monomer and lower parts of Y arms. Fc region: Important because this part can bind to FC receptors on cells or activate complement pathway resulting in cell lysis. 10/22/2009 8
Antibody classes (or Isotypes) Antibodies are classified based on differences in their heavy chains There are five types of heavy chains, Mu, Gamma, Alpha, Epsilon, and Delta Therefore there are five classes of antibodies: IgM, IgG, IgA, IgE, and IgD These antibodies differ in their physiochemical properties, their distribution, concentration, and their functions Within some antibody classes, there are also subclasses 10/22/2009 9
Classes of Antibodies 10/22/2009 10
Antibody Fragments Antibodies when treated with certain enzymes are cleaved into a Fab fragment and an Fc fragment The roles of the Fab and Fc regions reflect the two main roles of the antibody. First to bind specifically to the antigen and secondly to stimulate its elimination. Enzyme digestion Fab fragment responsible for binding antigen Fc region responsible for fixing complement and binding to Fc receptors on monocytes 10/22/2009 11
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Antibodies important for the blood bank The most important antibody classes in the blood bank are IgM and IgG This is because these antibodies are capable of destroying transfused antigen-positive RBCs and causing anaemia and transfusion reactions 10/22/2009 13
IgMantibodies IgM form most of the anti-a and anti-b antibodies. IgM antibodies against A or B blood group antigens are known as naturally occurring antibodies. They are called naturally occurring because they are present without previous exposure to A or B antigens. IgM antibodies appear during the first few months after birth, probably as a result of exposure to ABH antigen-like substances in the diet or the environment. IgM is a very effective class in destroying the antigen because it can fix complement. 10/22/2009 14
IgM antigen binding sites IgM is a pentamer molecule (has five Ig units). Each Ig unit (labeled 1) has two epitope binding fab regions, so IgM theoretcally is capable of binding up to 10 epitopes = however only five sites are readily available to combine with antigen. therfore IgM referred to as a pentavalent antibody. 15
IgG antibodies IgG antibodies are known as immune antibodies IgG antibodies are important because they are the class of antibody that is formed in response to transfusion with nonself (or incompatible) RBCs IgG is a monomer Ig which is much smaller than IgM IgG is the only class of antibody that can cross the placenta IgG exists in four subclasses (IgG1-4) 10/22/2009 16
IgMversus IgG IgM IgG Heavy chain type Mu Gamma MW(kd) 900 150 Placental transfer Complement fixation No ++++ + Yes 10/22/2009 17
IgMversus IgG IgM IgG 10/22/2009 18
Effector roles of antibodies 1) Induction of physical changes 2) Cell lysis 3) Phagocytosis 4) Inflammation 10/22/2009 19
Consequences of RBC Antigen-Antibody reaction Agglutination Lysis by phagocytosis 20
Effector roles of antibodies Induction of physical changes: For example binding of antibody to antigen on the surface of cells result in their clumping together or agglutination Cell Lysis: 1) Antibodies of IgM and IgG can bind to complement, which activate a series of reactions resulting in the lysis of cells. IgM is a much more efficient activator of complement than IgG. In blood transfusion reactions, much of the pathology involves the activation of complement 2) through binding of the Fc region of IgG to certain type of lymophyctes and this results in the activation of the lymophycte and its release of toxins that results in the lysis of cells. This is called antibody-dependent cellular cytotoxicity (ADCC) and is only mediated by IgG 10/22/2009 21
Detection of antibody-antigen reactions Antibody-Antigen (Ab-Ag) reactions are used in the blood bank to: - To determine the blood group - To perform antibody screening - For compatibility testing The visualization of the Ab-Ag reactions are usually achieved by either haemagglutination or haemolysis These are simple tests and techniques to perform but are governed by many physical and chemical factors that must be always taken into account to ensure correct results 10/22/2009 22
Haemagglutination The major technique used in the blood bank Haem= blood, and agglutination= clumping, or Haemagglutination is red cell agglutination Agglutination of red cells by antibodies occurs in two steps: 1) Sensitization= antibody binds to the antigen 2) Agglutination= when sensitized cells clump together resulting in the formation of visible agglutination. 10/22/2009 23
