PRINCIPLES OF PRECIPITATION REACTIONS | BSMLS-3C

PRINCIPLES OF PRECIPITATION REACTIONS

OVERVIEW OF PRECIPITATION
Definition of Terms:
❖ Precipitation
 It involves the combination of soluble antigen with soluble antibody to form visible insoluble
complexes.
❖ Precipitinogen
 participating soluble antigens (e.g., proteins or polysaccharides of bacterial capsules)

❖ Agglutinins
 participating soluble antibodies

Precipitation Curve
❖ Zone of Equivalence
 number of binding sites of multivalent antigen and antibody are approximately equal
 the point where maximum precipitation occurs
 Lattice hypothesis by Marrack: bivalent (at least) antibody + multivalent antigen →
multimolecular lattice → insoluble complexes (precipitates)
 Any deviation from the zone of equivalence decreases the efficiency of the reaction resulting in
diminished agglutination formation or false-negative reactions.
❖ Prozone
 occurs when the concentration of antibody exceeds the concentration of antigen (region of
antibody excess)
 will result in weak reactions or no precipitate formed
 Solution: serum may be diluted with appropriate buffer
❖ Postzone
 occurs when the concentration of antigen exceeds the concentration of antibody (region of
antigen excess)
 will result in weak reactions or no precipitate formed
 Solution: increase the amount of patient’s serum

Two Methodologies in Precipitation:

❖ Dean and Webb or Alpha () Procedure
 precipitation test in which antigen is serially diluted (antibody or antiserum is constant)
 Prozone: region of antigen excess
 Postzone: region of antibody excess

❖ Ramon or Beta (β) Procedure

 precipitation test in which antibody is serially diluted (antigen is constant)
 Prozone: region of antibody excess

PREPARED BY: KAREN B. ROSETE, RMT 1

 PRINCIPLES OF PRECIPITATION REACTIONS | BSMLS-3C

 Postzone: region of antigen excess

Take Note:
❖ IgG is a much better precipitating antibody than IgM
❖ IgA is a poorer precipitin than IgM
❖ IgE is nonprecipitating
❖ Zoning phenomenon (postzone, prozone) → major deterrent to the use of fluid precipitation tests
❖ Ramon procedure is the methodology employed in serology since for most serology tests, it is the
serum (source of antibody) that is concentrated and needs to be diluted

TYPES OF PRECIPITATION REACTIONS

A. Fluid-Phase Precipitation
❖ Measurement of precipitation by light scattering
• Turbidimetry
• Nephelometry
❖ Interfacial (Ring) Test

B. Precipitation Reactions in Gel Medium

❖ Antigen and antibody diffuse through the agar gel and precipitate when they reach the zone of
equivalence
❖ Gel/support medium: agarose (most widely used medium), gelatin, cellulose acetate, polyacrylamide,
starch
❖ Molecules of different sizes tend to diffuse through gels at different rates: ↑ MW = ↓ Diffusion rate
❖ Reactants:
 single diffusion → one reactant is moving through the medium
 double diffusion → two reactants are moving through the medium
❖ Medium:
 single dimension → gel matrix on test tube; diffusion is vertical
 double dimension → gel matrix on a petri dish with circular holes; diffusion is both vertical and
horizontal (or reactants diffuse radially)
❖ Two Techniques:
 Passive Immunodiffusion Techniques
• Antigen-antibody combination by means of diffusion in the gel without the use of
electric current
• Factors affecting diffusion rate: size of the particle, temperature, gel viscosity, amount of
hydration

 Immunoelectrophoresis Techniques
• Diffusion of reactants through the gel is caused by electrophoresis
• Electrophoresis → separates molecules according to differences in their electric charge
when placed in an electric field

❖ Based on the number of reactants moving and movement of diffusion, there are 4 combinations of
Precipitation Reactions:
 Single diffusion-single dimension

PREPARED BY: KAREN B. ROSETE, RMT 2

 PRINCIPLES OF PRECIPITATION REACTIONS | BSMLS-3C

 Single diffusion-double dimension

 Double diffusion-single dimension
 Double diffusion-double dimension
TECHNIQUES OF IMMUNOLOGICAL PRECIPITATION IN GELS
Number of reactants 1 1 2 2
moving
Number of effective 1 2 1 2
dimensions
Passive Oudin* Radial Oakley and Ouchterlony*
immunodiffusion immunodiffusion Fulthorpe*
techniques (RID) †
Immunoelectrophor ▪ Rocket ▪ Crossed ▪ Double ▪ Immuno-
esis immuno- immuno- electro- electrophor
electrophores electrophores immunodiffus esis (IEP)*
is† is* ion (double ▪ Grabar and
▪ Laurell ▪ Double- IED)* Williams
technique crossed ▪ Crossed
▪ Single immuno- antigen-
electro- electrophores antibody
immunodiffus is electrophores
ion ▪ Two- is
dimensional ▪ Counter
immuno- electrophores
electrophores is
is ▪ Crossing over
▪ Ressler electrophores
is
▪ Counter
immuno-
electrophores
is (CIE)
▪ Counter
current
immuno-
electrophores
is
▪ Immuno-
osmophoresis
(IOP)
▪ Electrosynere
sis
▪ Immunoelect
ro-
osmophoresis
(IEOP)

