Evaluation of Protein Polymorphism: Immunofixation Electrophoresis

This assignment focuses on the evaluation of protein polymorphism using Immunofixation Electrophoresis (IFE). The introduction provides background information on the detection of paraproteins, the use of serum protein electrophoresis (SPEP) and urine protein electrophoresis (UPEP), and the clinical significance of gamma globulin protein. The aim of the study was to identify monoclonal proteins (M-proteins) in human serum using IFE. The materials and methods section describes the protocols followed. The results section presents the different patterns of immunoglobulin detected in the IFE of the test serum samples, contrasting them with a normal control pattern. The discussion interprets the results, highlighting the use of IFE for detecting specific proteins of interest, diagnosing multiple myeloma, and addressing potential errors. The conclusion summarizes the findings, indicating the presence of multiple myeloma in the abnormal serum sample. The assignment references several relevant sources to support the analysis and methodology.

IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
Name of the Student
Name of the University
Author’s Note

1IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
Table of Contents
Introduction................................................................................................................................2
Aim of the study.........................................................................................................................5
Material and methods.................................................................................................................5
Result..........................................................................................................................................6
Discussion:.................................................................................................................................7
Conclusion..................................................................................................................................8
References................................................................................................................................10

2IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
Introduction
The detection of paraprotein, which might be found in the blood due to malignancy or
other diseases, could be achieved by the use of SPEP or the serum protein electrophoresis
along with UPEP or the Urine protein electrophoresis. These methods are used to separate the
protein from a complex mixture, based on the charge, shape and size in the buffered of gel
agarose, after exposing the protein samples to certain buffering medium along with electronic
current (van de Donk et al. 2016). The gel is then stained in order to capture the
electropherogram so as to identify the pattern of the bands formed and evaluate it
accordingly. Albumin, alpha-1, alpha-2, beta 1, beta 2, and gamma globulin protein can be
measured and analysed by serum protein electrophoresis. The gamma globulin protein is
clinically significant as immunoglobulins migrate to it and the pattern of the gamma globulin
band observed in the conventional electrophoresis of the serum protein comprises of a total of
five immunoglobulins, namely immunoglobulin G, immunoglobulin A, immunoglobulin M,
immunoglobulin D and immunoglobulin E. In the normal human serum, eighty percent of
gamma globulin is IgG (immunoglobulin G) approximately, about fifteen percent is IgA
(immunoglobulin A), and five percent is IgM (immunoglobulin M) along with traces of IgD
(immunoglobulin D) and IgE (immunoglobulin E). The amount of the M protein quantified
by other tests along with the diagnostic results of the bone marrow biopsy analysed in
addition with the other characteristics could help in differentiating the multiple myeloma
anomaly from other causal agents of monoclonal gammopathy. However, the polyclonal
gammopathies might be due to inflammation (Papadea and Schlosser 2015). Therefore, the
immunofixation electrophoresis must be conducted with precision and executed with
adequate care as it is sensitive towards the small monoclonal (M) protein (Kuriakose,
Cheppayil, Narayanan and Vasudevan 2019).

3IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
The serum protein electrophoresis generally is conducted for the identification of the
individuals with multiple myeloma along with other disorders of the serum protein. The
proteins in the serum are separated by the electrophoresis on the basis of the physical
properties in addition with the subsets of the proteins which are then evaluated to analyse the
pattern and interpreting the presence of the anomaly or diseased condition if any (Papadea
and Schlosser 2015). The plasma protein levels respond to the acute inflammation, trauma,
malignancy, infarction, necrosis, chemical injury and burns and these responses alter the
pattern of the protein levels. The monoclonal protein or the M protein associated with a wide
range of disorders.
The homogeneous spike kind of a peak amidst the focal region in gamma-globulin
zone signifies monoclonal gammopathy (Keren 2017). These anomaly includes the
monoclonal gammopathy (MG) ,which is classified when a circulation of paraprotein is
present, B cell malignancy such as multiple myeloma (MM), malignancy plasmocytes, and B
lymphocytes, which normally produces an excessive amount of free light chain namely kappa
and lambda (Winter 2012). The amount of the M protein quantified by other tests along with
the diagnostic results of the bone marrow biopsy analysed in addition with the other
characteristics could help in differentiating the multiple myeloma anomaly from other causal
agents of monoclonal gammopathy. However, the polyclonal gammopathies might be due to
inflammation (Papadea and Schlosser 2015). Therefore, the immunofixation electrophoresis
must be conducted with precision and executed with adequate care as it is sensitive towards
the small monoclonal (M) protein (Kuriakose, Cheppayil, Narayanan and Vasudevan 2019).
The classification along with the characterisation of specific paraproteins is obtained
by the immunofixation electrophoresis (IFE) technique. The immunofixation electrophoresis

4IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
can be performed on the human serum samples or/ and urine samples. The IFE protocol
allows visualization of the specific proteins of significance by fixating them on the gel with
the addition of the specific antiserums against the IgG, the IgA, the IgM, the IgD, the IgE,
along with the two light chains the Kappa molecule, and the lambda molecule, followed by
sufficient washing to discard all the other protein molecules, prior to the staining step before
initialising the electrophoresis protocol (Jenner 2014). As a result, the precipitation band
pattern will be formed in the presence of the M-protein. The IgG, the IgA, the IgM along
with the two light chains namely the Kappa, and the lambda light chains are the most
common M-protein which can be found in an individual’s serum.
Aim of the study
The aim of the research was identification of the monoclonal proteins (M-protein) in
the human serum using immunofixation electrophoresis (IFE) technique.
Material and methods
The material and the protocols to perform the immunofixation electrophoresis (IFE)
technique are followed precisely as described in the laboratory practical Manuel clinical
chemistry.

5IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
Result
Figure 1: The figure illustrates the different patterns of immunoglobulin detected in the
immunofixation electrophoresis the test serum samples and is contrasted with a normal
control pattern image of the immunofixation electrophoresis.
The test serum shows the presence of IgG kappa with free kappa along with extra
band in the IgG lane which might be due to formation of IgG complex with alpha 1 and alpha
2 in vitro. The figure also shows presence of IgM kappa along with poorly defined IgG
Lambda. Presence of albumin is seen in both the normal control pattern and the pattern
formed by the test serum sample after immunofixation electrophoresis.
Normal control pattern Test serum sample

6IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
Discussion:
In this experiment the test serum sample was analysed and the results of the
immunofixation electrophoresis were interpreted using the pattern formed by the control
serum sample. Immunofixation electrophoresis (IFE) has become the method of choice to
detect specific protein of interest in immunoglobulin as it easy to interpret and analyse for M-
protein. IFE normally done when there is abnormal results are shown in serum
electrophoresis.
Referring to normal pattern in serum protein electrophoresis as it shows in Figure (2), and
compare it with test anomalous serum protein electrophoresis pattern figure (3) of the patient
sample, there is a large spike in gamma region can be interpreted to be multiple myeloma
diagnosis in the individual.
Figure (2) illustrated the normal serum protein electrophoresis pattern.
The immunofixation electrophoresis of test serum illustrates the presence of IgG
kappa with free kappa along with extra band in the IgG lane which might be due to formation
of IgG complex with alpha 1 and alpha 2 in vitro. The figure also shows presence of IgM
kappa along with poorly defined IgG Lambda. Presence of albumin is seen in both the normal
control pattern and the pattern formed by the test serum sample after immunofixation
electrophoresis.

7IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
Figure (3) highlights the abnormal serum protein electrophoresis pattern
Figure (1) abnormal serum sample shows a defined limited band in protein lane with
conforming band at the light chain Kappa and heavy chain lane M (Jenner 2014). This
indicate the present of IgM kappa. This typically indicate multiple myeloma as plasma cell
secrete paraprotein accumulate in the bone marrow and can be detected in the serum
Evaluation of the total light chain along with the free immunoglobulin assays of the serum
and the samples of the urine with immunofixation electrophoresis for the detection of the
monoclonal proteins in patients with multiple myeloma form monoclonal gammopathy. And
in figure (3), the anomalous serum protein electrophoresis pattern of the patient sample with
numerous myeloma where there is a large peak in gamma region. However, some errors
might be done during the procedure of IFE which might generate false results. For instance,
there is a band shown in IgA correlation to lambda in light chain. This can happen due to the
co migrate of monoclonal immunoglobulin (IgA) with other serum proteins.
Conclusion
The test serum shows the presence of IgG kappa along with the free kappa along with
extra band in lane of IgG which might be due to formation of IgG immunecomplex with
alpha 1 and alpha 2 in vitro. Additionally, this might be caused by partial proteolysis of

8IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
immunoglobulin complex owing to presence of thepolyclonal IgG. This might also be due to
IgG compound’s post translational modification resulting only in the Fc section of the IgG
compound which will react with the anti-IgG anti serum. In conclusion, the conclusion of
immunofixation electrophoresis (IFE) test of the two serum samples (normal and abnormal)
that abnormal serum sample indicate the present of multiple myeloma.

9IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
References
Jenner, E., 2014. Serum free light chains in clinical laboratory diagnostics. Clinica Chimica
Acta, 427, pp.15-20.
Katzmann, J.A., Dispenzieri, A., Kyle, R.A., Snyder, M.R., Plevak, M.F., Larson, D.R.,
Abraham, R.S., Lust, J.A., Melton III, L.J. and Rajkumar, S.V., 2016, December. Elimination
of the need for urine studies in the screening algorithm for monoclonal gammopathies by
using serum immunofixation and free light chain assays. In Mayo Clinic Proceedings (Vol.
81, No. 12, pp. 1575-1578). Elsevier.
Keren, D.F., 2017. High-resolution electrophoresis and immunofixation: techniques and
interpretation. Elsevier.
Kuriakose, E., Cheppayil, S.N.U., Narayanan, S.K. and Vasudevan, A., 2019. A Study on
Free Light Chain Assay and Serum Immunofixation Electrophoresis for the Diagnosis of
Monoclonal Gammopathies. Indian Journal of Clinical Biochemistry, 34(1), pp.76-81.
Nowrousian, M.R., Brandhorst, D., Sammet, C., Kellert, M., Daniels, R., Schuett, P., Poser,
M., Mueller, S., Ebeling, P., Welt, A. and Bradwell, A.R., 2015. Serum free light chain
analysis and urine immunofixation electrophoresis in patients with multiple
myeloma. Clinical Cancer Research, 11(24), pp.8706-8714.
Papadea, C. and Schlosser, R.J., 2015. Rapid method for β2-transferrin in cerebrospinal fluid
leakage using an automated immunofixation electrophoresis system. Clinical
chemistry, 51(2), pp.464-470.
van de Donk, N.W., Otten, H.G., El Haddad, O., Axel, A., Sasser, A.K., Croockewit, S. and
Jacobs, J.F., 2016. Interference of daratumumab in monitoring multiple myeloma patients

10IMMUNOFIXATION ELECTROPHORESIS: EVALUATION OF PROTEIN POLYMORPHISM
using serum immunofixation electrophoresis can be abrogated using the daratumumab IFE
reflex assay (DIRA). Clinical Chemistry and Laboratory Medicine (CCLM), 54(6), pp.1105-
1109.
Winter, W.E., 2012. Monoclonal Gammopathies. Multiple Myeloma and Related Serum
Protein Disorders: An Electrophoretic Guide, p.51.