GROUP THREE MEMBERS

 FELIX MUCHANGA 22010531

 PATRICK CHIBBAMULILO 22010600
 AARON HANG’ANDU 22010198
 CAREEN YAMBA 22010172
 KELLY MUNKOMBWE 22010947
 HAGAI MUTUNA 22010706
 LUWI MUPATA 22010225
 KAYOMBO MUNGILI 22010561
 AARON NKHUWA 22010280
LAURELL(ROCKET), CROSS-
IMMUNOELECTROPHORESIS
 Immunoelectrophoresis
Is a laboratory technique used to separate and
identify proteins based on their electrical
charge and their reaction with specific
antibodies. It combines two methods, which
allows for the detection and analysis of specific
proteins or antigens in a complex mixture:
Electrophoresis and immunodiffusion
 LAURELL IMMUNOELECTROPHORESIS
 It is a quantitative technique used to detect the
concentration of specific proteins or antigens in a given
sample.
 It is called “rocket Immunoelectrophoresis” because of
the presence of precipitin bands in the shape of rocket-
like structures at the end of the reaction.
 This method involves migration of antigens in a layer of
agarose gel containing specific antibodies to the antigens
of interest. This happens under the influence of electric
field on the antigens and gives rise to the rocket-shaped
patterns of precipitation.
 The length of the precipitin arc and area under the
rocket are directly proportional to the protein (antigen)
concentration.
 THE PRINCIPLE
 In this method, an agarose gel is prepared in a semi-solid form with
an incorporated appropriate antibodies at a pH value that keeps
them (antibodies) in immobile state.
 Thereafter, antigens are introduced into the wells cut in the gel
facing cathode terminal of the electrophoresis tank and the
experiment is run through two processes:
 Electrophoresis: This is the application of electric current, which
produces electric fields and these fields put influence on the
antigens, forcing them to migrate or move through the gel meeting
their specific antibodies.
 Immunodiffusion: This is the migration or diffusion of antigens out
of the wells after electric current is applied. As the antigens enter
the agarose gel, they combine with their specific antibodies to form
immune complexes which become visible. The gel is then incubated
and stained at the end of these two processes for proper
visualization.
 Materials

Electrophoresis Apparatus
Gel Support Materials(agarose or agar and
glass slides)
Quality Control Materials( antiserum and
buffers)
Samples(antigens or proteins)
Micro-pipettes and well puncher
THE ELECTROPHORESIS
materials
 Procedure
 Preparation of Gel: Prepare an agarose gel matrix containing
specific antibodies suitable for electrophoresis. Mix it well
with antiserum for uniform distribution of antibodies within
the gel. Pour the gel into a glass slide and allow it to solidify.
 Sample Preparation: Prepare the samples containing the
proteins or antigens desired to be analyzed. These samples
may be serum, plasma, or tissue
 Sample loading: load samples into evenly spaced wells being
cut at the end of the gel.
 BufferSystem: A buffer (Barbital buffer) is used to maintain
the pH and conductivity of electrophoresis system, ensuring
optimal separation conditions.
 Electrophoresis: Subject the gel to an electric current,
allowing the proteins to migrate through the gel matrix
according to their charge.
 Incubation:Incubate the gel to allow the protein and the
antibody at 37℃ to form immune complexes.
 Visualization:Stain the gel to visualize the separated
proteins and any complexes formed between the protein
of interest and the antibody. Common stains include
Coomassie Brilliant Blue or silver staining.
Note: the absence of precipitin arcs in the gel shows that
there is no antigen-antibody reaction.
1) Prepare agarose and pour on a
glass slide and wait for it to
solidify

2) After the gel solidifies on the

glass slide make evenly spaced
wells.

3) Place the glass slide in the gel

tank that contains a buffer.
4) And insert the antigens in the
wells
1) Run the electrophoresis tank at 100V
for 2hours for the migration of
antigens.

2) Incubate the glass slide after

electrophoresis at 37℃ over night

3) After staining precipitin lines can be

easily visualized.
 Applications
 Quantification of Specific Proteins
- Used to measure the concentration of specific proteins
in biological fluids such as serum, urine, and cerebrospinal
fluid.
 Clinical Diagnostics
- Helpful in diagnosing and monitoring diseases associated
with abnormal protein levels, such as multiple myeloma,
autoimmune diseases, and immunodeficiencies.
 Vaccine Development
- Used to quantify antigen levels in vaccine formulations
to ensure consistency and efficacy.
 Research Applications
- Employed in basic and applied research to study protein
 Applications cont.
Quality Control in Biopharmaceuticals
- Used to monitor the purity and concentration of protein
therapeutics during production.
 Allergy Testing
- Can be used to measure specific IgE antibodies in allergic
responses.
 Agricultural and Environmental Testing
- Applied in the detection and quantification of proteins in plant
and animal samples, as well as in environmental monitoring of
allergens or contaminants.
 Biotechnology
- Utilized in the characterization of recombinant proteins and
monoclonal antibodies produced in biotechnological processes.
 Advantages

