Explain the general process that is used to produce monoclonal antibodies (in mice and humans).

Understand the difference between Mabs that are fully mouse, chimeric, humanized, and fully human. Also be familiarwith the proper naming of these types of Mabs when used as drugs.

Monoclonal is an antibody made by one B-cell.

The process of producing monoclonal antibodies (MAbs) typically involves several steps:

1. Immunization: In the case of mice, they are injected with the antigen (the target molecule) against which the desired antibody is to be generated. For humans, if the antibodies are needed for therapeutic purposes, human donors may be immunized. The immune system of the mouse or human then produces a diverse array of antibodies against the antigen.
2. Cell Fusion: B cells, which produce antibodies, are harvested from the immunized mouse or human donor. In the laboratory, these B cells are fused with immortalized cells (like myeloma cells) to create hybridoma cells. These hybridomas have the ability to continuously produce the desired antibody.
3. Screening: The hybridoma cells are screened to identify those producing the desired monoclonal antibody. This usually involves testing the culture supernatants for the presence of the specific antibody using techniques like enzyme-linked immunosorbent assay (ELISA).
4. Cloning: Once the hybridomas producing the desired antibody are identified, they are cloned to ensure that each clone produces the same antibody.
5. Expansion and Harvesting: The selected hybridoma clones are then cultured in large quantities to produce the monoclonal antibody. The antibodies are harvested from the culture supernatant or from the cells themselves.

Now, regarding the different types of monoclonal antibodies:

1. Fully Mouse Monoclonal Antibodies: These antibodies are entirely derived from mouse cells and are composed solely of mouse-derived components. Usually indicated by the suffix "-momab" (e.g., Rituximab).
2. Chimeric Monoclonal Antibodies: Chimeric antibodies are engineered to contain both mouse and human components. Typically, the variable regions (responsible for antigen binding) are derived from mouse antibodies, while the constant regions are replaced with human counterparts. This reduces the immunogenicity of the antibody in humans, as it is less likely to trigger an immune response.Typically indicated by the suffix "-ximab" (e.g., Infliximab).
3. Humanized Monoclonal Antibodies: Humanized antibodies are primarily human, with only a small portion of the molecule derived from mouse cells. Usually, only the complementarity-determining regions (CDRs), which are responsible for antigen binding, are of mouse origin. The remaining portions of the antibody are human. Often indicated by the suffix "-zumab"(e.g., Trastuzumab).
4. Fully Human Monoclonal Antibodies: These antibodies are entirely derived from human cells and do not contain any mouse components. They are less likely to trigger an immune response in humans compared to chimeric or humanized antibodies. Typically indicated by the suffix "-umab" (e.g., Adalimumab).

Explain what a hybridoma is, and explain which key phenotypes of the hybridoma are contributed by the B cell and which are contributed by the myeloma cell.

A hybridoma is a cell line generated by fusing a specific type of B cell, known as a plasma cell, with a myeloma cell, which is a cancerous B cell. This fusion results in a hybrid cell with characteristics of both parent cells.

Here's a breakdown of the key phenotypes contributed by each parent cell:

1. B Cell (Plasma Cell):
   • Antibody Production: B cells, particularly plasma cells, are responsible for producing antibodies. This ability is retained by the hybridoma, allowing it to continuously produce the desired monoclonal antibody.
   • Antigen Specificity: B cells produce antibodies that are specific to a particular antigen. This specificity is crucial in generating monoclonal antibodies that target a specific molecule or antigen of interest.
2. Myeloma Cell:
   • Immortality: Myeloma cells are cancerous and have the ability to divide indefinitely. This trait is crucial for the continuous production of the hybridoma.
   • Selection Marker: Myeloma cells often carry genetic mutations that confer resistance to certain selective agents, such as drugs or growth factors. These markers can be utilized to select for hybridomas that have successfully fused with myeloma cells.

By fusing these two cell types, the resulting hybridoma cell line combines the antibody-producing capability and antigen specificity of the B cell with the immortality and selection markers of the myeloma cell. This allows for the continuous production of monoclonal antibodies with the desired specificity.

Explain the fundamental process of flow cytometry. What is a fluorescent antibody? What is the laser used
for?

How does the flow cytometer determine which antibody(s) are bound to which cells? Why do cells move up the Y axis, and why do cells move laterally along the X axis (what do the movements tell us about the cells)?

Flow cytometry is a powerful technique used to analyze and sort cells based on various physical and chemical characteristics as they flow in a fluid stream through a beam of light. Here's an overview of the fundamental process:

1. Sample Preparation: Cells are first isolated from a biological sample (such as blood or tissue) and suspended in a fluid medium. They may undergo various treatments, including labeling with fluorescent antibodies or dyes, to selectively bind to specific cell surface markers or intracellular components.
2. Flowing the Sample: The prepared cell suspension is then introduced into the flow cytometer, where it is hydrodynamically focused into a single-cell stream. This stream passes through a flow cell, where it intersects with one or more laser beams.
3. Laser Excitation: The flow cytometer uses one or more lasers to illuminate the cells as they pass through the flow cell. The laser(s) emit a specific wavelength of light that interacts with the fluorescent dyes or antibodies bound to the cells. This interaction causes the dyes or antibodies to emit fluorescent light at different wavelengths.
4. Detection and Analysis: Detectors in the flow cytometer measure the emitted fluorescent light, capturing data on the intensity and wavelength of fluorescence emitted by each cell. This data is then processed by the flow cytometer's software to generate detailed information about the cells' characteristics, such as size, granularity, and the presence or absence of specific surface markers.

