Antibody based detection Chem 4630 Lesson #4 2/5/11

Uses of antibodies in forensics -Blood typing - Landsteiner A, B, AB, O and Rh - Specific molecular recognition (usually of a protein) - recognition of certain biomolecules

Antibodies - general form Antigen-binding site Heavy chain variable region Hinge region Heavy chain constant region Stem region Light chain variable region Light chain constant region Disulfide bond (cysteine bridge) (a)

Antibody – another view - variable regions of the light chain (grey) and the heavy chain (yellow) form the antigen binding site - light chain constant region is blue while heavy chain constant region is red. The two chains are joined by carbohydrate (purple). This is a space-filling model of immunoglobulin. Yellow regions in the heavy chain constant region represent amino acids that may be variable –they are not part of the antigen binding site. ©Mike Clark, www.path.cam.ac.uk/~mrc7/

How Antibody Binds to Antigen On this slide, the top panel of images is a schematic representation of the binding of antigen to antibody. The blue portion corresponds to the antibody molecule, with VH representing the variable portion of the heavy chain and VL representing the variable portion of the light chain, that come together to form the antigen binding site. The red dot (orange cylinder, yellow wedge) symbolizes a single determinant of an antigen. On the previous slide of the pollen grain, entitled “Antigens”, each dot on the pollen grain represents a potential antibody binding site. Complex antigens like pollen grains have many binding sites and can bind to more than one antibody. In fact, most antigens stimulate more than one type of B cell. But the antibody that each B cell produces is specific for only for its antigen.   The different orientations in the top panel of images simply represent some of the various spatial interactions that may occur between antigen and antibody. The lower panel of images represents the same interaction, but as a molecular model of the antigen/antibody interaction as might be seen if one were looking downward, directly into the antigen binding site. The top part of this figure shows how different shaped antigens can fit into the binding site of antibodies: left, pocket; center, groove; right, extended surface. The panels below show space-filling or computer-generated models indicating where contact between the peptide antigen and antibody occurs.

Precipitation Tests Precipitation Immunodiffusion

Agglutination Tests Agglutination Hemagglutination

Antigens and Antibodies of ABO Blood Types

Typing Blood Single drops of blood are mixed with different antisera Agglutination with an antisera indicates the presence of that antigen on the RBC

ELISA Tests

Immunological Tests (cont.) Neutralization Immuno- fluorescence

The chemistry of binding Ab + Ag <==> AbAg K = Ab-Ag/[Ab][Ag] 104 to 1012 L/mol

More complicated On rate = K Off rate = Kd = [P] [L]/[C]

How tight is tight? Biotin and avidin bind with a dissociation constant of roughly 10 - 15 M = 1 fM = 0.000001 nM Range of antibodies 3 uM to 3 nM

Quenching

Western Blots Protein profiling