analytical bioc lec 3

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Questions and Answers

What is the primary function of the variable sequences in B-cells?

To produce a uniform antibody across different cells

To stabilize the antibody structure

To create unique antibodies for antigen recognition (correct)

To enhance the recycling of antibodies

What is a major structural characteristic of IgG antibodies?

They form dimers

They have both heavy and light chains (correct)

They are homodimeric proteins

They are made of two heavy chains

What are the two types of fragments produced when an antibody is cleaved by papain?

Hc and Lc

VDJ and Fc

Fab and Fc (correct)

Fv and Fc

What do complementarity determining regions (CDRs) allow antibodies to do?

Recognize specific epitopes on antigens Signup and view all the answers

How do nanobodies differ from traditional antibodies?

They are single chain molecules Signup and view all the answers

What is the typical procedure for producing polyclonal antibodies?

Administering a recombinant protein at intervals Signup and view all the answers

What does the term 'monomeric' describe in relation to IgG antibodies?

The hetero-tetrameric configuration of the antibody Signup and view all the answers

Why is the immune serum obtained from immunized animals beneficial?

It can specifically recognize the immunized antigen Signup and view all the answers

What role do B-cells play in the immune system?

They produce antibodies for recognizing foreign molecules. Signup and view all the answers

What is the primary function of antibodies in the immune response?

To bind tightly and specifically to their target proteins. Signup and view all the answers

How are antibodies generated in B-cells?

Through random recombination of gene modules. Signup and view all the answers

What are epitopes?

Subsets of antigens recognized by specific antibodies. Signup and view all the answers

What typically happens to B-cells that recognize host molecules?

They are eliminated from the immune system. Signup and view all the answers

What characterizes the binding affinity of antibodies?

It remains within a range of $10^{-7}$ to $10^{-10} M$. Signup and view all the answers

How can distinct antigens lead to cross-reactivity?

By having a single epitope recognized by multiple antibodies. Signup and view all the answers

Which statement accurately reflects the role of antigens in the immune response?

Antigens are foreign substances that stimulate antibody production. Signup and view all the answers

What is the primary purpose of using secondary antibodies in laboratory experiments?

To recognize and label primary antibodies for detection. Signup and view all the answers

Which technique is primarily used to quantify the presence of a specific antigen in a sample?

ELISA Signup and view all the answers

What does the process of immunohistochemistry allow researchers to determine?

The location and presence of specific proteins in tissue sections. Signup and view all the answers

What is a common issue associated with polyclonal antibodies?

They may recognize similar epitopes in different proteins. Signup and view all the answers

In an ELISA assay, what is the role of the capture antibody?

To immobilize the antigen of interest on the solid surface. Signup and view all the answers

What problem can arise from using different vendors for antibodies targeting the same epitope?

Inconsistency in batch quality of antibodies. Signup and view all the answers

What is typically required to visualize the signal in an antibody-based assay?

A fluorophore or enzyme-linked detection method. Signup and view all the answers

What is an essential first step in preparing tissue for immunohistochemistry?

Fixation of the tissue. Signup and view all the answers

What is the primary difference between polyclonal and monoclonal antibodies?

Monoclonal antibodies are less likely to cross-react with undesired proteins compared to polyclonal antibodies. Signup and view all the answers

What is the significance of the hybridoma process in monoclonal antibody production?

It allows for the creation of immortal cells that produce a single antibody. Signup and view all the answers

How many antibodies are registered across all companies mentioned?

2.5 million Signup and view all the answers

Which application of antibodies is NOT mentioned in the provided content?

Detection of specific proteins on mass spectrometry Signup and view all the answers

What component constitutes the majority of the volume after centrifugation of rabbit blood?

Serum Signup and view all the answers

What is the purpose of heat treatment and chromatography steps applied to antibodies?

To inactivate and remove contaminating proteins. Signup and view all the answers

What is the initial step in the process of producing hybridoma cells?

Immunizing a mouse with a specific antigen. Signup and view all the answers

What are antibodies generally used for in laboratory applications?

Detecting and quantifying specific proteins in assays. Signup and view all the answers

Study Notes

Antibodies

Antibodies are proteins created by the immune system to specifically bind to foreign substances, known as antigens.
They are highly specific and bind tightly to their targets, making them incredibly useful for detecting and labeling proteins.
Antibodies have significant applications in both diagnostics and medicine.

