Chapter 8: Precipitation Reactions Law of Mass Action

 Antigen + Antibody  Law of Mass Action

- Important role in diagnosing many different diseases - Govern all reversible antigen–antibody binding
- States that free reactants are in equilibrium with bound reactants
 Immunoassays
- Developed to detect either antigen or antibody  Equilibrium constant
- Maybe Manual or Automated - Represents difference in the rates of the forward and reverse
- Based on 2 principles: reaction according to the following equation:
o Precipitation
K1 [AgAb]
 Combining soluble antigen with soluble antibody to produce K= =
insoluble complexes that are visible - Where: K2 [Ab][Ag]
 First noted by Kraus (1897), culture filtrates of enteric
bacteria would precipitate when mixed with antibody o [AgAb] = conc. Antibody-antigen complex (mol/L)
o Agglutination o [Ab] = conc. of Antibody (mol/L)
 Process by which particulate antigens (cells) aggregate to o [Ag] = conc. of Antigen (mol/L)
form larger complexes when specific antibody is present - can be seen as a measure of the goodness of fit.
- ↑ Strength of bind/Avidity = ↓ tendency of complexes to
Antigen-Antibody Binding dissociate
- ↓ K2 = ↑K1
- ↑ K = ↑ amount of antigen-antibody complex
 The primary union of binding sites on antibody with specific
 Reaction more visible ore detectable
epitopes on an antigen depends on affinity and avidity.

Precipitation Curve
 Affinity
- Initial force of attraction between a single Fab site on an antibody
molecule and a single epitope

 Epitope – Paratope: held together by non-covalent bonds:

1. Ionic bonds
- Occur between oppositely charged particles
2. Hydrogen bonds
- Attraction between polar molecules that have a slight
charge separation
- Positive charge resides on hydrogen atom
3. Hydrophobic bonds
- Occur between nonpolar molecules that associate with
another and exclude molecules of water
4. Van der Waals forces
 Zone of Equivalence (ZOE)
- Interaction between electron clouds of oscillating dipoles
- Ab = Ag
- Where optimum precipitation occurs
** These are weak bonds that occur over a 1x10-7 mm distance, so there
- Number of multivalent sites of antigen and antibody are
must be a very close fit between antigen and antibody.
approximately equal
- Precipitation at ZOE
** Strength of attraction depends on the specificity of antibody for a
o Antibody binds to more than one antigen and vice
particular antigen. The more specific the Ag-Ab to each other, the stronger
versa forming a lattice (Reversible reaction)
their bond (attraction).
 Lattice Hypothesis
- Formulated by Marrack
 Cross-reactivity
1. Each antibody molecule must have at least two binding
- Antibodies are capable of reacting with antigens that are
sites
structurally similar to the original antigen that induced antibody
2. Antigen must be multivalent
production
- Antigen + Antibody = Multimolecular lattice that increases in
- High resemblance to original antigen, stronger the bond
size unti it precipitates
- Perfect lock-and-key relationship = maximal affinity
- Heidelberger and Kendall
o Performed quantitative precipitation reactions
 Avidity
that established proof for this theory.
- Sum of all the attractive forces between antigen and antibody
- Involves the strength with which a multivalent antibody binds a
↑ soluble antigen = ↑ amount of precipitation up to the ZOE
multivalent antigen
- Measure of overall stability of an antigen-antibody complex
↑ amount of antigen overwhelms the number of antibody combining
- High avidity can compensate for low affinity
sites present = ↓ precipitation
 Prozone Phenomenon (PrP) - Used in:
- Ab > Ag o Quantification of immunoglobulins
- Antigen combines with only one or two antibody molecules (IgG, IgA, IgM, and IgE)
- No cross-linkages or visible end result o Kappa and Lambda chains
- Many free antibody molecules remain in solution o Complement proteins
- False Negative reactions o C Reactive Protein
o due to high antibody concentration o Clotting Factors
o Dilute antibody and perform the test again
PASSIVE IMMUNODIFFUSION TECHNIQUES
 Postzone Phenomenon (PsP)
- Ab < Ag  Agar
- Small aggregates are surrounded by excess antigen - High-molecular-weight complex polysaccharide derived from
- No lattice is formed seaweed
- Excess antigen may obscure presence of small amount of antibody - Has negative charge
 Paired sera
 Test is repeated with an additional patient specimen  Agarose
- Purified agar
** For precipitation to be detectable, they must be run in the zone of - Used as support medium (used to read/determine precipitation
equivalence. results)
- More preferred than Agar because has almost no charge
MEASUREMENT OF PRECIPITATION BY LIGHT SCATTERING
 Agar and agarose:
Precipitation in a Fluid Medium  Stabilize diffusion process
 Allow visualization of precipitin bands
1. Turbidimetry
- Measure of the turbidity or cloudiness of a solution Precipitation by Passive immunodiffusion
- Measures the reduction in light intensity due to:
Reflection, Absorption, Scatter  Passive Immunodiffusion
- When no electrical current is used to speed up the antigen-
**Scattering of light occurs in proportion to the size, shape, and antibody combination by means of diffusion.
concentration of molecules in a solution. - Combination of Ag and Ab occurs by allowing them to diffuse
towards each other (in a medium).
 Absorbance units
- measure of the ratio of incident light to that of transmitted light  Factors that affect Rate of Diffusion
1. Size of particles
**Measurements are made using spectrophotometer or automated 2. Temperature
clinical chemistry analyzer. 3. Gel viscosity
4. Amount of hydration
2. Nephelometry
- Measures the light that is scattered at a particular angle from the **0.3 to 1.5% of agar concentration allows diffusion of most reactants
incident beam as it passes through a suspension.
- can be used to detect either antigen or antibody. 1. One-Dimensional
- Amount of light scattered = index of the solution’s concentration
- Recorded as: a) Single Diffusion technique
o arbitrary units of relative light scatter - Pioneered by James Oudin
o mg/dL – milligram per deciliter - Oudin Single Diffusion (Single-linear Diffusion)
o IU/mL – international units per mL - Antibody was incorporated into agarose in a test tube
- Measure light scatter at 10° to 90° - Only one reactant migrates (either Ag or Ab)
- Medium: Agarose gel in a test tube
** Nephelometry is more sensitive than Turbidity with a lower limit of - Positive result: Precipitin band
detection of 1-10 mg/dL **If after 24hrs incubation and NO PRECIPITIN LINE appeared, DO NOT
IMMEDIATELY REPORT as Negative. Incubate again for another 24hrs
a) End point nephelometry at 37C.
- Reaction is allowed to run essentially to completion, but large
particles tend to fall out of solution and decrease the amount of b) Radial Immunodiffusion
scatter. - Modification of single-diffusion technique
- used to measure IgG, IgM, IgA, and complement components.
b) Kinetic/ Rate nephelometry - Antibody is uniformly distributed in the support gel, and antigen is
- Rate of scattering increase is measured immediately after the applied to a well cut into the gel
reagent is added. - Antigen-antibody combination changes proportions until the zone
- Rate change is directly related to antigen concentration of of equivalence is reached and stable lattice is formed
antibody concentration is constant. - Area of the ring = measure of Antigen concentration
- provides accurate and precise quantitation of serum proteins, and - Disadvantage: fairly expensive to run
the cost per test is typically lower than other methods.
 Two techniques for the measurement of Radial Immunodiffusion: ELECTROPHORETIC TECHNIQUES

i. End-point method Precipitation by Electrophoretic Techniques

- Developed by Mancini
- Antigen is allowed to diffuse to completion
- When equivalence is reached, no further change in the ring
diameter (occurs between 24 and 72 hours)
- Square of diameter is directly proportional to antigen
concentration
- Disadvantage: takes time to obtain results
ii. Kinetic method
- aka Fahey and MacKelvey Method
- uses measurements taken before point of equivalence is
reached
- Diameter is proportional to the log of concentration
- Readings are taken at about 18 hours
 Electrophoresis
- Separates molecules according to differences in their electric
charge when they are place in an electric field
- Can be applied to both single and double diffusion
1. Rocket Immunoelectrophoresis
- Just like RID but uses electrophoresis to facilitate migration of the
antigen into the agar
- End result: Conical precipitin line resembling a rocket
- Height of rocket (well to apex) directly proportional to the
amount of antigen
- Much more rapid than RID
- Used to quantitate immunoglobulins using pH 8.6 buffer.

