ACTIVITY 13

BIOCHEMISTRY OF IMMUNITY: IMMUNOCHEMISTRY

Immunochemistry is the study of the chemistry of the immune system. This involves the study of
the properties, functions, interactions and production of the chemical components (antibodies/
immunoglobulins, toxin, epitopes of proteins like CD4, antitoxins, cytokines/chemokines, antigens)
of the immune system, immune responses and determination of immune materials by
immunochemicals.
In addition, immunochemistry is the study of the identities and functions of the components of the
immune system. Immunochemistry is also used to describe the application of immune system
components, in particular antibodies, to chemically labelled antigen molecules for visualization.

Various methods in immunochemistry have been developed and refined, and used in scientific
study, from virology to molecular evolution. Immunochemical techniques include: enzyme-linked
immunosorbent assay, immunoblotting (e.g., Western blot assay), precipitation and agglutination
reactions, immunoelectrophoresis, immunophonotyping, immunochromatographic assay and
cyflometry.

One of the earliest examples of immunochemistry is the Wasserman test to detect syphilis. Svante
Arrhenius was also one of the pioneers in the field; he published Immunochemistry in 1907 which
described the application of the methods of physical chemistry to the study of the theory of toxins
and antitoxins.

Immunochemistry is also studied from the aspect of using antibodies to label epitopes of interest in
cells (immunocytochemistry) or tissues (immunohistochemistry).
1. Discuss the various immunochemistry techniques.
a. enzyme-linked immunosorbent assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA) is a widely used immunological method for
detecting and quantifying antibodies, antigens, proteins, and glycoproteins in biological samples. It
has various applications, such as diagnosing HIV infection, conducting pregnancy tests, and
measuring cytokines. ELISA assays are typically performed in 96-well plates, enabling multiple
samples to be analyzed simultaneously. Each ELISA targets a specific antigen, and there are kits
available for detecting various antigens. Also, there is a variation of ELISA called sandwich ELISA,
where two sets of antibodies are employed to detect secreted products like cytokines.

b. Agglutination
Agglutination is a process that involves the clumping together of cells. This clumping occurs as a
result of a specific interaction between an antibody and an antigen present on the cells. The
reaction takes place when antibodies, such as IgG or IgM, which form connections with the
antigenic particles that possess multiple epitopes. Although some antibodies can bind to the
antigen, they may not necessarily lead to agglutination. However, the introduction of an anti-
immunoglobulin, known as the Coombs test, can trigger the agglutination process. The
agglutination technique encompasses various approaches, including direct or indirect agglutination,
agglutination inhibition, and haemagglutination. Agglutination reactions can be performed on slides,
in test tubes, or on microtiter plates, and they generally offer higher sensitivity compared to
immunoprecipitation methods. These techniques provide either qualitative or quantitative
outcomes.

c. Immunoprecipitation
Immunoprecipitation (IP) is commonly used in laboratories for the selective purification of antigens.
This process involves utilizing a specific antibody that is attached to a solid support, such as
magnetic particles or agarose resin. By immobilizing the antibody, it becomes capable of binding to
the desired antigens present in a sample, allowing for their isolation. The main objective of
immunoprecipitation is to obtain just enough protein for subsequent analysis. This can be achieved
by comparing treated and untreated samples to determine the relative amount of the protein of
interest. It takes place in a microcentrifuge tube, where a small amount of resin is used. This allows
for efficient and effective batch-wise incubation steps to occur. Also, the purpose of
immunoprecipitation is to extract proteins and other biomolecules from cell or tissue lysates, which
can then be further analyzed using techniques like western blotting or other assays.

d. immunoelectrophoresis
Immunoelectrophoresis involves the precipitation of substances in agar using an electric field. The
term "immunoelectrophoresis" was coined by Grabar and Williams in 1953. This method combines
the principles of immuno-diffusion and electrophoresis to separate and analyze a mixture of
antigens. Initially, the antigen mixture undergoes electrophoresis, where it is divided into its
individual components. Afterward, a double immuno-diffusion test is conducted, where the antigens
are placed in wells within a gel and an electric current is applied. By applying an electric current to a
gel-coated slide, the antigen mixture placed in wells can be separated into its individual
components based on their size and charge. This test plays a crucial role in identifying and
approximately quantifying various proteins present in the serum.

e. Immunofixation
Immunofixation electrophoresis involves running an electric current through a sample to separate
proteins, followed by the use of antiserum to cause the target protein to precipitate. By ensuring
that the antibodies are slightly greater in quantity or equivalent to the antigens, the resulting
complex of antigen and antibody remains insoluble. This is used for detecting and identifying
monoclonal immunoglobulins in various bodily fluids, such as serum, urine, and cerebrospinal fluid.
The process consists of two stages: first, the separation of serum proteins through electrophoresis,
and second, the identification of monoclonal immunoglobulins through immunoprecipitation using
specific antibodies.

f. immunoturbidimetry
Immunoturbidimetry, also known as turbidimetric immunoassay, is used for analyzing urinary
proteins. It relies on the measurement of turbidity in a sample to accurately determine the
concentration of a specific analyte. It is particularly useful in quantifying antigen-antibody
complexes, as the formation of such complexes leads to an increase in turbidity within the sample.
It provides valuable information about the levels of urinary proteins, enabling healthcare
professionals to make informed clinical decisions.

g. Immunonephelometry
Immunonephelometry is used in the immunoassay technique, which is for the precise determination
of specific proteins. It involves measuring the scattered light and allows for the assessment of the
size, shape, and concentration of the particles causing the scattering (in theory, at least). In
immunoassays utilizing nephelometry, the scatterers are the complexes formed by the antigen-
antibody interaction.