 Quantitative Analysis
 Provides precise quantification of specific proteins in complex biological
samples.
 Specificity
- High specificity due to the use of antigen-antibody reactions, reducing the
likelihood of cross-reactivity.
 Speed
- Faster than traditional immunodiffusion techniques, as it combines
electrophoresis with immunodiffusion.
 Simplicity
- Relatively straightforward technique that does not require sophisticated
equipment or extensive sample preparation.
 Multiple Samples
- Allows for the simultaneous analysis of multiple samples on the same gel,
enhancing throughput.
 Disadvantages
Time consuming
Reproducibility limitations
Technical expertise
Instrumentation
Detection limitations
 CROSS IMMUNOELECTROPHORESIS
 is a variation of immunoelectrophoresis used to
identify and characterize antigens in a sample.
It combines two-dimensional electrophoresis
with immunodiffusion in a single gel.
 Itincludes two methods ; first dimension and
second dimension
 It is also called two dimension technique.
 Principle
Cross Immunoelectrophoresis is a two-dimensional
technique that combines electrophoresis and
immunodiffusion to analyze complex mixtures of
antigens. It provides both qualitative and
quantitative information about the antigen
composition of a sample
 Procedure
 First Dimension Electrophoresis
 The sample is subjected to electrophoresis on an agarose gel to
separate the antigens based on their charge and size.
 The goal of the first dimension is to separate the mixture of
proteins in the sample into distinct bands. This separation is
based on the proteins' electrophoretic mobility.
 Use an agarose gel, as it provides a suitable medium for
electrophoresis of proteins.
 Buffer Solution: Prepare an appropriate buffer to maintain the
pH and ionic strength during electrophoresis. This buffer is also
used to prepare the gel and fill the electrophoresis tank.
 The second dimension this is where the separated proteins interact with
specific antibodies, leading to the formation of precipitin arcs.
 The second dimension aims to identify and quantify individual proteins.
 Agarose Gel: Prepare another agarose gel for the second dimension, similar to the one
used in the first dimension.
 Antibodies: Incorporate specific antibodies against the proteins of interest into the
agarose gel. These antibodies will bind specifically to their target proteins.
 Gel Strip Placement: place the gel strip containing the separated proteins from the first-
dimension perpendicular onto the agarose gel containing the antibodies.
 Perform electrophoresis perpendicular to the direction of the first dimension. This allows
the proteins to migrate into the gel containing the antibodies.
 Precipitin Arc Formation: As the proteins migrate through the gel, they encounter
antibodies specific to their respective antigens.
 Protein-antibody complexes form and precipitate, resulting in the formation of arcs
along the gel.
 Visualization: After electrophoresis, the gel is typically stained to visualize the precipitin
arcs.
 Application of immunoelectrophoresis
Characterization of Complex Mixtures
- Used to analyze complex antigen mixtures, such as those found in
biological fluids (e.g., serum, urine) or tissue extracts.
- Allows the separation and identification of multiple antigens in a single
sample.
Detection of Antigen-Antibody Reactions
- Used to study the specificity and affinity of antigen-antibody
interactions.
- Helps in understanding the immune response and the molecular basis of
antigenicity.
Clinical Diagnostics
- Used to diagnose diseases by detecting specific antigens or antibodies
in patient samples.
- Provides precise identification of pathogens or immune disorders,
aiding in accurate diagnosis and treatment planning.
Purity Assessment of Biological Products
 Advantages
Identification of multiple samples:
It is useful for identifying multiple antigens within the same
sample, as it allows for simultaneous separation and detection of
multiple antigens.
Comparative analysis:
it enables the comparative analysis of antigenic patterns between
different samples, facilitating the study of antigenic similarities
and differences.
Highly discriminative:
It is highly discriminative, allowing for the differentiation of
closely related antigens based on their migration patterns.
 Disadvantages
Complexity and Technical Expertise
Requires a high level of technical skill and expertise to perform accurately.
May not be suitable for all laboratories, particularly those with limited
technical resources or inexperienced personnel.

Time-Consuming
The process can be lengthy, involving multiple steps including
electrophoresis, incubation, and detection.
May not be ideal for situations where rapid results are needed.
Limited Sensitivity Compared to Modern Techniques
While sensitive, CIE may not be as sensitive as newer techniques like
enzyme-linked immunosorbent assay (ELISA) or mass spectrometry.
May miss low-abundance antigens or proteins that could be detected by
more advanced methods.
 Disadvantages
Limited Dynamic Range
The dynamic range of CIE is limited compared to some modern analytical
techniques.
May not effectively measure very high or very low concentrations of
antigens.
Interpretation of Results
Interpreting the results can be subjective and requires experience.
Potential for variability in interpretation between different operators.
Cost
Can be relatively expensive due to the need for specific reagents, high-
quality antibodies, and equipment.
May not be cost-effective for all laboratories, particularly those with
limited budgets.
 Interpretation of results
 In rocket Immunoelectrophoresis, the height of the precipitin arcs
(rockets) formed during electrophoresis is measured to determine the
concentration of antigens, with taller rockets indicating higher
concentrations. These heights are compared to a standard curve made
from known antigen concentrations to quantify the antigen in unknown
samples. In cross Immunoelectrophoresis, the resulting precipitin arcs
provide information on the number, size, shape, and intensity of antigens.
The position of each arc corresponds to the antigen's electrophoretic
mobility, while the shape and intensity reflect the antigen's concentration
and distribution. By comparing these arcs to reference patterns, one can
identify and quantify the antigens present in the sample. Combining the
quantitative precision of rocket Immunoelectrophoresis with the detailed
antigenic profiling of cross Immunoelectrophoresis allows for a
comprehensive analysis of antigen mixtures in terms of both concentration
and identity.
Interpretation of results
 In conclusion

immunoelectrophoresis is a technique for
protein separation and identification.