 Monospecific antiserum a) One-dimension electroimmunodiffusion

- Must be used to increase clarity of precipitation (High o Adaptation of radial immunodiffusion (RID)
affinity) o Developed by Laurell (early 1960)
-
 Sources of Error
1. Overfilling/Under filling the wells 2. Countercurrent Immunoelectrophoresis
2. Nicking the side of the wells when filing
3. Spilling sample outside the wells 3. Immunoelectrophoresis
4. Improper incubation time and temperature - Double diffusion technique that occurs at right angles
5. Incorrect measurement - Incorporates electrophoresis current to enhance results
- Introduced by Grabar and Williams (1953)
2. Two - Dimensional - Two-step process
- Source of Antigen: SERUM
a) Ouchterlony Double Diffusion - Antiserum is placed in the trough, and the gel is incubated for 18-
- Old, classic immunochemical techniques 24 hours.
- Both Antigen and Antibody diffuse independently through a semi- - Has been used for detection of:
solid medium in two dimensions o Myelomas
- Incubation conditions: o Waldenstrom’s macroglobulinema
 12 to 48 hours o Malignant lymphoma
 Moist chamber o Immunodeficiencies – no precipitin band formed
- Density of the lines reflects the amount of immune complex o Lymphoproliferative disorders
formed - Limit of Detection: 3-20 uL
- Used to identify:
o Aspergillus, Blastomyces, Coccidioides, Candida 4. Immunofixation Electrophoresis
- Most Ouchterlony plates are set up with a central well - First described by Alper and Johnson
surrounded by 4 - 6 equidistant outer wells. - Similar to Immunoelectrophoresis EXCEPT that after
 The position of the precipitin bands between wells allows electrophoresis takes place, antiserum is applied directly to the
for the antigens to be compared with one another. Several gel’s surface.
patterns are possible: - Uses Agarose or Cellulose Acetate
o Arc: Represents serological identity - Requires shorter time and results higher resolution
o Cross-line: Two separate reactions, Compared antigens - Gel thickness: Approx. 1mm
share no common epitopes - Hypogammaglobulinemia = faintly staining bands
o Fusion of two lines with a spur: Indicates partial - Polyclonal hypergammaglobulinemia = darkly staining bands in
identity, Spur always points to the simpler antigen gamma region
- Waldenstrom’s macroglobulinemia = Monoclonal bands with dark
 Factors affecting accuracy of results and narrow bands in specific lanes
1. Overfilling the wells - Useful in demonstrating antigens present in:
2. Irregular hole punching o Serum
3. Nonlevel incubation o Urine – for Bence-Jones proteins (Multiple myeloma)
4. Drying out of gel o CSF – Specimen of choice for Multiple Sclerosis
5. Inadequate time for diffusion
Western Blot
b) Double-linear Ouchterlony - Adaptation of immunofixation electrophoresis
- Confirmatory tests to detect HIV-1
c) Double-radial Ouchterlony - Uses Nitrocellulose paper
Sources of Error in Electrophoresis

1. Applying current in wrong direction – samples may run off the gel
or not separated
2. Incorrect pH of buffer
3. Incorrect electrophoresis time
4. Concentrations of antigen and antibody
5. Amount of current
a. Weak – incomplete separation
b. Too strong – generation of heat leading to
denaturation of proteins

COMPARISON OF PRECIPITATION TECHNIQUES

TECHNIQUE APPLICATION SENSITIVITY COMMENT

(μg ab/mL)

Nephelometry Immunoglobulins, 1–10 Automated,

complement, sensitive,
C-reactive protein, expensive
other serum proteins equipment
needed

Radial Immunoglobulins, 10–50 Quantitative,

immunodiffusion complement slow, not as
sensitive as
nephelometry

Ouchterlony Complex antigens such 20–200 Semiquantitativ,

double diffusion as fungal antigens slow, may be
difficult to
interpret

Rocket Immunoglobulins, 2 Fast,

electrophoresis complement, alpha- quantitative,
fetoprotein technically
demanding

Immuno Differentiation of 20–200 Slow,

electrophoresis serum proteins semiquantitative
difficult to
Interpret

Immunofixation HIV, Lyme disease, Variable Fairly rapid,

electrophoresis syphilis semiquantitative,
sensitive