h. Immunoassay
Immunoassays are used to quantify molecules with biological significance by utilizing specialized
antibodies that exhibit both specificity and selectivity. These assays are particularly utilized in high-
throughput screening (HTS) and lead optimization projects where the objective is to identify and
measure molecules that are either generated within cells or released as a response to the
compounds being screened. In order to obtain the most precise and reliable measurement of an
unknown concentration, the development of an immunoassay must consider not only the typical
criteria used in assay development, such as the standard deviation or optimal signal window, but
also its ability to accurately predict the value of an unknown sample.

i. Competitive binding
A competitive binding assay is used to analyze the interaction between a labeled ligand and a target
protein. In this assay, an unlabeled ligand is introduced as a competitor to measure the binding
affinity of the labeled ligand. This technique not only provides qualitative information about the
binding but also allows for the comparison of the relative affinities of different molecules towards
the same target. Also, it is particularly useful in quantifying interactions with high affinity, which are
typically challenging to measure directly. In order for the competitive binding assay to be effective,
the competing ligand and the labeled ligand must both bind to the same specific site on the target
protein.

j. C-reactive protein
C-Reactive Protein (CRP), which is also referred to as Pentraxin 1, belongs to a group of non-
glycosylated proteins known as the Pentraxin family. CRP acts as an acute-phase reactant that is
produced by the liver and released into the bloodstream in response to tissue damage. An elevated
concentration of CRP present in the bloodstream serves as an indicator that there might be an
ongoing inflammatory response taking place within the body. Consequently, a CRP test, which is
specifically designed to quantify the quantity of CRP present in the blood sample, becomes
essential in assessing and monitoring this inflammatory marker. It operates by utilizing the concept
of latex agglutination, where latex particles that have been combined with human anti-CRP are
mixed with a sample of a patient's serum that contains C-reactive proteins. As a result, a visible
reaction of agglutination occurs within a short span of just 2 minutes.

k. Sphectrophometric Techniques
Spectrophotometry is used for measuring the absorption of light or the concentration of chemicals
in a solution. A spectrophotometer is utilized, which is composed of two main instruments: a
spectrometer and a photometer. The spectrometer generates light of a particular wavelength, while
the photometer quantifies the intensity of light by determining the amount that passes through the
sample. This technique involves passing a beam of light through the sample, and different
compounds in the solution will absorb or transmit light at specific wavelengths.

l. Immunofluorescence
Immunofluorescence (IF) involves the utilization of specific antibodies that have been chemically
linked to fluorescent dyes. These antibodies are used to label and identify particular target antigens
with the help of fluorescent dyes like fluorescein isothiocyanate or cyanine dye.
Immunofluorescence can be combined with other methods of fluorescent staining, such as DNA
labeling using DAPI, to enhance the visualization of different targets in various samples, like tissue
sections or cultured cells. This visualization is achieved by observing the samples under a
specialized microscope that is capable of detecting and capturing fluorescence, such as an
epifluorescence or confocal microscope. Also, there are two main approaches to
immunofluorescence: direct and indirect. In direct immunofluorescence, a single antibody that
specifically recognizes the target antigen is directly attached to a fluorophore. On the other hand,
indirect immunofluorescence involves the use of two antibodies.

m. Immunoelectronmicroscopy
Immunoelectronmicroscopy refers to a diverse range of techniques that use antibodies or molecules
that interact with antibodies, such as protein A or protein G, in combination with electron
microscopy. The objective of this is to precisely locate antigens or antibodies within cells and
tissues at the ultrastructural level. Moreover, various other biological macromolecules, such as
lectins, which possess well-established ligand-binding properties, can also be utilized in this
process. Initially, immunoelectronmicroscopy refers to studies conducted using the transmission
electron microscope. However, it has expanded to encompass investigations carried out with the
scanning electron microscope as well.

n. Immunostaining
Immunostaining is a widely used technique in which antibodies are employed to identify and
measure levels of antigens. This process involves using a primary antibody that is designed to bind
to a specific molecule, allowing it to detect the presence of that molecule. A secondary antibody,
labeled with a fluorescent or enzymatic tag, is then used to bind to the primary antibody, allowing
the visualization and quantification of the marker. This can be done using a fluorescence
microscope or by adding a colored substrate. Also, the quality of the staining is influenced by the
specificity and quality of the primary antibody. Therefore, different primary antibodies targeting the
same molecule can produce different staining results. In addition, there are various immunostaining
techniques available, and the selection of a specific technique should be based on the desired
information, such as determining the location or expression levels of a protein.

o. Immunodetection
Immunodetection techniques operate on the fundamental principle of a primary antibody identifying
a specific epitope on the target protein, while a secondary antibody is employed to identify and bind
to the primary antibody. In order to obtain a distinct and reliable signal, the primary antibody needs
to possess the ability to accurately identify and bind solely to the target protein.

p. Immunoblotting
Immunoblotting allows for the identification of specific target proteins amidst a mixture of unrelated
protein species. This involves the use of antibodies or other specific ligands to facilitate the
recognition of the protein of interest through antigen-antibody or protein-ligand reactions. To
perform immunoblotting, proteins are first separated by electrophoresis and then transferred onto a
membrane, typically made of nitrocellulose. This membrane is then exposed to a primary antibody
that is specific to the target protein, followed by a secondary antibody that is labeled with enzymes
or radioisotopes. In cases where the ligand used is not an antibody, the reaction can be visualized
by utilizing a directly labeled ligand. Another variation of this technique is the dot blot, which
simplifies the process by directly spotting protein samples onto the membrane without the need for
electrophoresis separation.

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