PRECIPITATION REACTIONS

 Soluble antigen + Soluble antibody =

PRECIPITATION A. Radial Immunodiffusion
 Insoluble complexes - modification of SINGLE-DIFFUSION
 Visible to the eyes technique
 PRECIPITATION CURVE - Commonly used in Clinical Laboratory
1. Zone of Equivalence – optimum precipitation - Measures IgG, IgM, IgA and complement
2. Prozone – excess AB is present component
3. Postzone – excess AG is present - Requires instrumentation
METHODS
I. Slide Precipitation AB – uniformly AG – applied into
- Simple distributed in a well cut into gel
- Defected visually by using LIGHT support gel
MICROSCOPE
- *Colored Cards
- COARSE = NEGATIVE
- FINE = POSITIVE AG-AB Combination- (changing
II. Tube Reaction proportions until the zone of
- Simple and widely used equivalence is reached and
- PRINCIPLE: Layering of an AG over an AB stable lattice of network is
solution formed in the gel
- Formation of precipitate = NEGATIVE
- Ring Formation (Brown Ring) = POSITIVE Area of Ring: measure of AG concentration
III. Immunodiffusion - Compared to standard curve using AGs of
- Precipitation is BEST demonstrated unknown concentration
- Random movement of AG/AB to form Techniques for Measurement of (RID)
AG-AB complexes in medium such as gel. I. Mancini/Endpoint Method
Medium: - AG is allowed to diffuse completely
 AGAR: high molecular weight complex - NO further change in ring diameter
polysaccharide, from seaweed - 24-72 hrs
 AGAROSE: purified agar - Diameter is directly proportional to
- Used to help stabilize the diffusion concentration of AG
process and allow visualization of - Graph:
precipitation bondings X-axis: Concentration
Y-axis: Diameter squared smooth
PASSIVE IMMUNODIFFUSION curve into the points
- Diffusion of reactants to form an AG-AB - Drawback: TIME
reactions without electrocurrent to speed II. Fahey & McKelvey Method
up reaction - Kinetic method
Rate of Diffusion: - Measurement is taken BEFORE the point
1. Size of praticles of equivalence is reached
2. Temperature - Diameter is proportional to log of
3. Gel viscosity concentration
- 0.3 – 1.5% Agar concentration: Diffusion - 18 hours
of most reactants - Graph: Semilog paper
4. Amount of hydration Log axis: AG concentration
5. Interference between matrix and Arithmetic axis: Diameter
reactants III. Ouchterlony Double Diffusion
- AB and AG diffuse independently through
a semi solid medium (agar)

