*2.4.4 Cell spreading assay*

In this type of assay, the spreading process of individual cells is seen and recorded with the help of Differential Interference Contrast microscopy (DIC). The spreading state is recorded every 5 seconds with a Charge-Coupled Device (CCD) of the camera, producing high-quality grayscale images. The process of taking images could extend to several hours [28].

#### **2.5 Hybridoma technology and monoclonal antibodies**

Antibodies, one of the major elements of the immune system are the glycoproteins produced by the immunoglobulins; B-cells provide protection against invading pathogens. The antibodies are highly specific and selective, thus have been used as an extraordinary tool in bioengineering and biomedical research for many years. The antibodies are majorly classified into two categories, Monoclonal Antibodies (mAbs) and Polyclonal Antibodies (pAbs) are based on their origin from the lymphocytes. mAbs are produced by only B lymphocyte or B cells and are monospecific. Due to this property, they possess high specificity and affinity towards a single epitope of an antigen whereas pAbs are produced by different B-cells and possess different affinities for multiple epitopes of a specific antigen. Since mAbs are highly specific, they are produced on a large scale through culturing of antibodies-producing cells widely known as 'Hybridomas', which are commonly derived from mice, and the method is known as 'Hybridoma Technology [29].

Hybridoma technology was discovered and developed by two eminent scientists, Georges Kohler and Cesar Milstein in 1975 and is considered to be one of the biggest breakthroughs. It has proved to be a robust, effective, and successful methodology employed in the field of biotechnology and biomedical research that solely deals with mAb isolation. The B cells go through the antibody maturation process in the germinal centers of secondary lymphoid tissues (for example, lymph nodes, spleen, tonsils, and Peyer's patches). Upon proliferation, certain mutations are experienced by the B cells, specifically in the genes encoding the variable region of the antibodies that helps in the selection for high-affinity tight binding to the corresponding antigen. The overall resulting antibodies by B cells consist of a natural pairing of the light chain and variable heavy chain genes with constant region genes. This region contains Class Switch Recombination (CSR) differentiates from the hybridoma technology in which CSR is absent [29].

Following are the steps employed for the production of monoclonal antibody by hybridoma technology.

## *2.5.1 Isolation of antibody-producing B lymphocyte*

The mouse/mice is/are immunized every 2–3 weeks with red blood cells taken from sheep in order to produce the B cells. These antibodies are isolated from the spleen cells of mice.

#### *2.5.2 Screening of mouse for production of antibody*

After the process of immunization, the blood samples are taken from the mouse to determine the serum antibody titer. When the titer reaches the optimal level, the mouse is boosted by injecting antigen 3 days prior to fusion with myeloma cells [30].

#### *2.5.3 Fusion of B cells with myeloma cells*

Fusion of isolated spleen cells (limited life span) with tumor lymphocytes (immortal) with the help of PEG (Polyethylene Glycol) leads to the development of hybridomas with an unlimited life span.
