immunochemical methods in clinical laboratory

• particle method: precipitation using immunodiffusion and immunoelectrophoresis
• light scattering: nephelometry and turbidimetry

label methods: non competitive, competitive, heterogenous, homogeneous

• affinity: the strength of a single Ab and Ag bond to the epitope
• avidity: total strength of all the bonds of the Ab and Ag

detection of Ag and Ab complex without labels

• when there are excess Ag 
• cannot detect ppt

• when there are excess Ab
• cannot detect ppt

gel provides a medium which allows soluble antigen and antibody to pass. The precipitated complexes are easier to separate in gel than liquid suspension

• single radial immunodiffusion (RID)
• Ab is in the gel

double RID

• Advantages: simple, accurate, adaptable
• disadvantages: slow

assays that detect Ab-Ag complex by using labels

• easily attached to antigen/antibody
• easily measured
• does not interfere with Ab-Ag reaction
• inexpensive and non toxic

• radioisotopes
• enzymes
• fluorophors
• luminescent substance

• advantages: sensitivity, size, flexibility
• disadvantages: toxicity, shelf life, disposal cost

• labeled Ag* competes with unlabeled Ag for binding sites
• as Ag increases it binds to Ab resulting in less binding of Ag*. Ag* is limited and constant in amount
• one step process: Ag*, Ag, and reagent Ab are incubated simultaneously
• sequential step: Ag* is incubated with Ab then Ag is added

• Ab* is used to detect Ag
• excess Ab* is required to ensure labeled Ab reagent does not limit reaction
• Concentration of Ag is directly proportional to bound Ab*