Now, to address your specific questions:

• Fluorescent Antibody: A fluorescent antibody is an antibody that has been labeled with a fluorescent dye. When this antibody binds to its target antigen on the surface of a cell, the fluorescent dye emits light upon excitation by a laser. This allows for the visualization and quantification of the bound antibody and, consequently, the presence of the target antigen on the cell.
• Laser Usage: The laser in a flow cytometer is used to excite the fluorescent dyes or antibodies bound to the cells passing through the flow cell. By emitting a specific wavelength of light, the laser induces fluorescence in the labeled cells, which is then detected by the flow cytometer's detectors.

Analyze two-dimensional flow histograms. You should be able to view and analyze the histograms to the level that you can extract information about the cells and answer the questions that are posed in the practice problems
included in this lecture packet.

To analyze a two-dimensional flow histogram, you typically examine the distribution of cells based on two parameters, usually represented on the X and Y axes. Here's a step-by-step approach:

1. Identify the Axes: Determine what each axis represents. For example, the X-axis might represent forward scatter (related to cell size or granularity), while the Y-axis might represent side scatter (related to internal complexity or granularity), or they might represent fluorescence intensity for different fluorophores.
2. Examine Cell Populations: Look for distinct clusters or populations of cells on the histogram. Each cluster represents a subpopulation of cells with similar characteristics.
3. Analyze Distribution: Pay attention to the distribution of cells within each population. Are they tightly clustered, or is there a broad distribution? This can provide information about the homogeneity or heterogeneity of the cell population.
4. Interpretation: Based on the information obtained from the histogram, you can draw conclusions about the cell populations present in the sample. For example, if there are distinct clusters with different levels of fluorescence intensity, it suggests the presence of cells expressing different levels of the target antigen.

Now, let's address practice problems typically associated with two-dimensional flow histograms:

1. Identifying Cell Populations: Look for clusters or peaks on the histogram and determine how many distinct populations are present. Are there any outliers or unusual distributions?
2. Comparing Samples: If you have multiple samples, compare their histograms to identify differences or similarities in cell populations. Are there any shifts or changes in the distribution of cells between samples?
3. Quantifying Parameters: Measure specific parameters such as the mean fluorescence intensity or the proportion of cells within a particular population. This can provide quantitative information about the sample.
4. Correlation Analysis: Examine the relationship between the parameters represented on the X and Y axes. Is there a correlation between cell size and granularity, or between fluorescence intensity and cell complexity?

By carefully analyzing two-dimensional flow histograms, you can extract valuable information about the cellular characteristics and composition of your samples.

When making monoclonal antibodies in mice, the mouse is given repeated booster shots with the antigen of interest (fact). Which one of the following statments about these booster shots is false?

a.The booster shots enhance the liklihood that the monoclonal antibody you develop will have high affinity for its epitope.

b.The booster shots increase the number of effector TH cells specific for peptides derived from the antigen of interest.

c.The booster shots expand the number of antigen-specific B cells released by the bone marrow.

d.The booster shots result in the production of more plasma cells secreting the antibody of interest.

e.The booster shots increase the likelihood that the the monoclonal antibody you develop will not be IgM (enhanced class-switching).

c. The booster shots expand the number of antigen-specific B cells released by the bone marrow.

Hybridomas are produced when a plasma cell fuses its plasma membrane with the plasma membrane of a myeloma cell (fact). Which two (2) of the following phenotypes of the hybridoma are contributed by the myeloma cell?

a.The ability to secrete antibody.

b.The ability to survive in cell culture.

c.The ability to divide indefinitely.

d.The ability to class-switch.

e.The ability to participate in ADCC.

b.The ability to survive in cell culture.

c.The ability to divide indefinitely.

In FLOW cytometry histograms, individual cells move along the X and Y axis based on the fluorescent antibodies that bind them (fact). True/False: The distance that an individual cell moves along either the X or Y axis is directly related to the amount of the antibody that is bound to its surface.

True

VP7 is a protein produced by virus X. You infect a mouse with virus X, then collect a blood sample 12 hours later and analyze the white blood cells using flow cytometry. You include a monoclonal antibody that binds to HLA-DP proteins and a monoclonal antibody that binds to VP7 (this antibody will bind to virus-infected cells). Based on the data presented in the histogram, what cell type is the most likely to be infected by virus X?

a.B lymphocytes

b.T lymphocytes

c.Dendritic cells

d.Liver cellseMacrophages

b. T lymphocytes

You infect separate mice with virus Z and virus Y. One day later you collect a sample of leukocytes from the blood of each mouse. Cells infected with virus Z express a virus protein called VP4 on their surfce, cells infected with virus Y express a virus protein called VP8 on their surface. You analyze the two samples of cells by flow cytometry using the monoclonal antibodies shown below. Do you think that CTLs are better at killing cells infected with Virus Z or cells infected with Virus Y?

a.Virus Z

b.Virus Y

a.Virus Y