AGGLUTINATION

 Clumping of antigen-coated cells or beads by specific antibody (normally use

IgM)

 Methods enhancing agglutination to detect incomplete Ab (like IgG)

o Adding a second antibody in the indirect Coomb’s test
o Adding of colloid to the suspension
o Incubation in LISS (lower ionic strength solution)
o Treat RBC with proteolytic enzymes (zeta potential)
o Use substance (dextrin, BSA) to increase the viscosity of reaction medium

Classification of agglutination reactions:

1. Direct agglutination reactions: RBC (Ag) + specific Ab isohemagglutinins (anti A
or anti B) = IgM (agglutination)
Common agglutination based assays:
 ABO blood group typing
 Rheumatoid factor assay
 Rh (D) Ag
 Qualitative pregnancy test
 Mono spot test

2. Passive (indirect) agglutination

Patient serum, Ab (IgM) + soluble Ag adsorbed particle = agglutination
Methods to increase efficiency of Ag adsorption to cells:
 Mild treatment of RBC with tannic acid or other oxidizing agents
 Using covalent cross-linking agents (glutaraldehyde)

3. Direct Coomb’s or direct anti globulin test (DAT)

 Detection of incomplete Ab (anti Rh IgG)
 Incomplete Ab (anti Rh IgG Ab) + RBC (Rh IgG Ag) = bind (no
agglutination)
 Incomplete Ab (specific anti Rh IgG Ab) (first Ab) + RBC (Rh IgG Ag) +
rabbit anti human IgG (second Ab against first Ab) = agglutination

Clinical agglutination assay:

 Rose Wanler test:
o Rheumatoid arthritis patient serum (IgM / Ab) + sheep RBC coated with
rabbit IgG (Ag) = agglutination.
o Rheumatoid factor (RF) (IgM-anti IgG) agglutinate latex beads coat with
IgG

 Pregnancy test: assay to measure hCG

o Agglutination assays
o RIA (very quantitative and sensitive)
 o Direct latex particle agglutination (pregnancy) tests:
 Anti hCG (Ab) coated latex (known) + sample with hCG (Ag) =
agglutination
o Hemagglutination inhibition latex agglutinate – inhibition
o Particles coated with hCG (Ag) + anti hCG (Ab) + patient sample with β-
hCG (Ag) = no agglutination (added free hCG blocks agglutination)
o Particles coated with hCG (Ag) + anti hCG (Ab) + patient sample with no
β-hCG (Ag) = agglutination

 Mono spot test.

o Rapid, specific, and sensitive test for heterophil Ab or IM.
o Patient serum (IM) Ab + sheep RBC (Ag) = agglutination
o Infectious mononucleosis (IM):
 Heterophile Ab: Ab reacting with Ag or cells from various
mammals