Immunology

ANTIBODIES

• we have ~1012 antibodies made against foreign viruses, bacteria, parasites (vaccines)• antibodies combine with foreign antigens to inactivate foreign species• antigenic determinants (epitopes) recognized by antibody (unique shape, 5-10 AA sequence)• antibody containing serum called ANTISERUM

Antibody (IgG) - antigen (small peptide from HIV)

ANTIBODIES

• structure “Y shape”• two identical light chains, two identical heavy chains• N-termini of light and heavy come together to form antigen binding site (unique AA here!)

light

heavy

Hypervariable region antigen binds here

Immunology

Western Blot

IMMUNE SYSTEM

• antibodies found in the gamma globulin fraction of serum• called immunoglobulins (Ig) - have IgG, IgA, IgD, IgE, IgM• major class of Ig is IgG• purified foreign proteins can act as antigens and after injection into a differing species they can stimulate antibody formation

EX: inject rabbit IgG into goat to cause goat to make anti-rabbit IgG antibodies

EX: vaccines - inject infant with attenuated, non-infective polio virus to cause baby to make antibodies to the antigen and acquire immunity to POLIO

Immunoassays - double diffusion

• specific Ag-Ab complexes undergo precipitation rxns that are visible in agar gel

• cut small wells in agar and place small amounts of Ag and Ab in wells

• Ag and Ab diffuse outward toward each other at rates related to [], size and shape

• milky white line of precipitation is seen where Ag contacts Ab

Immunoassay - Western Blot• Abs used in Western blots to identify specific protein • use of protein gels to identify purification steps• purified foreign proteins can serve as antigens because they will stimulate Ab formation when injected into animals

Western Blot

Immunoassay - ELISA

(enzyme linked immunosorbent assay)

ELISA (enzyme linked immunosorbent assay)

ELISAPrep of microtiter plate

Adsorb Ag to platemake serial dilutionsblock addition sites with TBS-gelatin (blocking agent)remove liquid in specific order!!! Pay attention to the details!!reapply TBS-gelatin - specific order!!!

Ab reactionadd Ab to each wellblocking stepremove liquid in specific order!!addition of solutions with subsequent removal of liquid in specific order!!

Color developmentadd color development to each wellobserve blue colorchange pH of solution in well by adding HCl - observe yellow color

ELISA

TMBSolubleColorlesssubstrate

TMBTMBInsolubleBlueProduct

TMBInsolubleYellowProduct

HCl