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Questions and Answers

What is a characteristic of homogenous immunoassays?

They can be either competitive or non-competitive. (correct)

They are only applicable for large proteins.

They require multiple washing steps.

They necessitate separation of components before analysis.

What is the primary purpose of an immunoassay?

To exploit the properties of antibody-antigen complexes. (correct)

To detect and quantify antibody concentrations.

To separate proteins from each other.

To measure the volume of a solution.

Which statement best describes heterogenous immunoassays?

They can be performed with no separation steps.

They require multiple steps of washing or blocking. (correct)

They involve a single-step filtration process.

They are faster than homogenous assays.

What does the term 'conjugation' in immunoassays refer to?

The binding of a label to either antibody or antigen. Signup and view all the answers

Which of the following is NOT a consideration for immunoassay design?

Determining the concentration of the antibodies. Signup and view all the answers

What type of standard curve is generated when plotting the concentration of bound label against analyte concentration?

Inverse standard curve Signup and view all the answers

In a homogeneous competitive immunoassay, what do both the labeled and unlabeled analytes compete for?

A limited amount of antibody binding sites Signup and view all the answers

Which of the following is NOT a component that can be conjugated to antibodies or antigens for detection?

Cellular organelles Signup and view all the answers

Which technique involves linking antibodies or antigens to another molecule for detection and quantitation?

Conjugation Signup and view all the answers

What is the purpose of washing in the competitive immunoassay process?

To remove unbound materials Signup and view all the answers

What is a characteristic of non-competitive immunoassays?

An excess amount of antibody is used to capture analyte from the sample. Signup and view all the answers

How does the signal in a non-competitive immunoassay correlate with the antigen concentration?

The signal generated is directly proportional to the concentration of antigen present. Signup and view all the answers

Which of the following statements about competitive immunoassays is true?

They use either labeled antigen or labeled antibody. Signup and view all the answers

What is an advantage of non-competitive immunoassays?

They provide excellent detection in complex sample types. Signup and view all the answers

What is a limitation of non-competitive immunoassays?

They need carefully matched antibody pairs. Signup and view all the answers

Which solid phases can be used in heterogeneous assays?

Agarose, polystyrene beads, and magnetic particles. Signup and view all the answers

What is a key factor in the effectiveness of competitive immunoassays?

They function by measuring the amount of label present. Signup and view all the answers

What potential issue is associated with using micro-titre plates in assays?

They may cause edge effects due to thermal conductivity. Signup and view all the answers

Study Notes

Introduction to Immunoassays

Immunoassays use immunochemical methods to exploit the properties of antibody-antigen (Ab-Ag) complexes
Antibodies (Abs) are used as reagents to detect and quantify antigens (Ags)
A wide range of compounds, from small molecules to large proteins, can be measured using immunoassays

Learning Objectives

Describe the characteristics of homogenous and heterogeneous assays
Outline the principles of non-competitive and competitive immunoassays
Understand the meaning of conjugation
List components of immunoassays that can be conjugated to antibodies and antigens

Immunoassay Design

Careful selection of antibodies (Abs) with appropriate binding strength and specificity is crucial
Abs should exhibit high affinity and high specificity for the target antigen
Choosing the correct label and detection method is essential for measuring Ab:Ag binding
Labelled Abs and Ag complexes are combined through covalent attachments (conjugation)
Enzyme-substrate interactions generate signals that are detected
Signal detection is a critical step in immunoassay design

Homogenous vs. Heterogenous Assays

Homogenous immunoassays: Do not require separation steps before analysis; reagents are added simultaneously; can be competitive or non-competitive
Heterogenous immunoassays: Require separation steps (e.g., filtration, washing, blocking) before analysis; typically multiple steps; can be competitive or non-competitive; are the most common type of immunoassay

Heterogenous Assay Characteristics

Can be either competitive or non-competitive
Immuno-reagents are attached to a solid phase (e.g., agarose, polystyrene beads, microtiter plates) via covalent or non-covalent (adsorption) methods
Factors to consider during heterogeneous assay design include:
- Immunoreaction or Ab-Ag binding should not affect signal generation
- The enzyme label should retain its activity after the immunoreaction
- Non-specific adsorption of components can affect the results
- Solid phase material and edge effects can impact the results

Non-competitive Immunoassays

An excess of unlabelled antibody captures the analyte from the sample
A labelled secondary antibody binds to the captured analyte forming a 'sandwich' complex
The signal generated, measured spectrophotometrically, is directly proportional to the analyte concentration
Non-competitive techniques are generally more sensitive and reproducible than competitive assays

Non-competitive Immunoassays (Sandwich ELISA)

Unlabeled capture antibody is immobilzed on a solid surface
Sample (analyte) is added
Capture antibody binds to its target antigen within the sample
Detection antibody (linked to an enzyme) is added, binding to the second region of the antigen to form a 'sandwich'
An enzyme substrate is added to generate a signal (colorimetric changes are commonly measured spectrophotometrically)
Wash steps are done to remove unbound components

Non-competitive Immunoassays: Advantages and Limitations

Advantages: Excellent detection in complex samples such as plasma and serum; high specificity and sensitivity
Limitations: Requires carefully matched antibody pairs; difficult to test very small antigens

Heterogenous Competitive Immunoassays

Labeled and unlabeled analytes (antigens) compete for a limited number of binding sites on the antibody
Less label measured in the assay means more unlabeled competitor antigen is present (inverse relationship)
Concentration of bound label is plotted against the analyte concentration resulting in an inverse standard curve

Homogenous Competitive Immunoassays

Both the labeled antigen and the unlabeled antigen (from the sample) compete for a limited amount of antibody
Fewer labeled complexes found when analyte concentrations are higher

Competitive Immunoassays

Capture antibody is immobilized on a solid phase
Analyte (antigen) and enzyme-labelled analyte compete for limited antibody binding sites
Washing removes unbound material
Enzyme substrate is added to produce a signal

Activity - Comparison of ELISA Procedures

Sandwich ELISA: Antibody-coated well → analyte addition → enzyme-conjugated antibody addition
Competitive ELISA: Antibody-coated well → analyte and labelled analyte addition → enzyme-conjugated antibody addition (signals inversely correlate with analyte concentration)

Conjugation & Detection Techniques

Conjugation is linking either antigen or antibody to another molecule for detection or quantitation
Antigens or antibodies can be conjugated to various molecules including:
- Enzymes (e.g., alkaline phosphatase, horseradish peroxidase)
- Fluorescent labels (e.g., fluorescein isothiocyanate)
- Biotin/avidin
- Beads (e.g., polystyrene, magnetic)
- Isotopic labels (e.g., ³H, ¹²⁵I, ¹⁴C)

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Description

This quiz focuses on the principles and design of immunoassays, including the roles of antibodies and antigens in detecting and quantifying compounds. Learn about the characteristics of different assay types, conjugation techniques, and the importance of affinity and specificity in antibody selection.

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