PREPARED BY: KAREN B. ROSETE, RMT 3

 PRINCIPLES OF PRECIPITATION REACTIONS | BSMLS-3C

▪ Counter
migration
electrophores
is
FLUID-PHASE PRECIPITATION
A. Turbidimetry
❖ measure of the turbidity or cloudiness of a solution
❖ detector is placed in line with the incident light
❖ detection device → measures the reduction in light intensity due to reflection, absorption, or scatter
❖ Scattering of light occurs in proportion to the size, shape, and concentration of molecules present in
solution
B. Nephelometry
❖ The amount of light scattered is an index of the solution’s concentration.
❖ detector is placed at a particular angle from the incident light
❖ Nephelometers measure light scatter at angles ranging from 10 degrees to about 90 degrees
❖ More sensitive than turbidimetry
❖ End point nephelometry → light scattering is measured after the completion of the reaction (causes
falsely decreased result)
❖ Kinetic or rate nephelometry → rate of scattering increase is measured immediately after the
reagent is added
❖ Reagent: source of antibody | Patient’s sample: source of antigen
❖ Applications: quantification of immunoglobulins (IgG, IgA, IgM and IgE, kappa and lambda light chains),
quantification of other serum proteins (complement components, CRP, haptoglobin, ceruloplasmin)
C. Interfacial or Ring Test
❖ Simple qualitative precipitation test
❖ Undiluted antibody + diluted antigen solution → incubated at RT (usually 30 minutes) → observe for
development of hazy, cloudy, or milky layer (5-minute interval) → ring formation
❖ Negative result: No precipitin | Positive result: Precipitin at the interface
❖ Application: C-reactive protein (CRP), Lancefield grouping of β-hemolytic streptococci, Ascoli’s
thermoprecipitin test for anthrax diagnosis

PASSIVE IMMUNODIFFUSION TECHNIQUES

A. Oudin
❖ single diffusion-single dimension method
❖ antibody was incorporated into agarose in a test tube → overlaid with an antigen → mobile antigens
diffuse through the medium → insoluble Ag-Ab complexes → ↑ size of the complex → band of precipitate
forms

B. Radial Immunodiffusion (RID)

❖ single diffusion-double dimension method
❖ antibody is uniformly distributed in the support gel → antigen is placed in a well → antigens diffuse out
from the well → insoluble Ag-Ab complexes → ring forms → ring diameter is measured against a
standard curve
❖ Two Techniques for the Measurement of RID
 Mancini Method/End-point Method

PREPARED BY: KAREN B. ROSETE, RMT 4

 PRINCIPLES OF PRECIPITATION REACTIONS | BSMLS-3C

•antigen is allowed to diffuse to completion; measurements are taken after equivalence is

reached
• equivalence occurs between 24 and 72 hours
 Fahey Method/Kinetic Method
• Measurements are taken before the point of equivalence is reached (at about 18-19 hours
or as specified by the manufacturer)
❖ Sources of Error: overfilling/underfilling the wells, nicking the side of the wells, improper incubation time
and temperature, incorrect measurement
❖ Application: used to measure IgG and IgA subclasses, complement components

C. Oakley and Fulthorpe

❖ Double diffusion-single dimension method
❖ antibody was incorporated into agarose in a test tube → overlaid with a plain/neutral agar → overlaid
with antigens → both antigens and antibodies diffuse through the gel → insoluble Ag-Ab complexes →
band of precipitate forms

D. Ouchterlony Double Diffusion

❖ Double diffusion-double dimension method
❖ Both antigen and antibody diffuse independently through a semisolid medium in two dimensions
❖ Wells are cut in a gel → reactants are added to the wells → incubated for 12-48 hours in a moist
chamber →precipitin lines form
❖ Density of precipitin lines = amount of Ag-Ab complexes formed
❖ Ouchterlony plates consist of:
 One central well = where multi-specific antibody is placed
 4-6 equidistant wells = where different antigens are placed

❖ Patterns:
 Serological Identity
• indicated by a precipitation line that forms a continuous arc
• antigens share identical epitopes; unknown antigens are serologically identical
 Partial Identity
• indicated by the formation of precipitation line with a spur
• antigens share a common determinant; one antigen is simpler, the other is a
more complex antigen
• Spur always points to the simpler antigen (has fewer antigenic determinants)
 Nonidentity
• Indicated by formation of precipitin lines that cross one another
• Antigens share no common epitopes; antigens are serologically distinct

❖ Application:
 antigen detection (Aspergillus, Blastomyces, Coccidioides, and Candida)
 antibody identification (e.g., antibodies to nuclear antigens)

PREPARED BY: KAREN B. ROSETE, RMT 5

 PRINCIPLES OF PRECIPITATION REACTIONS | BSMLS-3C

REFERENCES:
❖ Stevens, C.D. (2010). Clinical Immunology and Serology: A Laboratory Perspective. (3rd Ed.). Philadelphia:
F.A Davis Company
❖ Stevens, C.D. and Miller, L.E. (2017). Clinical Immunology and Serology: A Laboratory Perspective. (4th
Ed.). Philadelphia: F.A Davis Company
❖ Turgeon. M.L. (2009). Immunology and Serology in Laboratory Medicine. (4th Ed.). Singapore: Mosby

PREPARED BY: KAREN B. ROSETE, RMT 6