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Styles ‘18
- Horizontal and vertical - 1D electroimmunodiffusion
i. Wells are cut in the gel - Adaptation of RID
ii. Reactants are added in the well - Electrophoresis is used to facilitate
iii. Incubate (12-48 hrs) in a moist migration of the AG into the agar
chamber - AG diffuses out of the well: precipitation
iv. Precipitin lines will form (where the begins.
moving front of the AG meets the AB) - Change in AG concentration: dissolution
1. AG-AB applied to holes punched in and reformation of precipitate
agar gel - END RESULT: Precipitin line= CONICAL
a. Precipitin band SHAPED resembles ROCKET
b. Precipitin band - The height of the rocket is measured:
2. Leave to diffuse i. HEIGHT is directly proportional to
3. Wash and stain the AMOUNT OF AG PRESENT
- The density of line reflect the amount of - Rocket Electrophoresis is more rapid than
immune complex (Oudin Assay) RID.
- AB that is multispecific is placed in the
Uses of Rocket Electrophoresis
central well
- Different AGs are placed in the Quantitate IG (buffer= pH 8.6)
surrounding wells Assay of proteins
*position of precipitin bands between When the concentrastion is too low
wells allow for the AGs to be compared to be detected by Nephelometry and
with one another. too high for RID
- Double diffusion assay in 2D Ex: Alpha feto protein, IgG in urine,
- A quality assay spinal fluid, complement component
- PRECIPITATE shows the relationship of the body
between the AGs II. Immunoelectrophoresis
- NOT very accurate as molecules of some - Double diffusion
molecular weight may overlap, giving - Utilizes electric current to enhance result
misleading results - Introduced by Grabar & Williams in 1963
- IDENTITTY, NON IDENTITY, PARTIAL - Two-step process
IDENTITY (refer to book) - Can be used for SEMIAGGLUTINATION of
wide range antigen
Diffusion Patterns
- Qualitative and semiqualitative technique
1. Fusion lives at their junction to form an
- AG source: SERUM
arc-serologic identity/presence of
Application:
common epitope
Scramming tool for the differentiation of
2. Crossed lines – demonstrates 2 seprate
more than 30 serum proteins
reactions, the compared AG shared are
Major Classes of IG
common epitopes
Used for detection of:
3. Fusion of 2 lives with spur – partial
i. Myelomas
identity
ii. Waldentrom’s macroglubinemia
Uses of Ouchterlony DD iii. Malignant melanomas
Identification of fungal antigens iv. Other lymphoproliferative disorder
Detection of ABs to extractable nuclear Immunodeficiencies can be detected in this
bodies procdure, if no precipitin band is formed for a
particular immunoglobulin
ELECTROPHORETIC TECHNIQUES
Deficiencies in complement can also be
Principle: separation of molecules according to detected
electric charge. Replaced by immunofixation electrophoresis
- Gives quicker results and easier to
I. Rocket Immunoelectrophoresis 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
Styles ‘18