Haemagglutination First stage: Sensitization Stage 1 of agglutination: binding of antibody to antigen Antigen 10/22/2009 24
Haemagglutination Second stage: Agglutination Stage 2 of agglutination: clumping of red cells by antibody bridges 10/22/2009 25
No agglutination agglutination 10/22/2009 26
IgMand IgG Only IgMcan bring about the two stags of agglutination, so the reaction is visible That's why IgM is called complete antibody IgGcan bind (sensitize) to the red cells but can not bring about agglutination That's why IgG is called incomplete antibody 10/22/2009 27
Repulsive force between red cells (zeta potential) Red cells carry a negative charge on their surface which results in a repulsive force (known as zeta potential) This repulsion cause the RBCs to stay apart This is important in vivo to prevent red cell from clumping together and from binding to the cell wall In the lab we need to reduce this force to facilitate agglutination Dr. Albanyan, CLS 441, KSU 10/22/2009 28
Zeta Potential Because of its small size, IgG can not bridge between red cells because of the repulsive force (zeta potential) that keeps the red cells apart Dr. Albanyan, CLS 441, KSU 10/22/2009 29
Zeta potential Because of its large size, IgM can bridge between red cells despite the zeta potential that keeps red cells apart 10/22/2009 30
Zeta Potential Dr. Albanyan, CLS 441, KSU 10/22/2009 31
How to help IgGin accomplishing the second stage of agglutination? 32
Centrifugation Centrifugation is an effective way to enhance agglutination reactions because it decreases reaction time by brining the reactants together It allows RBCs to overcome their repulsive force (zeta potential) for each other and therefore it facilitates agglutination It places antigen and antibody in close proximity 10/22/2009 33
Enhancement media Agglutination reactions for IgM antibodies and their corresponding RBC antigens are easily accomplished in saline medium as these antibodies usually do not need enhancement to react strongly with antigens Detection of IgG, however, may require enhancement techniques Many of the enhancement media increase the reactivity of IgG by decreasing the zeta potential of RBC membranes Examples are: Albumin, low ionic strength solution (LISS), Polyethylene Glycol, Proteolytic enzymes, and Antihuman Globulin (AHG) reagents 10/22/2009 34
Antihuman Globulin (AHG) 35
Addition of Enzymes 10/22/2009 36
Factors that influence Agglutination reactions As any other biochemical reaction, agglutination reaction is influenced by many factors including: Concentration of the reactants Centrifugation ph Temperature Ionic strength Antibody isotype The surface charg RBC antigen dosage The use of various enhancement media Antihuman globulin reagents (AHG) 10/22/2009 37
Antibody Antigen Ratio The optimal concentration of antibody and antigen is very important Any excess of unbound antibody leads to a prozon effect Any excess of unbound antigen leads to a postzone effect In either situation the formation of agglutination may not occur leading to a false negative results Another reason for the antigen being in a non optimal concentration is due to the weak expression of the antigen on the surface of the RBC (dosage effect) 10/22/2009 38
Antibody Antigen Ratio 10/22/2009 39
Effect of ph The ideal ph is in the range of 6.5-7.5 (similar to the ph of normal plasma or serum) 10/22/2009 40
Temperature Different antibody classes may exhibit optimal reactivity at different temperatures IgM usually react best at ambient tempratures or below 22 C IgG antibodies usually require 37 C Its therefore important to do testing with a range of temperatures 10/22/2009 41
Ig Type IgM antibodes are generally capable of agglutinating RBCs suspended in saline medium The IgM is larger than IgG and for this reason it is more efficient in bridging the distance between two RBCs Another reason why IgM is more efficient than IgG is because it has 10 antigen binding sites, whereas IgG has only 2 10/22/2009 42
Detection of RBC antibody-antigen reactions The most important step is obtaining the right sample Different tests may require different sample types For example to measure complement fixation you have to use serum Complement activity requires the presence of divalent Ca 2+ and Mg 2+ ions which are chelated by some anticoagulants 10/22/2009 43