Monoclonal Antibody and its Diagnostic Application- Review

Monoclonal antibodies are immunoglobulins which have great advantages over the field of biochemistry, immunology, and biotechnology and also known as mouse antibodies because they are produced by mouse. As they are difficult to produce in contrast to polyclonal antibodies, they are precious (expensive) than polyclonal. They are also produced by hybridoma technology which was discovered by Georges Kohler and Cesar Milstein by first immunize the mouse, then extract the plasma cells from the spleen and fused with myeloma cells (cancer cells), finally produce hybridoma cells. During vaccination (immunization) of mice, animal welfare is taken into consideration. They have prominent applications in diagnosis, so monoclonal antibodies are incorporated in diagnostic techniques (Western immunoblotting,ELISA, Immunofluorescence test, and immunohistochemistry) in the diagnosis of livestock diseases. Monoclonal antibodies in diagnostic tests are important for detecting an antigen or antibody against microbes.


Introduction
An antibody, also known as an immunoglobulin [1], is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen, via Fab's variable region [2]. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen. Based on affinity antibodies are classified; one as Monoclonal antibodies are antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity, in that they bind to the same epitope, and the other is polyclonal antibodies bind to multiple epitopes (https://en.wikipedia.org/ wiki/Monoclonal_antibody). Monoclonal antibodies are also produced by an old immunological technique with great applications in the fields of biochemistry, immunology, and biotechnology among others [3]. The production of monoclonal antibody using hybridoma technology was first invented by Georges Kohler and Cesar Milstein [4].
One unique Merit of hybridoma production is that a mixture of antigens can be used to generate specific antibodies. Additionally, this also enables screening of antibodies of choice from a mixture of antibody populations generated by purified antigen where single-cell clones can be isolated [5]. MABs can be used in immunodiagnostic techniques as reagents to identify the antigen of the causative agents or indirectly for serological detection of antibodies against the causative agents [6,7]. They have also been reported to be used in experimental purposes ranging from molecular detection of antigenic epitopes to monoclonal antiidiotype antibody for utilization as a vaccine to induce protective immunity [8]. Development in biotechnology has contributed to the large scale production of mAbs which forms an integral part of many diagnostic techniques. These assays are frequently employed either for the detection of infectious agents or any of its structural components or even the antibodies generated against the infectious agents [9].
An additional role of monoclonal antibodies in disease prediction and detection is promising. Monoclonal antibody technology plays a significant role in the development of specific serologic reagents towards antigen in limited amounts. They provide both highly specific and reproducible immunological assay for rapid and accurate diagnosis of different types of infectious diseases [10]. The advantages and disadvantages of the use of monoclonal antibodies in several immunoassays need to be evaluated. This will help in the specific diagnosis of infectious diseases in different laboratories [11]. Thus, this review aims to highlight monoclonal antibodies and their Diagnostic applications including their Production technology.

Structure and Function
Antibodies are large heterodimeric molecules and are composed of two types of polypeptide chains, called the heavy and the light chain. The two types of light chains are kappa and lambda.
By cleavage with the enzyme papain, the fragment-antigen binding part can be separated from the fragment constant part of the molecule. The Fab fragments contain the variable domains, which consist of three antibody hypervariable amino acid domains responsible for the antibody specificity embedded into constant regions [12]. Antibodies are a key component of the adaptive immune response, playing a central role both in the recognition of foreign antigens and the stimulation of an immune response to them. The advent of monoclonal antibody technology has made it possible to raise antibodies against specific antigens presented on the surfaces of tumors. Monoclonal antibodies can be acquired in the immune system through passive immunity or active immunity. The merits of active monoclonal antibody therapy are the fact that the immune system will produce antibodies long-term, with only a short-term drug administration to induce this response. However, the immune response to certain antigens may be inadequate, especially in the elderly. Additionally, adverse reactions from these antibodies may occur because of long-lasting responses to antigen. Passive monoclonal antibody therapy can ensure consistent antibody concentration and can control for adverse reactions by stopping administration. However, repeated administration and consequently higher costs for this therapy are major disadvantages [13]. . This technology is essential to produce a hybrid cell. These hybrid cells are produced by fusing B-lymphocyte with tumor cell and they are called myeloma cells. As the result, these hybrid Cells produced from hybridoma technology are cultured in a laboratory and passaged using a mouse peritoneal cavity and these cells produce monoclonal antibodies, and this technology is known as hybridoma technology (https://www.biotecharticles.com). To produce monoclonal antibodies by Hybridoma technology; mice are first exposed to the antigen that an antibody is to be generated  These cells produce antibodies and are immortal. The incubated medium is then diluted into multi-well plates to such an extent that each well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, are thus monoclonal antibodies (https://en.wikipedia.