Styles ‘18
 - Horizontal and vertical
i. Wells are cut in the gel
ii. Reactants are added in the well
iii. Incubate (12-48 hrs) in a moist
chamber
iv. Precipitin lines will form (where the
moving front of the AG meets the AB)
1. AG-AB applied to holes punched in
agar gel
a. Precipitin band
b. Precipitin band
2. Leave to diffuse
3. Wash and stain
- The density of line reflect the amount of
immune complex (Oudin Assay)
- AB that is multispecific is placed in the
central well
- Different AGs are placed in the
surrounding wells
*position of precipitin bands between
wells allow for the AGs to be compared
with one another.
- Double diffusion assay in 2D
- A quality assay
- PRECIPITATE shows the relationship
between the AGs
- NOT very accurate as molecules of some
molecular weight may overlap, giving
misleading results
- IDENTITTY, NON IDENTITY, PARTIAL
IDENTITY (refer to book)
Diffusion Patterns
1. Fusion lives at their junction to form an
arc-serologic identity/presence of
common epitope
2. Crossed lines – demonstrates 2 seprate
reactions, the compared AG shared are
common epitopes
3. Fusion of 2 lives with spur – partial
identity
Uses of Ouchterlony DD
 Identification of fungal antigens
 Detection of ABs to extractable nuclear
bodies
ELECTROPHORETIC TECHNIQUES
Principle: separation of molecules according to
electric charge.
I. Rocket Immunoelectrophoresis
- 1D electroimmunodiffusion
- Adaptation of RID
- Electrophoresis is used to facilitate
migration of the AG into the agar
- AG diffuses out of the well: precipitation
begins.
- Change in AG concentration: dissolution
and reformation of precipitate
- END RESULT: Precipitin line= CONICAL
SHAPED resembles ROCKET
- The height of the rocket is measured:
i. HEIGHT is directly proportional to
the AMOUNT OF AG PRESENT
- Rocket Electrophoresis is more rapid than
RID.
Uses of Rocket Electrophoresis
 Quantitate IG (buffer= pH 8.6)
 Assay of proteins
 When the concentrastion is too low
to be detected by Nephelometry and
too high for RID
 Ex: Alpha feto protein, IgG in urine,
spinal fluid, complement component
of the body
II. Immunoelectrophoresis
- Double diffusion
- Utilizes electric current to enhance result
- Introduced by Grabar & Williams in 1963
- Two-step process
- Can be used for SEMIAGGLUTINATION of
wide range antigen
- Qualitative and semiqualitative technique
- AG source: SERUM
Application:
 Scramming tool for the differentiation of
more than 30 serum proteins
 Major Classes of IG
 Used for detection of:
i. Myelomas
ii. Waldentrom’s macroglubinemia
iii. Malignant melanomas
iv. Other lymphoproliferative disorder
 Immunodeficiencies can be detected in this
procdure, if no precipitin band is formed for a
particular immunoglobulin
 Deficiencies in complement can also be
detected
 Replaced by immunofixation electrophoresis
- Gives quicker results and easier to
interpret
Styles ‘18
  Identification of monoclonal protein  Leaving only the AG-AB complex
- Free Kappa and Lambda LC protein reference pattern, & AG-
 May be used to identify urine proteins AB precipitate: stained with
III. Immunofixation Electrophoresis protein sensitive stain
- First described by Alper & Johnson
- Similar to immunoelectrophoresis Reporting of Results:
- *exception: Antiserum is applied 1. Monoclonal proteins migrate in the CATHODE
DIRECTLY to the surface of the gel other region
than being placed in a trough 2. Monoclonal protein band will copy the same
- Replaced IEP in the evolution of migration & shape as the monoclonal band on
monoclonal gammopathies – because of the reference protein electrophoresis pattern
rapidity and ease of interpretation 3. Abnormality:
TWO STAGES PROCEDURE - Monoclonal proteins may migrate
i. Agarose Gel Protein Electrophoresis anywhere within the GLOBULIN region
ii. Immunoprecipitation - Identified by the corresponding
Test Specimes: antiserum used
 Urine Sources of Error:
 Serum - Evaporation of uncovered specimens:
 CSF inaccurate test results
 Other Body Fluids - Plasma: Band in all patterns across the gel
Primary Use: Characterization of Monoclonal IG *Fibrinogen may adhere to the gel matrix
- Can determine 3 variables of protein Limitations:
i. Antigenic Specificity - Excess antigens: not slight AB excess/ AG-AB
ii. Electrophoretic mobility equivalency.
iii. Quantity/Ratio of test & control *Caused by a very high level of Ig in
patients the sample*
- Principle: - Monoclonal proteins may adhere to the gel
Titan Gel Immunofix: intended for the matrix especially IgM. Appear in all 5 antisera
identification of monoclonal areas of the gel.
gammopathies in serum, urine or CSF. - Urine monoclonal protein level: Total protein
- High-resolution protein electrophoresis quantification
*Single specimen: Agarose Gel (6 IEP Immunofixation
different positions) Ease of use Easy More complex
*Proteins: separated according to their Sensitivity Less More
net charge by electrophoresis Monoclonal Better for Used for
IMMUNOFIXATION gammopathies typing ;arge different
- Monospecific antisera: 5 electrophoresis MCGP characterization
patterns (IgG, IgA, IgM and Kappa & of anomalous
Lambda) proteins
- Protein fixative solution: 6th pattern Interpretation Challenging Easier
- Plate: incubated (10mins)
Precipitation: NEPHELOMETRY
 Presence of complementary AG - Is a lab assay that is based on the
 stable AG-AB precipitate fixes the protein in measurement of the turbidity of the
the gel. suspension
AFTERFIXATION - Can be used to measure:
- Gel (precipitated protein; washed out in  Immunoglobulins
deproteination solution (eg. Dil. NaCl) of  C-reactive proteins
agarose, leaving only  Other proteins
- NON PRECIPITATED: washed out of the Principle:
agarose
Styles ‘18
 - The protein being measured is reacted with
specific antiserum human protein forming
insoluble complexes
- Light is passed through the suspension
- Complexes scatter the incident light that is
detected by the PHOTODIODE
- Can be measured by comparing the unknown
values with the standard of known protein
concetrations
- AMOUNT OF LIGHT SCATTERED is
proportional to the amount of INSOLUBLE
COMPLEXES

TURBIDIMETRY
- Measurement of the turbidity/cloudiness of a
solution
- Measures the reduction in light intensity due
to reflective absorption or scatter
- Uses Spectrophotometer to detect
concentration of particulate matter in a
sample
FLOW CYTOMETRY
- Is a technique for counting and examining
microscopic particulates, such as cells &
chromosomes by suspending them in a
stream of fluid & passing them by an
electronic detection apparatus
- Measures the properties of single cells
- Routinely used for diagnosis of disorders,
especially blood cancers
- Measure particle-scatter as well as innate
fluorescence
- Enumerate particulates in suspension
- Separate “live” frim “dead” particles
- Evaluation: 105 to 5*1o particles in less than 1
minute
- Physical properties of the cells
- Chemical properties of the cells
Principle:
- Light scattered by a laser or arc lamp
- Specific fluorescence detection
- Hydrodynamically focused in stream of
particles
- Electrostatic particles, separation for sorting
I. Forward Scatter
 Correlates with the cell volume
II. Side Scatter
 Depends on the inner complexity of
the particle shape of the nucleus,
mount and type of cytoplasmic
granules, membrane roughness
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