Antigens and Epitopes

Antigens are substances that trigger the production of antibodies, often proteins or specific sugars.
Epitopes are specific regions of the antigen that are recognized by individual antibodies.
Different antibodies can recognize different epitopes on the same antigen.
Antigens may share similar epitopes, leading to cross-reactivity between antibodies.

Antibody Production

B-cells are the specialized immune cells that produce antibodies.
Each B-cell produces a single antibody, and there are millions of different B-cells, each with its own unique antibody.
B-cells with antibodies that recognize self-molecules are normally eliminated to prevent the immune system from attacking its own cells.
When a foreign molecule - an antigen - is detected, B-cells with antibodies that recognize that antigen proliferate, increasing the amount of that specific antibody.

Antibody Gene Recombination

Antibody genes are generated through a complex process of recombination within B-cells.
The genome doesn’t contain the exact gene sequences for each antibody, but rather multiple interchangeable modules.
B-cells randomly assemble these modules, resulting in a vast diversity of unique antibody genes.

Antibody Structure

Antibodies are composed of two chains: a heavy chain and a light chain.
Antibodies can be classified based on their structure, with some being monomeric (IgG), dimeric (IgA), or pentameric (IgM).

Antibody Fragments

Antibodies can be cleaved into smaller fragments using enzymes like papain.
This process yields Fab fragments, which retain antigen-binding specificity but are smaller.
Fabs are often used in diagnostics and therapeutic applications.

Complementarity Determining Regions (CDRs)

CDRs are hypervariable regions located on both the heavy and light chains of antibodies.
These regions form loops at the tip of the Fab fragment and are responsible for recognizing and binding specific epitopes on antigens.

Nanobodies

Nanobodies are small, single-chain antibody fragments derived from camelids and sharks.
Nanobodies are much smaller than Fabs and can be readily produced in bacteria, making them useful for various applications.

Immunization

Immunization involves injecting an animal with a specific antigen to stimulate antibody production.
The animal's serum, which contains a mixture of antibodies, can then be collected.

Polyclonal Antibodies

Polyclonal antibodies are derived directly from the immune serum of an immunized animal.
They recognize multiple epitopes on a target antigen.
Polyclonal antibodies can be purified to increase specificity but can still exhibit cross-reactivity with other proteins.

Monoclonal Antibodies

Monoclonal antibodies are produced by immortalized hybridoma cells, which are created by fusing an antibody-producing B-cell with a tumor cell.
These antibodies are highly specific, targeting a single epitope on an antigen.
Monoclonal antibodies are widely used in research, diagnostics, and therapeutics.

Production of Monoclonal Antibodies

The process involves immunizing an animal with an antigen, isolating B-cells from the spleen, and fusing them with myeloma cells.
Hybridoma cells are selected for their ability to produce antibodies against the target antigen and are then cloned for large-scale antibody production.

Applications of Antibodies

Antibodies have a wide range of applications, including:
- Detection and quantification of antigens (ELISA assays)
- Detection of specific proteins in gels (Western blotting)
- Labeling specific proteins in cells or tissues (immunostaining)
- Purification of proteins by affinity chromatography
- Inhibiting the biological activity of proteins
- Cell sorting based on surface antigens
- Disease diagnostics and therapeutic treatment

Secondary Antibodies

Secondary antibodies are antibodies that recognize and bind to antibodies from a different species.
They are often used for detection and signal amplification in antibody-based assays.
Secondary antibodies are commonly labeled with fluorophores or enzymes for visualization.

ELISA

ELISA (enzyme-linked immunosorbent assay) is a technique used to detect and quantify specific antigens in samples.
It involves immobilizing an antigen on a solid surface and using antibodies to detect its presence.
ELISA can be used for a wide variety of purposes, including medical diagnostics and research.

Immunohistochemistry

Immunohistochemistry (IHC) is a technique that uses antibodies to detect specific proteins in tissue sections.
Thin slices of tissue are stained with antibodies, which bind to their target antigens, allowing for visualization of the protein's distribution within the tissue.

Antibody Considerations

It's important to be aware of potential challenges when using antibodies:
- Polyclonal antibodies can sometimes cross-react with other proteins.
- Antibodies targeting the same epitope from different suppliers may vary in their sensitivity and specificity.
- Commercial antibodies sometimes fail to recognize the target they are supposed to bind, and polyclonal antibodies may show batch variation.

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Description

Test your understanding of antibodies, antigens, and their complex relationship in the immune system. This quiz covers the specifics of antibody production, the role of B-cells, and how antigens trigger immune responses. Gain insights into diagnostics and clinical applications of antibodies.