org/wiki/Hybridoma_technology#Applications).
The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of proper specificity. The first screening technique used is called ELISA. The hybridoma culture supernatant, secondary enzyme-labeled conjugate, and the chromogenic substrate is then incubated, and the formation of a colored product indicates a Positive hybridoma. Alternatively, immunocytochemical screening can also be used [15]. The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Supplemental media containing interleukin-6 are essential for this step. Once a hybridoma colony is established, it will continually grow in culture medium and produce antibodies [16]. Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to larger tissue culture flasks. This maintains the well-being of the hybridomas and provides enough cells for cryopreservation and supernatant for subsequent investigations. The culture supernatant can produce 1 to 60 μg/ml of the monoclonal antibody, which is maintained at -20°C or lower temperature [17]. By using culture supernatant or a purified immunoglobulin preparation, further analysis of a potential monoclonal antibody-producing hybridoma can be made in terms of reactivity, specificity, and cross-reactivity

Identification and Isolation of the Hybridoma Cells:
Hybridoma cells producing specific antibodies for the antigen used This includes the scope of the core curriculum and the relevant core competencies, such as specific workshops for animal procedures [21][22][23][24].

Diagnostic Application of Monoclonal Antibody
The importance of monoclonal antibodies is numerous and includes the prevention, diagnosis, and treatment of disease

Western Immunoblotting
The inception of the protocol for protein transfer from an electrophoresis gel to a membrane, protein blotting has evolved greatly. Western blotting analysis can detect one protein in a solution that contains any number of proteins and giving the protein information, and it is widely used in protein detection. It is a method in molecular biology or biochemistry or immunogenetics to detect protein in a given sample of tissue homogenate or extract, which is normally used with a high antibody directed against the desired antigen [30].

Monoclonal antibody in Immunohistochemistry
Immunohistochemistry is one of the diagnostic methods which such as peroxidase, that can catalyze a color-producing reaction.
Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein [33].
Immunofluorescence Test: Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This method uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope [34]. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary [35]. Additionally, Immunofluorescence can also be used on tissue sections, cultured cell lines, and may be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules. This technique can even be used to visualize structures such as intermediate-sized filaments (Franke et al, 1978). If the topology of a cell membrane has yet to be determined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures [36].
To make fluorochrome-labeled antibodies, a fluorochrome must be conjugated to the antibody. Likewise, an antigen can also be The following explanation will focus primarily on these classes in terms of conjugated antibodies [37]. The first class is Primary immunofluorescence uses a one, primary antibody, chemically linked to a fluorophore. The primary antibody recognizes the target antigen and binds to a specific region called the epitope. The attached fluorophore can be detected via fluorescent microscopy; will emit a specific wavelength of light once excited.
Direct immunofluorescence, although somewhat less common, has notable advantages over the secondary procedure. The direct attachment of the messenger to the antibody reduces the number of steps in the procedure, saving time, and reducing non-specific background signals [38].
The second class also called Secondary immunofluorescence uses two antibodies; the unlabeled first antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognizes the primary antibody, and binds to it.

Multiple secondary antibodies can bind a single primary antibody.
This provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the primary (or direct) protocol above but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.

Monoclonal Antibodies in Diagnostic Histopathology
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Conclusion and Recommendation
It can be concluded that monoclonal antibody production is a very sophisticated process. It requires immunization of antigens against a specific antigen, collection of spleen cells from immunized mice, and fusing the spleen cells with myeloma cell with an immortal myeloma cell. The technology is referred to as hybridoma technology which targets to produce a monoclonal antibody against a specific epitope of antigen as opposed to a polyclonal antibody which is specific to multiple epitopes in an antigen. Monoclonal antibodies have been utilized for diagnostic applications like western blotting, immunohistochemistry, and ELISA. In conclusion, the technology is not widely used in Ethiopia, training with this regard should be intensified. Moreover, it should be used for different diagnostic and therapeutic purposes in our country.